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Ka ārai te arai toto-roro me te arai toto-roro i ngā rongoā koiora ki te tae ki ō rātou whāinga i roto i te pūnaha io matua, ā, ka ārai i te maimoatanga whai hua o ngā mate io. Hei kimi i ngā kaikawe roro hou i roto i te tinana ora, i whakauruhia e mātou he whare pukapuka peptide phage T7, ā, i kohia te toto me te wai cerebrospinal (CSF) mā te whakamahi i tētahi tauira puna nui o ngā kiore kua whakapūmautia. I tino whakanuia ngā tārua phage motuhake i roto i te CSF i muri i ngā wāhanga e whā o te kōwhiringa. I whakaatuhia e te whakamātautau i ngā peptide kaitono takitahi neke atu i te 1000 ngā wā whakanui i roto i te CSF. I whakaūhia te mahi koiora o te tuku peptide-mediated ki te roro e te 40% hekenga o te taumata o te amyloid-β i roto i te wai cerebrospinal mā te whakamahi i tētahi aukati peptide BACE1 e hono ana ki te peptide transit hou kua tautuhia. E kī ana ēnei hua ko ngā peptide i tautuhia e ngā tikanga kōwhiringa phage in vivo he waka whai hua pea mō te tuku pūnaha o ngā macromolecules ki te roro me te pānga rongoā.
Ko te rangahau whakaora i arotahi ki te pūnaha io matua (CNS) kua aro nui ki te tautuhi i ngā rongoā me ngā kaihoko kua arotauhia e whakaatu ana i ngā āhuatanga arotahi ki te CNS, me te iti ake o te kaha ki te kimi i ngā tikanga e akiaki ana i te tuku rongoā kaha ki te roro. Kei te tīmata tēnei ki te huri i nāianei i te mea ko te tuku rongoā, inā koa ko ngā ngota nui, he wāhanga nui o te whanaketanga rongoā neuroscience hou. Ko te taiao o te pūnaha io matua e tiakina pai ana e te pūnaha arai toto-roro, kei roto ko te arai toto-roro (BBB) ​​​​me te arai toto-roro (BCBB)1, e uaua ai te tuku rongoā ki te roro1,2. E kiia ana ko te nuinga o ngā rongoā ngota nui me te neke atu i te 98% o ngā rongoā ngota iti ka tangohia mai i te roro3. Koia te take he mea tino nui ki te tautuhi i ngā pūnaha kawe roro hou e whakarato ana i te tuku rongoā whakaora whai hua me te motuhake ki te CNS 4,5. Heoi, ko te BBB me te BCSFB he whai wāhitanga pai hoki mō te tuku rongoā i a rātou e uru ana ki roto i ngā hanganga katoa o te roro mā roto i ōna toto whānui. Nō reira, ko ngā mahi o nāianei ki te whakamahi i ngā tikanga tuku kore-whakaeke ki te roro e ahu mai ana i te tikanga o te kawe a te kaiwhakawhiwhi (PMT) mā te whakamahi i te kaiwhakawhiwhi BBB6 taketake. Ahakoa ngā whanaketanga nui o nāianei mā te whakamahi i te ara kaiwhakawhiwhi transferrin7,8, me whakawhanake tonu i ngā pūnaha tuku hou me ngā āhuatanga kua whakapaitia. Hei whakatutuki i tēnei, ko tā mātou whāinga he tautuhi i ngā peptide e kaha ana ki te whakahaere i te kawe CSF, i te mea ka taea te whakamahi i ēnei hei tuku i ngā macromolecules ki te CNS, hei whakatuwhera rānei i ngā ara kaiwhakawhiwhi hou. Ina koa, ko ngā kaiwhakawhiwhi me ngā kaikawe motuhake o te pūnaha roro (BBB me BSCFB) ka taea te mahi hei whāinga pea mō te tuku kaha me te motuhake o ngā rongoā koiora. Ko te wai roro (CSF) he hua huna o te choroid plexus (CS) ā, kei te pā tika ki te wai interstitial o te roro mā te wāhi subarachnoid me te wāhi ventricular4. Nō nā tata nei kua whakaaturia ko te wai roro subarachnoid ka horapa nui ki roto i te interstitium o te roro9. Ko tā mātou e tumanako nei kia uru atu ki te wāhi parenchymal mā te whakamahi i tēnei ara rerenga subarachnoid, mā te BBB rānei. Hei whakatutuki i tēnei, i whakatinanahia e mātou he rautaki whiriwhiri phage in vivo pakari e tino tautuhi ana i ngā peptide e haria ana e tētahi o ēnei ara motuhake e rua.
Ka whakaahuahia e mātou ināianei tētahi tikanga tirotiro whakaaturanga phage in vivo me te tauira CSF me te raupapatanga tere (HTS) hei aroturuki i ngā rauna kōwhiringa tuatahi me te kanorau whare pukapuka teitei rawa atu. I mahia te tirotiro ki ngā kiore mohio me te cannula cisterna nui (CM) kua whakatōkia tuturu hei karo i te poke o te toto. He mea nui, ka tīpakohia e tēnei huarahi ngā peptide e arotahi ana ki te roro me ngā peptide me te mahi kawe puta noa i te arai roro. I whakamahia e mātou ngā phage T7 nā te iti o te rahi (~60 nm)10 me te kī he pai mō te kawe i ngā vesicles e taea ai te whiti i te arai endothelial me/ranei epithelial-medulla. Whai muri i ngā rauna e whā o te panning, i wehea ngā taupori phage e whakaatu ana i te whakanui kaha o te CSF in vivo me te hononga microvascular roro. He mea nui, i taea e mātou te whakaū i ā mātou kitenga mā te whakaatu ko ngā peptide kaitono pai rawa atu me te hanga matū ka taea te kawe i te kawenga pūmua ki roto i te wai roro. Tuatahi, i whakatūria ngā pānga pharmacodynamic o te CNS mā te whakakotahi i tētahi peptide kawe matua me tētahi aukati o te peptide BACE1. Haunga te whakaatu ka taea e ngā rautaki tirotiro mahi in vivo te tautuhi i ngā peptide kawe roro hou hei kawe utanga pūmua whai hua, e tumanakohia ana ka noho hei mea nui anō hoki ngā huarahi kōwhiringa mahi rite ki te tautuhi i ngā ara kawe roro hou.
I runga i ngā waeine hanga-papa (PFU), i muri i te taahiraa tākai phage, i hoahoatia, i hangaia hoki he whare pukapuka o ngā peptide phage T7 rārangi 12-mer matapōkere me te kanorau o te 109 pea (tirohia ngā Rauemi me ngā Tikanga). He mea nui kia mōhio i ata tātarihia e mātou tēnei whare pukapuka i mua i te panning in vivo. Ko te whakanui PCR o ngā tauira whare pukapuka phage mā te whakamahi i ngā primers kua whakarerekētia i whakaputa i ngā amplicons e tika ana ki te HTS (Pikitia Tāpiri 1a). Nā te a) ngā hapa raupapatanga HTS11, b) te pānga ki te kounga o ngā primers (NNK)1-12, me te c) te aroaro o te phage momo-mohoao (ngā whakaurunga angaanga) i roto i te whare pukapuka tūtatari, i whakatinanahia he tikanga tātari raupapatanga hei tango i ngā mōhiohio raupapatanga kua manatokohia anake (Pikitia Tāpiri 1b). Ka pā ēnei taahiraa tātari ki ngā whare pukapuka raupapatanga HTS katoa. Mō te whare pukapuka paerewa, i whiwhihia he 233,868 pānui katoa, 39% o ēnei i eke ki ngā paearu tātari, ā, i whakamahia mō te tātari whare pukapuka me te kōwhiringa mō ngā rauna e whai ake nei (Pikitia Tāpiri 1c–e). Ko te nuinga o ngā pānui he maha o ngā takirua turanga e 3 te roa me te tihi i te 36 nucleotides (Pikitia Tāpiri 1c), e whakaū ana i te hoahoa whare pukapuka (NNK) 1-12. He mea nui, tata ki te 11% o ngā mema o te whare pukapuka kei roto he whakauru PAGISRELVDKL tuara momo-mohoao (wt) 12-ahu, ā, tata ki te haurua o ngā raupapatanga (49%) kei roto he whakaurunga, he mukunga rānei. I whakaū te HTS o te whare pukapuka i te kanorau nui o ngā peptides i roto i te whare pukapuka: neke atu i te 81% o ngā raupapatanga peptide i kitea kotahi noa iho, ā, 1.5% anake i puta i roto i te ≥4 tārua (Pikitia Tāpiri 2a). Ko ngā auau o ngā waikawa amino (aa) i ngā tūranga 12 katoa i roto i te repertoire i hono pai ki ngā auau e tumanakohia ana mō te maha o ngā codon i hangaia e te repertoire NKK kua heke (Pikitia Tāpiri 2b). Ko te auau i kitea o ngā toenga aa i whakakōrerohia e ēnei whakaurunga i hono pai ki te auau i tatauhia (r = 0.893) (Pikitia Tāpiri 2c). Ko te whakarite i ngā whare pukapuka phage mō te werohanga ko ngā mahi whakanui me te tango i te endotoxin. Kua whakaaturia i mua ko tēnei ka taea te whakaiti i te kanorau o ngā whare pukapuka phage12,13. Nō reira, i raupapahia e mātou he whare pukapuka phage kua whakanuia e te pereti kua tangohia te endotoxin, ā, i whakaritea ki te whare pukapuka taketake hei whakatau tata i te auau o te AA. I kitea he hononga kaha (r = 0.995) i waenga i te puna taketake me te puna kua whakanuia me te purea (Pikitia Tāpiri 2d), e tohu ana kāore te whakataetae i waenga i ngā ira i whakanuia i runga i ngā pereti mā te whakamahi i te phage T7 i puta ai he pānga nui. Ko tēnei whakataurite e hangai ana ki te auau o ngā tohu tripeptide i roto i ia whare pukapuka, nā te mea kāore e taea te hopu katoa i te kanorau o ngā whare pukapuka (~109) ahakoa me te HTS. I whakaatuhia e te tātari auau o te aa i ia tūranga he pānga iti e whakawhirinaki ana ki te tūranga i roto i ngā tūranga whakamutunga e toru o te repertoire i whakaurua (Pikitia Tāpiri 2e). Hei whakamutunga, i whakatau mātou he pai te kounga me te kanorau o te whare pukapuka, ā, he iti noa ngā huringa o te kanorau i kitea nā te whakanui me te whakarite o ngā whare pukapuka phage i waenga i ētahi rauna kōwhiringa.
Ka taea te tango tauira wai cerebrospinal raupapa mā te pokanga i te cannula ki roto i te CM o ngā kiore mohio hei whakahaere i te tautuhi i te phage T7 i werohia mā te uaua (iv) mā te BBB me/ranei te BCSFB (Pikitia 1a-b). I whakamahia e mātou ngā ringa whiriwhiri motuhake e rua (ngā ringa A me B) i ngā rauna tuatahi e toru o te kōwhiringa in vivo (Pikitia 1c). I whakanuia haeretia e mātou te kaha o te kōwhiringa mā te whakaiti i te nui katoa o te phage i whakaurua i ngā rauna tuatahi e toru o te kōwhiringa. Mō te rauna tuawhā o te panning, i whakakotahihia e mātou ngā tauira mai i ngā peka A me B, ā, i mahia ētahi atu kōwhiringa motuhake e toru. Hei ako i ngā āhuatanga in vivo o ngā matūriki phage T7 i roto i tēnei tauira, i werohia te phage momo-mohoao (PAGISRELVDKL master insert) ki roto i ngā kiore mā te uaua hiku. Ko te whakaoranga o ngā phage mai i te wai cerebrospinal me te toto i ngā wā rerekē i whakaatu ko ngā phage icosahedral T7 iti he wāhanga whakakorenga tuatahi tere mai i te wāhanga toto (Pikitia Tāpiri 3). I runga i ngā taitā i tukuna me te nui o te toto o ngā kiore, i tatauhia e mātou tata ki te 1% te taumaha o te phage mai i te horopeta i tukuna i kitea i roto i te toto 10 meneti i muri i te werohanga uaua. Whai muri i tēnei hekenga tere tuatahi, i inehia he whakakorenga tuatahi puhoi ake me te haurua-ora o te 27.7 meneti. He mea nui, he iti noa iho ngā phage i tangohia mai i te wāhanga CSF, e tohu ana i te iti o te papamuri mō te hekenga o te phage momo-mohoao ki roto i te wāhanga CSF (Pikitia Tāpiri 3). I te toharite, tata ki te 1 x 10-3% noa iho ngā taitā o te phage T7 i roto i te toto me te 4 x 10-8% o ngā phage i whakaurua tuatahitia i kitea i roto i te wai cerebrospinal puta noa i te wā tauira katoa (0-250 meneti). He mea nui, ko te haurua-ora (25.7 meneti) o te phage momo-mohoao i roto i te wai cerebrospinal he rite ki tera i kitea i roto i te toto. E whakaatu ana ēnei raraunga kei te ora tonu te arai e wehe ana i te wāhanga CSF mai i te toto i roto i ngā kiore i whakauruhia ki te CM, e āhei ai te kōwhiringa in vivo o ngā whare pukapuka phage ki te tautuhi i ngā tāruarua ka taea te kawe ngāwari mai i te toto ki te wāhanga CSF.
(a) Te whakatū i tētahi tikanga mō te tango tauira anō i te wai cerebrospinal (CSF) mai i tētahi puna nui. (b) Te hoahoa e whakaatu ana i te wāhi pūtau o te ārai pūnaha io matua (CNS) me te rautaki kōwhiringa i whakamahia hei tautuhi i ngā peptide e whiti ana i te ārai toto-roro (BBB) ​​me te ārai toto-roro. (c) Te tūtohi rere o te whakaaturanga phage in vivo. I ia rauna kōwhiringa, i werohia ngā phage (ngā tohu kararehe i roto i ngā pere) mā te uaua. E rua ngā peka rerekē motuhake (A, B) e puritia motuhaketia ana tae noa ki te rauna 4 o te kōwhiringa. Mō ngā rauna kōwhiringa 3 me te 4, i raupapahia ā-ringa ia clone phage i tangohia mai i te CSF. (d) Te nekehanga o te phage i wehea mai i te toto (ngā porowhita whero) me te wai cerebrospinal (ngā tapatoru matomato) i te rauna tuatahi o te kōwhiringa i roto i ngā kiore e rua i muri i te werohanga uaua o te whare pukapuka peptide T7 (2 x 1012 phage/kararehe). Ko ngā tapawhā kikorangi e tohu ana i te kukū tīmatanga toharite o te phage i roto i te toto, i tatauhia mai i te nui o te phage i werohia, me te whakaaro ki te rōrahi toto katoa. Ko ngā tapawhā pango e tohu ana i te pūwāhi o te whakawhiti o te rārangi y i tangohia mai i ngā kukū phage toto. (e,f) Whakaatuhia te auau me te tohatoha whanaunga o ngā motika tripeptide taupatupatu katoa e kitea ana i roto i te peptide. Ka whakaaturia te maha o ngā motika i kitea i roto i te 1000 pānui. He mea nui (p < 0.001) kua tohua ngā motika whakarei ki ngā ira whero. (e) Kauwhata marara hononga e whakataurite ana i te auau whanaunga o te motika tripeptide o te whare pukapuka i werohia ki te phage i ahu mai i te toto mai i ngā kararehe #1.1 me #1.2. (f) Kauwhata marara hononga e whakataurite ana i ngā auau whanaunga o ngā motika tripeptide phage kararehe #1.1 me #1.2 i wehea i roto i te toto me te wai cerebrospinal. (g, h) Whakaaturanga ID raupapa o te phage i whakareihia i roto i te toto (g) ki ngā whare pukapuka i werohia me te phage i whakareihia i roto i te CSF (h) ki te toto i muri i te whiringa in vivo i roto i ngā kararehe e rua. Ko te rahi o te waehere reta kotahi e whakaatu ana i te auau o te puta o taua waikawa amino i taua tūranga. Kakariki = pōro, papura = kūpapa, kikorangi = taketake, whero = waikawa me te mangu = waikawa amino hydrophobic. Nā Eduard Urich i hoahoa, i whakaputa hoki te Pikitia 1a, b.
I werowerohia e mātou he whare pukapuka peptide phage ki roto i ngā kiore taputapu CM e rua (ngā clades A me B) ā, i wehea te phage mai i te wai cerebrospinal me te toto (Pikitia 1d). Ko te tere o te whakakorenga tuatahi o te whare pukapuka he iti ake te kitea ki te phage momo-mohoao. Ko te haurua-ora toharite o te whare pukapuka i werowerohia i roto i ngā kararehe e rua he 24.8 meneti i roto i te toto, he rite ki te phage momo-mohoao, me te 38.5 meneti i roto i te CSF. Ko ngā tauira phage toto me te wai cerebrospinal mai i ia kararehe i tukuna ki te HTS, ā, i tātarihia ngā peptide katoa i tautuhia mō te aroaro o te motif tripeptide poto. I whiriwhiria ngā motif tripeptide nā te mea he iti noa te pūtake mō te hanganga hanganga me ngā taunekeneke peptide-pūmua14,15. I kitea e mātou he hononga pai i roto i te tohatoha o ngā motif i waenga i te whare pukapuka phage i werowerohia me ngā clones i tangohia mai i te toto o ngā kararehe e rua (Pikitia 1e). E tohu ana ngā raraunga he iti noa te whakanui ake o te hanganga o te whare pukapuka i roto i te wāhanga toto. I tātarihia anō ngā auau waikawa amino me ngā raupapatanga whakaae i ia tūranga mā te whakamahi i te urutaunga o te pūmanawa Weblogo16. He mea whakamīharo, i kitea e mātou he nui te whakanui i ngā toenga glycine toto (Pikitia 1g). I te whakatauritea o te toto ki ngā ira i tīpakohia mai i te CSF, i kitea he kaha te kōwhiringa me te tango i ētahi tohu (Pikitia 1f), ā, ko ētahi waikawa amino i kitea i ngā tūranga kua whakaritea i roto i te 12-mema (Pikitia 1h). He mea nui, he rerekē te wai cerebrospinal o ia kararehe, engari i kitea te whakanui glycine toto i roto i ngā kararehe e rua (Pikitia Tāpiri 4a–j). Whai muri i te tātari pakari o ngā raraunga raupapa i roto i te wai cerebrospinal o ngā kararehe #1.1 me #1.2, i whiwhihia he peptide 12-mer ahurei 964 me te 420 katoa (Pikitia Tāpiri 1d–e). I whakanuia ngā ira phage motuhake, ā, i tukuna ki te tuarua o ngā whiringa in vivo. Ko ngā phage i tangohia mai i te tuarua o ngā whiringa i tukuna ki te HTS i roto i ia kararehe, ā, i whakamahia ngā peptide katoa i tautuhia hei whakauru ki tētahi kaupapa tautuhi tohu hei tātari i te putanga mai o ngā tohu tripeptide (Pikitia 2a, b, ef). Ki te whakataurite ki te huringa tuatahi o te phage i tangohia mai i te CSF, i kitea e mātou he kōwhiringa me te tango kōwhiringa anō o ngā motif maha i roto i te CSF i ngā peka A me B (Pikitia 2). I whakamahia he pūnaha tautuhi whatunga hei whakatau mena he rerekē ngā tauira o te raupapatanga ōrite. I kitea he ōritetanga mārama i waenga i ngā raupapatanga 12-ahu i tangohia e te CSF i roto i te clade rerekē A (Pikitia 2c, d) me te clade B (Pikitia 2g, h). I whakaatuhia e te tātaritanga whakakotahi i ia peka he rerekē ngā kōtaha kōwhiringa mō ngā peptide 12-mer (Pikitia Tāpiri 5c, d) me te pikinga o te ōwehenga CSF/titer toto i roto i te wā mō ngā clones whakakotahi i muri i te rauna tuarua o te kōwhiringa ki te whakataurite ki te rauna tuatahi o te kōwhiringa (Pikitia Tāpiri 5e).
Te whakarei ake i ngā tohu me ngā peptide i roto i te wai cerebrospinal mā te rua o ngā huringa o te kōwhiringa whakaaturanga phage mahi in vivo.
Ko ngā pūtau wai roro katoa i kohia mai i te rauna tuatahi o ia kararehe (kararehe #1.1 me te #1.2) i kohikohia, i whakanuia, i raupapahia te HT, ā, i werohia ngātahitia (2 x 1010 pūtau/kararehe) 2 kiore SM i whakapūtāhia (#1.1 → #). 2.1 me te 2.2, 1.2 → 2.3 me te 2.4). (a,b,e,f) Ngā kauwhata marara hononga e whakarite ana i te auau whanaunga o ngā tohu tripeptide o ngā pūtau CSF katoa i ahu mai i ngā rauna kōwhiringa tuatahi me te tuarua. Auau whanaunga me te tohatoha o ngā tohu e tohu ana i ngā tripeptide taupatupatu katoa i kitea i roto i ngā peptide i ngā aronga e rua. E whakaaturia ana te maha o ngā tohu i kitea i roto i te 1000 pānui. Ko ngā tohu i tīpakohia, i tangohia rānei (p < 0.001) i roto i tētahi o ngā whare pukapuka kua whakaritea, kua tohua ki ngā ira whero. (c, d, g, h) Te whakaaturanga o te tohu raupapa o ngā raupapatanga roa katoa o ngā waikawa amino 12 whai rawa i te CSF i runga i ngā rauna 2 me te 1 o te kōwhiringa in vivo. Ko te rahi o te waehere reta kotahi e tohu ana i te auau o te puta o taua waikawa amino i taua tūranga. Hei tohu i te tohu, ka whakaritea te auau o ngā raupapatanga CSF i tangohia mai i ngā kararehe takitahi i waenga i ngā rauna kōwhiringa e rua, ā, ka whakaaturia ngā raupapatanga whai rawa i te rauna tuarua: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 me (h) #1.2–#2.4. Ko ngā waikawa amino tino whai rawa i tētahi tūranga i roto i ngā kararehe nama 2.1 me nama 2.2 (g, h) rānei i roto i ngā kararehe nama 2.3 me nama 2.4 ka whakaaturia i roto i te tae. Kakariki = pōro, papura = kūpapa, kikorangi = taketake, whero = waikawa me te mangu = waikawa amino hydrophobic.
I muri i te tuatoru o ngā rauna kōwhiringa, i kitea e mātou he 124 ngā raupapa peptide ahurei (#3.1 me #3.2) mai i ngā ira phage kua whakahoutia ki te CSF e 332 i wehea mai i ngā kararehe e rua (Pikitia Tāpiri 6a). Ko te raupapa LGSVS (18.7%) te mea nui rawa atu te ōwehenga whanaunga, ā, i muri mai ko ngā whakaurunga momo-mohoao PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), me SARGSWREIVSLS (2.2%). I te rauna tuawhā whakamutunga, i kohikohia e mātou ētahi peka e rua kua tīpakohia motuhake mai i ngā kararehe motuhake e toru (Pikitia 1c). Mai i ngā ira phage raupapa 925 i whakahokia mai i te CSF, i te rauna tuawhā i kitea e mātou he 64 ngā raupapa peptide ahurei (Pikitia Tāpiri 6b), i roto i ēnei i heke te ōwehenga whanaunga o te phage momo-mohoao ki te 0.8%. Ko ngā tārua CSF tino kitea i te tuawhā o ngā rauna ko LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) me RLSSVDSDLSGC (3, 2%). %)). Ko te whānuitanga roa o ngā peptide kua tīpakohia nā ngā whakaurunga/whakakorenga nucleotide, ngā codon aukati wawe rānei i roto i ngā primers whare pukapuka i te wā e whakamahi ana i ngā codon kua pirau mō te hoahoa whare pukapuka NNK. Ka whakaputahia e ngā codon aukati wawe ngā peptide poto ake, ā, ka tīpakohia nā te mea kei roto i a rātou te kaupapa aa pai. Ka puta pea ngā peptide roa ake i ngā whakaurunga/whakakorenga i roto i ngā primers o ngā whare pukapuka hanga. Mā tēnei ka whakanohohia te codon aukati kua hoahoatia ki waho o te anga, ā, ka pānuihia kia puta rā anō he codon aukati hou ki raro iho. I te nuinga o te wā, i tatauhia e mātou ngā tauwehe whakarei mō ngā rauna kōwhiringa e whā mā te whakataurite i ngā raraunga whakauru ki ngā raraunga putanga tauira. Mō te wāhanga tuatahi o te tirotiro, i whakamahia e mātou ngā taitara phage momo-mohoao hei tohutoro papamuri kore-motuhake. He mea whakamīharo, he tino kaha te kōwhiringa phage kino i te huringa CSF tuatahi, engari kāore i roto i te toto (Pikitia 3a), tērā pea nā te iti o te tūponotanga o te horapa kore mahi o te nuinga o ngā mema o te whare pukapuka peptide ki roto i te wāhanga CSF, he pai ake rānei te pupuri, te tango rānei i ngā phage whanaunga mai i te toto i ngā bacteriophage. Heoi, i te wāhanga tuarua o te panning, i kitea te kōwhiringa kaha o ngā phage i roto i te CSF i ngā clade e rua, e kī ana ko te wāhanga o mua i whakanuia i roto i ngā phage e whakaatu ana i ngā peptide e whakatairanga ana i te tango CSF ​​(Pikitia 3a). Anō, kāore he whakanui nui i te toto. I roto anō i ngā wāhanga tuatoru me te tuawhā, i whakanuia nuitia ngā clone phage i roto i te CSF. I te whakataurite i te auau whanaunga o ia raupapa peptide ahurei i waenga i ngā wāhanga whakamutunga e rua o te kōwhiringa, i kitea e mātou he nui ake te whakanui o ngā raupapa i te wāhanga tuawhā o te kōwhiringa (Pikitia 3b). I tangohia he 931 ngā motif tripeptide katoa mai i ngā raupapa peptide ahurei 64 katoa mā te whakamahi i ngā whakatakotoranga peptide e rua. Ko ngā motika tino whai rawa i te tuawhā o ngā rauna i tirotirohia tata ake mō ō rātou kōtaha whakarei puta noa i ngā rauna katoa, ki te whakataurite ki te whare pukapuka i werohia (tapahi: 10% whakarei) (Pikitia Tāpiri 6c). I whakaatuhia e ngā tauira whānui o te kōwhiringa ko te nuinga o ngā motika i akohia i whakarei ake i ngā rauna katoa o mua o ngā peka kōwhiringa e rua. Heoi, ko ētahi motika (hei tauira, SGL, VSG, LGS GSV) i ahu mai i te momo rerekē A, ko ētahi atu (hei tauira, FGW, RTN, WGF, NTR) i whakarei ake i te momo rerekē B.
Te whakamana i te kawe CSF o ngā peptide e whakaatuhia ana e te phage kua whakareihia ki te CSF me ngā peptide ārahi biotinylated kua honoa ki ngā kawenga streptavidin.
(a) Ngā ōwehenga whakarei i tatauhia i ngā rauna e whā (R1-R4) i runga i ngā taitoru o te phage (PFU) i werohia (whakauru = I) me ngā taitoru o te phage CSF kua whakatauhia (putanga = O). I tatauhia ngā āhuatanga whakarei mō ngā rauna whakamutunga e toru (R2-R4) mā te whakataurite ki te rauna o mua me te rauna tuatahi (R1) me ngā raraunga taumaha. Ko ngā pae tuwhera he wai cerebrospinal, ko ngā pae atarangi he plasma. (***p<0.001, i runga i te whakamātautau-t a Student). (b) Rārangi o ngā peptide phage tino nui, i whakarōpūhia i runga i tō rātou ōwehenga whanaunga ki ngā phage katoa i kohia i roto i te CSF i muri i te rauna 4 o te kōwhiringa. Ko ngā clone phage e ono e tino kitea ana kua tohua ki te tae, kua tau me ō rātou āhuatanga whakarei i waenga i ngā rauna 3 me te 4 o te kōwhiringa (ngā whakaurunga). (c,d) Ko ngā clone phage e ono e tino whakareihia ana, ngā whare pukapuka peptide phage kau me te phage mātua mai i te rauna 4 i tātarihia takitahi i roto i te tauira tauira CSF. I kohia ngā tauira CSF me te toto i ngā wā kua tohua. (c) He rite tonu te rahinga o ngā tauira phage kaitono e 6 (2 x 1010 phage/kararehe), ngā tauira kau (#1779) (2 x 1010 phage/kararehe) me ngā whare pukapuka peptide phage kararehe (2 x 1012 phage/kararehe) Ka werohia kia 3 CM te iti rawa ka tukuna motuhake ki te kararehe kua werohia mā te uaua hiku. Ka whakaaturia te pharmacokinetics CSF o ia tauira phage me te whare pukapuka peptide phage i werohia i roto i te wā. (d) e whakaatu ana i te ōwehenga CSF/toto toharite mō ngā phage/mL katoa i whakahokia mai i roto i te wā tauira. (e) E whā ngā peptide kaiārahi hanga me te mana whakahaere kotahi i whakaranua ki te biotin ki te streptavidin mā roto i tō rātou N-terminus (whakaaturanga tetramer) ā, muri iho ka werohia (uaua hiku iv, 10 mg streptavidin/kg). Kia toru ngā kiore kua whakauruhia ki roto i te ngongo (N = 3). ). I kohia ngā tauira CSF i ngā wā kua tohua, ā, i inehia ngā kukū streptavidin mā te CSF anti-streptavidin ELISA (nd = kāore i kitea). (*p<0.05, **p<0.01, ***p<0.001, i runga i te whakamātautau ANOVA). (f) Te whakataurite i te raupapatanga waikawa amino o te phage peptide clone #2002 (papura) tino whai rawa me ētahi atu phage peptide clone kua tīpakohia mai i te 4 o ngā rauna o te kōwhiringa. Kua tohua ngā kongakonga waikawa amino ōrite me ngā kongakonga rite.
O ngā phage whakarei katoa i te tuawhā o ngā rauna (Pikitia 3b), e ono ngā tauira kaitono i tīpakohia mō ētahi atu tātaritanga takitahi i roto i te tauira tauira CSF. He rite tonu te nui o ngā whare pukapuka peptide phage kaitono e ono, phage kau (kāore he whakaurunga) me te prophage i werowerohia ki roto i ngā kararehe CM e toru kua whakapūtāhia, ā, i whakatauhia ngā pharmacokinetics i roto i ngā whakamātautau CSF (Pikitia 3c) me te toto (Pikitia Tāpiri 7). Ko ngā tauira phage katoa i whakamatautauria i aro ki te wāhanga CSF i te taumata 10-1000 ngā wā teitei ake i te phage whakahaere kau (#1779). Hei tauira, ko ngā tauira #2020 me #2077 he tata ki te 1000 ngā wā teitei ake ngā taitara CSF i te phage whakahaere. He rerekē te kōtaha pharmacokinetic o ia peptide i tīpakohia, engari he kaha teitei te kaha homing CSF o rātou katoa. I kite mātou i te hekenga tonu i roto i te wā mō ngā ira #1903 me #2011, engari mō ngā ira #2077, #2002 me #2009, ko te pikinga i roto i ngā meneti 10 tuatahi ka tohu pea i te kawe hohe engari me manatoko. I pumau ngā ira #2020, #2002, me #2077 i ngā taumata teitei, i te mea ka heke haere te kukū CSF o te ira #2009 i muri i te pikinga tuatahi. Kātahi ka whakaritea e mātou te auau whanaunga o ia kaitono CSF ​​me tōna kukū toto (Pikitia 3d). Ko te hononga o te titer toharite o ia kaitono CSF ​​me tōna titer toto i ngā wā tauira katoa i whakaatu ko te toru o ngā kaitono e ono i tino whai rawa i te CSF toto. He mea whakamīharo, i whakaatu te ira #2077 i te pumau toto teitei ake (Pikitia Tāpiri 7). Hei whakaū i te kaha o ngā peptide ki te kawe hohe i ngā utanga ehara i te matūriki phage ki roto i te wāhanga CSF, i hangaia e mātou e whā ngā peptide ārahi i ahu mai i te biotin i te N-terminus kei reira ngā peptide e piri ana ki te matūriki phage. I honoa ngā peptide biotinylated (nama 2002, 2009, 2020 me 2077) ki te streptavidin (SA) hei tiki i ngā āhua maha-ira e rite ana ki te āhua o te phage. Nā tēnei hōputu anō hoki i taea ai e mātou te ine i te whakaaturanga SA i roto i te toto me te wai cerebrospinal hei peptide pūmua kawe utanga. He mea nui, he maha ngā wā ka taea te tārua i ngā raraunga phage i te wā i tukuna ai ngā peptide hangai i roto i tēnei hōputu SA-hono (Pikitia 3e). He iti ake te whakaaturanga tuatahi o ngā peptide i whakaranua, ā, he tere ake te whakakorenga CSF me ngā taumata kāore e kitea i roto i ngā haora 48. Hei whiwhi māramatanga ki ngā ara tuku o ēnei peptide phage clones ki roto i te wāhi CSF, i tātarihia e mātou te taunga o ngā pānga peptide phage takitahi mā te whakamahi i te immunohistochemistry (IHC) hei kite tika i ngā matūriki phage 1 haora i muri i te werohanga intravenous in vivo. He mea nui, i kitea ngā tāruarua #2002, #2077, me #2009 mā te tae kaha i roto i ngā uaua roro, engari kāore i kitea te phage whakahaere (#1779) me te tāruarua #2020 (Pikitia Tāpiri 8). E whakaatu ana tēnei ka whai wāhi ēnei peptide ki te pānga ki te roro mā te whiti i te BBB. Me whai tātari taipitopito anō hei whakamātautau i tēnei whakapae, nā te mea ka uru pea te ara BSCFB. I te whakataurite i te raupapatanga waikawa amino o te tāruarua tino whai rawa (#2002) me ētahi atu peptide kua tīpakohia, i kitea ko ētahi o rātou he rite tonu te toronga waikawa amino, e tohu ana pea i te tikanga kawe rite (Pikitia 3f).
Nā tōna āhua plasma ahurei me te pikinga nui o te CSF i roto i te wā, i tirotirohia anōtia te clone whakaaturanga phage #2077 mō te wā roa ake o te 48-hāora, ā, i taea e ia te whakaputa anō i te pikinga tere o te CSF i kitea i roto i te hononga ki ngā taumata SA pumau (Pikitia 4a). Mō ētahi atu clone phage kua tautuhia, i pania te #2077 ki ngā capillaries roro, ā, i whakaatu i te hononga nui ki te capillary marker lectin ina tirohia i te taumira teitei ake, me te paninga pea i roto i te wāhi parenchymal (Pikitia 4b). Hei tirotiro mēnā ka taea te whiwhi pānga rongoā peptide-mediated i roto i te CNS, i whakahaerehia e mātou he whakamātautau i konatunatua ai ngā putanga biotinylated o i) te peptide whakawhiti #2077 me te ii) te peptide aukati BACE1 ki te SA i ngā ōwehenga rerekē e rua. Mō tētahi huinga i whakamahia e mātou te aukati peptide BACE1 anake, ā, mō tētahi atu i whakamahia e mātou he ōwehenga 1:3 o te aukati peptide BACE1 ki te peptide #2077. I tukuna ngā tauira e rua mā te uaua, ā, i inehia ngā taumata o te beta-amyloid peptide 40 (Abeta40) i roto i te toto me te wai cerebrospinal i roto i te wā. I inehia a Abeta40 i roto i te CSF nā te mea e whakaata ana i te aukati BACE1 i roto i te parenchyma roro. E ai ki te whakaaro, i whakaitihia e ngā matatini e rua ngā taumata toto o Abeta40 (Pikitia 4c, d). Heoi, ko ngā tauira anake kei roto he ranunga o te peptide nama 2077 me te aukati o te peptide BACE1 i honoa ki te SA i puta ai te hekenga nui o Abeta40 i roto i te wai cerebrospinal (Pikitia 4c). E whakaatu ana ngā raraunga ka taea e te peptide nama 2077 te kawe i te pūmua 60 kDa SA ki roto i te CNS, ā, ka puta hoki ngā pānga rongoā me ngā aukati SA-hono o te peptide BACE1.
(a) Werohanga clonal (2 × 10 phages/kararehe) o te T7 phage e whakaatu ana i ngā kōtaha pharmacokinetic roa o te CSF peptide #2077 (RLSSVDSDLSGC) me te phage whakahaere kāore i werohia (#1779) i roto i te iti rawa e toru ngā kiore i whakauruhia ki te ngongo CM. (b) Whakaahua microscopic confocal o ngā oko toto cortical māngai i roto i ngā kiore i werohia ki te phage (2 × 10 10 phages/kararehe) e whakaatu ana i te tae anō o te peptide #2077 me ngā oko toto (lectin). I tukuna ēnei clones phage ki te 3 kiore, ā, i tukuna kia rere mō te 1 hāora i mua i te toto. I tapahia ngā roro, ā, i pania ki ngā paturopi polyclonal FITC-tohu ki te capsid phage T7. Tekau meneti i mua i te toto me te whakapiringa i muri mai, i tukuna te lectin DyLight594-tohu mā te uaua. Ngā whakaahua kanapa e whakaatu ana i te tae lectin (whero) o te taha luminal o ngā oko toto iti me ngā phage (kakariki) i roto i te lumen o ngā capillaries me te kiko roro perivascular. Ko te pae tauine e rite ana ki te 10 µm. (c, d) Ko te peptide aukati BACE1 biotinylated anake, i honoa rānei ki te peptide whakawhiti biotinylated #2077 i honoa ki te streptavidin, ā, i muri mai ka werohia ki roto i te uaua o te iti rawa e toru ngā kiore CM cannulated (10 mg streptavidin/kg). Ko te whakahekenga i roto i te Aβ40 i raro i te aukati peptide BACE1 i inehia e te Aβ1-40 ELISA i roto i te toto (whero) me te wai cerebrospinal (karaka) i ngā wā kua tohua. Hei mārama ake, ka tuhia he rārangi iraira ki runga i te kauwhata i te tauine o te 100%. (c) Te whakahekenga ōrau o te Aβ40 i roto i te toto (ngā tapatoru whero) me te wai cerebrospinal (ngā tapatoru karaka) i roto i ngā kiore i rongoatia ki te streptavidin i honoa ki te peptide whakawhiti #2077 me te peptide aukati BACE1 i te ōwehenga 3:1. (d) Te whakahekenga ōrau o te Aβ40 toto (ngā porowhita whero) me te wai cerebrospinal (ngā porowhita karaka) o ngā kiore i rongoatia ki te streptavidin i honoa ki te peptide aukati BACE1 anake. Ko te kukū Aβ i roto i te mana whakahaere he 420 pg/ml (paerewa rerekētanga = 101 pg/ml).
Kua angitu te whakamahi i te whakaaturanga phage i roto i ētahi wāhanga o te rangahau koiora17. Kua whakamahia tēnei tikanga mō ngā rangahau kanorau toto in vivo18,19 me ngā rangahau e aro ana ki ngā oko toto roro20,21,22,23,24,25,26. I roto i tēnei rangahau, i whakawhānuihia e mātou te tono o tēnei tikanga whiriwhiri ehara i te mea ki te tautuhi tika i ngā peptide e aro ana ki ngā oko toto roro anake, engari ki te kitenga hoki i ngā kaitono me ngā āhuatanga kawe hohe hei whiti i te arai toto-roro. Inaianei ka whakaahuahia e mātou te whanaketanga o tētahi tukanga whiriwhiri in vivo i roto i ngā kiore CM kua whakauruhia he ngongo, ā, ka whakaatuhia tōna kaha ki te tautuhi i ngā peptide me ngā āhuatanga homing CSF. Mā te whakamahi i te phage T7 e whakaatu ana i tētahi whare pukapuka o ngā peptide matapōkere 12-mer, i taea e mātou te whakaatu he iti te phage T7 (tata ki te 60 nm te whānui)10 kia urutau ki te arai toto-roro, kia whiti tika ai i te arai toto-roro, i te plexus choroid rānei. I kitea e mātou ko te hauhaketanga o te CSF mai i ngā kiore CM kua whakakāhia he tikanga tirotiro mahi in vivo kua whakahaeretia pai, ā, ko te phage i tangohia kāore i te herea noa ki te toto toto engari i mahi hoki hei kaikawe puta noa i te arai toto-roro. I tua atu, mā te kohikohi toto me te whakamahi i te HTS ki te CSF me ngā phage i ahu mai i te toto, i whakaū mātou kāore tā mātou whiriwhiringa o te CSF i pāngia e te whakarei ake i te toto, te pai rānei mō te whakawhānui i waenga i ngā rauna kōwhiringa. Heoi, ko te wāhanga toto he wāhanga o te tukanga kōwhiringa, nā te mea me ora ngā phage e taea ana te tae atu ki te wāhanga CSF me te rere i roto i te toto kia roa ai te wā kia whakarei ake i a rātou anō i roto i te roro. Hei tango i ngā mōhiohio raupapa pono mai i ngā raraunga HTS mata, i whakatinanahia e mātou ngā tātari i whakaritea ki ngā hapa raupapatanga motuhake-pūhara i roto i te tukanga tātari. Mā te whakauru i ngā tawhā nekeneke ki roto i te tikanga tirotiro, i whakaū mātou i te tere o te pharmacokinetics o ngā phage T7 momo-mohoao (t½ ~ 28 min) i roto i te toto24, 27, 28 me te whakatau hoki i tō rātou haurua-ora i roto i te wai cerebrospinal (t½ ~ 26 min) ia meneti). Ahakoa ngā āhua pharmacokinetic ōrite i roto i te toto me te CSF, ko te 0.001% anake o te kukū toto o te phage i kitea i roto i te CSF, e tohu ana i te iti o te nekehanga papamuri o te phage T7 momo-mohoao puta noa i te arai toto-roro. E whakaatu ana tēnei mahi i te hiranga o te rauna tuatahi o te kōwhiringa ina whakamahia ngā rautaki panning in vivo, inā koa mō ngā pūnaha phage e tere ana te whakakore i te tohanga, nā te mea he iti noa ngā clone ka taea te tae ki te wāhanga CNS. Nō reira, i te rauna tuatahi, he tino nui te hekenga o te kanorau whare pukapuka, nā te mea he iti noa te maha o ngā clone i kohia i roto i tēnei tauira CSF tino pakari. I roto i tēnei rautaki panning in vivo he maha ngā mahi kōwhiringa pērā i te kohikohi kaha i roto i te wāhanga CSF, te oranga o te clone i roto i te wāhanga toto, me te tango tere i ngā clone phage T7 mai i te toto i roto i ngā meneti 10 tuatahi (Pikitia 1d me te Pikitia Tāpiri 4M). Nō reira, i muri i te rauna tuatahi, i tautuhia ngā clone phage rerekē i roto i te CSF, ahakoa i whakamahia te puna tuatahi kotahi mō ngā kararehe takitahi. E whakaatu ana tēnei he maha ngā mahi whiriwhiri pakari mō ngā whare pukapuka pūtake me te maha o ngā mema o te whare pukapuka ka hua ake he whakahekenga nui o te kanorau. Nō reira, ka noho ngā huihuinga matapōkere hei wāhanga nui o te tukanga whiriwhiri tuatahi, ka nui te awe ki te hua. He tino rite te āhua o te whakanui i te CSF o te nuinga o ngā tāruarua i roto i te whare pukapuka taketake. Heoi, ahakoa i raro i ngā āhuatanga whakamātautau ōrite, ka rerekē pea ngā hua whiriwhiri nā te iti o te maha o ia tāruarua motuhake i roto i te puna tuatahi.
He rerekē ngā kaupapa i whakanuia i roto i te CSF i ērā i roto i te toto. He mea whakamīharo, i kite mātou i te nekehanga tuatahi ki ngā peptide whai-glycine i roto i te toto o ngā kararehe takitahi. (Pikitia 1g, Pikitia Tāpiri. 4e, 4f). Ko ngā peptide glycine kei roto i te phage he pumau ake, ā, he iti ake te tūponotanga kia tangohia atu i te tohanga toto. Heoi, kāore i kitea ēnei peptide whai-glycine i roto i ngā tauira wai cerebrospinal, e kī ana i haere ngā whare pukapuka kua tohua i roto i ngā taahiraa kōwhiringa e rua: tētahi i roto i te toto, ā, ko tētahi i tukuna kia kohia i roto i te wai cerebrospinal. Kua whakamatautauria whānuitia ngā clone whai-CSF i puta mai i te tuawhā o ngā whiringa. Tata ki ngā clone katoa i whakamatautauria takitahi kua whakaūtia he whai-CSF ki te phage mana kore. Kotahi te peptide i pā ki te mata (#2077) i tirotirohia kia nui ake te taipitopito. I whakaatuhia he roa ake te haurua-ora plasma ki te whakataurite ki ētahi atu pānga (Pikitia 3d me te Pikitia Tāpiri 7), ā, he mea whakamīharo, kei roto i tēnei peptide he toenga cysteine ​​​​i te C-terminus. Kua whakaaturia tata nei ko te tāpiri o te cysteine ​​​​ki ngā peptides ka taea te whakapai ake i ō rātou āhuatanga pharmacokinetic mā te herea ki te albumin 29. Kāore i te mōhiotia tēnei mō te peptide #2077 i tēnei wā, ā, me rangahau anō. I whakaatuhia e ētahi peptides he whakawhirinakitanga valence ki te whakarei ake i te CSF (kāore i whakaaturia ngā raraunga), e pā ana pea ki te āhua mata o te T7 capsid i whakaaturia. Ko te pūnaha T7 i whakamahia e mātou i whakaatu i te 5-15 tārua o ia peptide mō ia matūriki phage. I mahia te IHC ki runga i ngā clone phage matua kaitono i werohia ki roto i te uaua ki roto i te cortex roro o ngā kiore (Pikitia Tāpiri 8). I whakaatuhia e ngā raraunga he iti rawa kia toru ngā clone (Nama 2002, Nama 2009 me Nama 2077) i taunekeneke ki te BBB. Kāore anō kia whakatauhia mēnā ka hua ake tēnei taunekeneke BBB i te kohikohinga o te CSF, i te nekehanga rānei o ēnei clone tika ki te BCSFB. He mea nui, ka whakaaturia e mātou ka mau tonu te kaha kawe CSF o ngā peptides kua tīpakohia i te wā i hangaia ai, ā, i herea ki te utanga pūmua. Ko te herenga o ngā peptide biotinylated N-terminal ki te SA ka rite ki ngā hua i whiwhihia me ā rātou clone phage i roto i te toto me te wai cerebrospinal (Pikitia 3e). Hei whakamutunga, ka whakaatuhia e mātou ka taea e te peptide matua #2077 te whakatairanga i te mahi roro o te aukati peptide biotinylated o BACE1 kua honoa ki te SA, ka puta he pānga pharmacodynamic nui i roto i te CNS mā te whakaiti nui i ngā taumata Abeta40 i roto i te CSF (Pikitia 4). Kāore i taea e mātou te tautuhi i tētahi homologue i roto i te pātengi raraunga mā te mahi i tētahi rapunga homology raupapa peptide o ngā pānga katoa. He mea nui kia mōhio ko te rahi o te whare pukapuka T7 he tata ki te 109, ko te rahi o te whare pukapuka ariā mō te 12-mers he 4 x 1015. Nō reira, i tīpakohia e mātou he wāhanga iti noa iho o te wāhi kanorau o te whare pukapuka peptide 12-mer, ko te tikanga pea ka taea te tautuhi i ngā peptide kua arotau ake mā te aromatawai i te wāhi raupapa tata o ēnei pānga kua tautuhia. E ai ki te whakapae, ko tētahi o ngā take kāore anō kia kitea he ōritenga tūturu o ēnei peptides ko te korenga o te kōwhiringa i te wā whanaketanga hei ārai i te urunga kore whakahaere o ētahi tohu peptide ki roto i te roro.
Ina huihuia, ka whakaratohia e ā mātou hua he pūtake mō ngā mahi ā muri ake nei ki te tautuhi me te whakaahua i ngā pūnaha kawe o te arai toto roro i roto i te tinana ora. Ko te whakatakotoranga taketake o tēnei tikanga e ahu mai ana i tētahi rautaki kōwhiringa mahi e kore e tautuhi noa i ngā tārua me ngā āhuatanga here toto roro, engari kei roto hoki he taahiraa nui e whai mahi ā-roto ai ngā tārua angitu ki te whiti i ngā arai koiora i roto i te tinana ora ki roto i te wāhanga CNS. Ko te whakamārama i te tikanga kawe o ēnei peptide me tō rātou hiahia ki te here ki te moroiti toto motuhake ki te rohe roro. Tērā pea ka arahi tēnei ki te kitenga o ngā ara hou mō te kawe o te BBB me ngā kaiwhakawhiwhi. E tumanako ana mātou ka taea e ngā peptide kua tautuhia te here tika ki ngā kaiwhakawhiwhi toto roro, ki ngā ligand e rere ana rānei e kawehia ana mā te BBB, BCSFB rānei. Ka rangahaua anōtia ngā kawe peptide me te mahi kawe CSF i kitea i roto i tēnei mahi. Kei te rangahauhia e mātou i tēnei wā te motuhaketanga o te roro o ēnei peptide mō tō rātou kaha ki te whiti i te BBB me/ranei te BCSFB. He taputapu tino whai hua ēnei peptide hou mō te kimi pea i ngā kaiwhakawhiwhi hou, i ngā ara hou rānei, me te whakawhanake i ngā tūāpapa hou tino whai hua mō te tuku i ngā macromolecules, pērā i ngā matū koiora, ki te roro.
Whakatōkia te cisterna nui (CM) mā te whakamahi i te whakarerekētanga o te tikanga kua whakamāramahia i mua. I whakanohoia ngā kiore Wistar kua whakamoea (200-350 g) ki runga i tētahi taputapu stereotaxic, ā, i tapahia te tumuaki waenga ki runga i te tumuaki kua heua, kua whakaritea kia kore e pangia kia kitea te angaanga. E rua ngā kōhao i te rohe o te whitiki o runga, ka whakau i ngā tīwiri whakau ki roto i ngā kōhao. I whakauhia tētahi atu kōhao ki te tihi occipital taha hei ārahi stereotactic o tētahi cannula maitai kore waikura ki roto i te CM. Tāpirihia he sima niho huri noa i te cannula, ā, whakauhia ki ngā tīwiri. Whai muri i te whakamaroke whakaahua me te whakapakeke sima, i katia te patunga kiri ki te tuitui supramid 4/0. Ka whakaūtia te whakatakotoranga tika o te cannula mā te turuturu ohorere o te wai cerebrospinal (CSF). Tangohia te kiore mai i te taputapu stereotaxic, whiwhi i te tiaki tika i muri i te pokanga me te whakahaere mamae, ā, tukua kia ora mō te iti rawa kotahi wiki tae noa ki te kitea ngā tohu toto i roto i te wai cerebrospinal. I riro mai ngā kiore Wistar (Crl:WI/Han) mai i Charles River (France). I puritia ngā kiore katoa i raro i ngā āhuatanga motuhake kāore he mate. I whakaaetia ngā whakamātautau kararehe katoa e te Tari Kararehe o te Tāone o Basel, Switzerland, ā, i mahia i runga anō i te Raihana Kararehe Nama 2474 (Aromatawai i te Kawe Roro Kaha mā te Ine i ngā Taumata o ngā Kaitono Whakaora i roto i te Wai Cerebrospinal me te Roro o te Kiore).
Kia tūpato te kiore me te pupuri i te cannula CM. Tangohia te Datura mai i te cannula ka kohia he 10 µl o te wai cerebrospinal e rere ana. Nā te mea i whakararuhia te mana o te cannula, ko ngā tauira wai cerebrospinal mārama anake, kāore he tohu o te poke o te toto, te tae rānei, i whakaurua ki tēnei rangahau. I te wā kotahi, tata ki te 10–20 μl o te toto i tangohia mai i tētahi tapahi iti i te pito o te hiku ki roto i ngā ngongo heparin (Sigma-Aldrich). I kohia te CSF me te toto i ngā wā rerekē i muri i te werohanga ā-uaua o te T7 phage. Tata ki te 5–10 μl o te wai i makahia i mua i te kohi i ia tauira CSF, e rite ana ki te rahinga mate o te catheter.
I hangaia ngā whare pukapuka mā te whakamahi i te wetereo T7Select 10-3b e ai ki te whakaahuatanga i roto i te pukapuka ā-pūnaha T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). I roto i te poto, i hangaia he whakaurunga DNA 12-mer matapōkere i te whakatakotoranga e whai ake nei:
I whakamahia te kōtona NNK hei karo i ngā kōtona aukati rua me te whakaputanga nui o te waikawa amino i roto i te whakaurunga. Ko te N he ōwehenga equimolar i konatunatua ā-ringa o ia nucleotide, ā, ko te K he ōwehenga equimolar i konatunatua ā-ringa o ngā nucleotide adenine me cytosine. I hurihia ngā rohe kotahi-whiri ki te DNA takirua mā te whakatipu anō me te dNTP (Novagen) me te whākōkī Klenow (New England Biolabs) i roto i te pāhare Klenow (New England Biolabs) mō ngā hāora e 3 i te 37°C. I muri i te tauhohenga, i whakahokia mai te DNA takirua-whiri mā te whakapūtanga EtOH. I keria te DNA hua me ngā whākōkī here EcoRI me HindIII (nō Roche). Ko te whakaurunga i tapahia me te purea (QIAquick, Qiagen) (T4 ligase, New England Biolabs) i herea ki roto i te anga ki roto i te weka T7 i mua i te tapahi i muri i te waikawa amino 348 o te ira capsid 10B. I whakatipuria ngā tauhohenga here i te 16°C mō ngā hāora 18 i mua i te takai in vitro. I mahia te takai phage in vitro i runga anō i ngā tohutohu i tukuna mai me te kete cloning T7Select 10-3b (Novagen) ā, i whakanuia kotahi te otinga takai kia pakaru mā te whakamahi i te Escherichia coli (BLT5615, Novagen). I poipoia ngā lysate, i titrated, ā, i tiohia ki te -80° C hei otinga taketake o te glycerol.
Whakareitanga PCR tika o ngā rohe taurangi phage i whakareitia i roto i te hupa, i te pereti rānei mā te whakamahi i ngā paraime whakakotahi 454/Roche-amplicon motuhake. Kei roto i te paraime whakakotahi whakamua ngā raupapatanga e taha ana i te rohe taurangi (NNK) 12 (motuhake ki te tauira), GS FLX Titanium Adapter A, me te raupapatanga kī whare pukapuka turanga-whā (TCAG) (Pikitia Tāpiri 1a):
Kei roto hoki i te paraime whakakotahi whakamuri te biotin e piri ana ki ngā pīrere hopu me te GS FLX Titanium Adapter B e hiahiatia ana mō te whakanui clonal i te wā o te emulsion PCR:
Kātahi ka tukuna ngā amplicons ki te 454/Roche pyrosequencing e ai ki te kawa 454 GS-FLX Titanium. Mō te raupapatanga ā-ringa a Sanger (Applied Biosystems Hitachi 3730 xl DNA Analyzer), i whakanuia te T7 phage DNA mā te PCR, ā, i raupapatia ki ngā takirua primer e whai ake nei:
I whakakahangia ngā whakaurunga mai i ngā papa takitahi mā te whakamahi i te Roche Fast Start DNA Polymerase Kit (e ai ki ngā tohutohu a te kaihanga). Me tīmata wera (10 meneti i te 95 °C) me te 35 huringa whakanui (50 hēkona i te 95 °C, 1 meneti i te 50 °C, me te 1 meneti i te 72 °C).
Ko ngā phage mai i ngā whare pukapuka, ngā phage momo-mohoao, ngā phage i whakaorangia mai i te CSF me te toto, ngā tārua takitahi rānei i whakanuia ki roto i te Escherichia coli BL5615 i roto i te hupa TB (Sigma Aldrich) i roto rānei i ngā rihi 500 cm2 (Thermo Scientific) mō te 4 hāora i te 37°C. I tangohia ngā phage mai i ngā pereti mā te horoi i ngā pereti ki te Tris-EDTA buffer (Fluka Analytical) mā te kohikohi rānei i ngā papa ki ngā pito pipette parakore. I wehea ngā phage mai i te wai whakapūkara ahurea, i te buffer tango rānei me te kotahi porowhita o te polyethylene glycol (PEG 8000) precipitation (Promega) ā, i whakatārewatia anō ki roto i te Tris-EDTA buffer.
I tukuna te phage kua whakanuia ki te 2-3 rauna o te tango i te endotoxin mā te whakamahi i ngā pire tango endotoxin (Miltenyi Biotec) i mua i te werohanga ā-roto (IV) (500 μl/kararehe). I te rauna tuatahi, i whakaurua ngā phage 2×1012; i te tuarua, ngā phage 2×1010; i te tuatoru me te tuawhā o ngā rauna kōwhiringa, 2×109 ngā phage mō ia kararehe. Ko te nui o ngā phage i roto i ngā tauira CSF me te toto i kohia i ngā wā kua tohua i whakatauhia mā te tatau i te papa e ai ki ngā tohutohu a te kaihanga (pukapuka pūnaha T7Select). I mahia te kōwhiringa phage mā te werohanga ā-roto o ngā whare pukapuka kua purea ki roto i te uaua hiku, mā te werohanga anō rānei i te phage i tangohia mai i te CSF mai i te rauna kōwhiringa o mua, ā, i mahia ngā hauhake i muri mai i te 10 meneti, 30 meneti, 60 meneti, 90 meneti, 120 meneti, 180 meneti, me te 240 meneti ia tauira CSF me te toto. E whā ngā rauna katoa o te panning in vivo i whakahaerehia, ā, i rongoa motuhaketia ngā peka e rua i tīpakohia, ā, i tātarihia hoki i ngā rauna tuatahi e toru o te kōwhiringa. Ko ngā whakaurunga phage katoa i tangohia mai i te CSF mai i ngā rauna tuatahi e rua o te kōwhiringa i tukuna ki te pyrosequencing 454/Roche, ko ngā ira katoa i tangohia mai i te CSF mai i ngā rauna whakamutunga e rua o te kōwhiringa i raupapa ā-ringa. Ko ngā ira toto katoa mai i te rauna tuatahi o te kōwhiringa i tukuna hoki ki te pyrosequencing 454/Roche. Mō te werohanga o ngā ira phage, i whakanuia ngā ira kua tīpakohia ki roto i te E. coli (BL5615) i runga i ngā pereti 500 cm2 i te 37°C mō ngā hāora e 4. Ko ngā ira kua tīpakohia takitahi, ā, kua raupapa ā-ringa i whakatipuria ki roto i te reo TB. Whai muri i te tangohanga phage, te purenga me te tango i te endotoxin (e ai ki te whakaahuatanga i runga ake nei), i werohia he 2×1010 ira/kararehe i roto i te 300 μl mā te uaua kotahi ki roto i te uaua hiku.
Te tukatuka mua me te tātari kounga o ngā raraunga raupapatanga. I hurihia ngā raraunga mata 454/Roche mai i te hōputu mahere awa paerewa rua (sff) ki te hōputu pānui tangata Pearson (fasta) mā te whakamahi i te pūmanawa kaihoko. I whakahaerehia te tukatuka anō o te raupapatanga nucleotide mā te whakamahi i ngā kaupapa me ngā tuhinga C motuhake (mōkihi pūmanawa kāore anō kia tukuna) e ai ki te whakaahuatanga i raro nei. Kei roto i te tātari raraunga tuatahi ngā tikanga tātari maha-wāhanga tino pakari. Hei tātari i ngā pānui kāore i roto i te raupapatanga DNA whakauru 12mer tika, i whakaritea ngā pānui kia rite ki te tapanga tīmatanga (GTGATGTCGGGGATCCGAATTCT), te tapanga mutu (TAAGCTTGCGGCCGCACTCGAGTA) me te whakauru papamuri (CCCTGCAGGGATATCCCGGGAGCTCGAC) mā te whakamahi i te whakamātautau Needleman-Wunsch ā-ao. te whakaritenga e āhei ana kia 2 ngā taupatupatu mō ia whakaritenga31. Nō reira, i tangohia mai i te whare pukapuka ngā pānui kāore he tohu tīmatanga me te tohu mutu me ngā pānui kei roto ngā whakauru papamuri, arā, ngā whakaritenga e nui ake ana i te maha o ngā taupatupatu kua whakaaetia. Mō ngā pānui e toe ana, i tangohia te raupapa DNA N-mer e toro atu ana mai i te tohu tīmatanga me te mutunga i mua i te tohu mutu mai i te raupapa pānui taketake, ā, i tukatukahia anō (e kiia nei ko te "whakauru"). Whai muri i te whakamāoritanga o te whakauru, ka tangohia te wāhanga i muri i te kōtona mutu tuatahi i te pito 5′ o te paraimere mai i te whakauru. Hei tāpiri, i tangohia hoki ngā nucleotide e arahi ana ki ngā kōtona kāore i oti i te pito 3′ o te paraimere. Hei whakakore i ngā whakauru kei roto ko ngā raupapa papamuri anake, i tangohia hoki ngā whakauru kua whakamāoritia e tīmata ana me te tauira waikawa amino "PAG". I tangohia mai i te whare pukapuka ngā peptide he roa muri-whakamāoritanga iti iho i te 3 ngā waikawa amino. Hei whakamutunga, tangohia te taurite i roto i te puna whakauru, ā, whakatauhia te auau o ia whakauru ahurei. Ko ngā hua o tēnei tātaritanga i whakaurua he rārangi o ngā raupapa nucleotide (whakauru) me ō rātou auau (pānui) (Ngā Pikitia Tāpiri 1c me te 2).
Ngā whakaurunga DNA rōpū N-mer mā te ōrite o te raupapatanga: Hei whakakore i ngā hapa raupapatanga motuhake 454/Roche (pēnei i ngā raruraru me ngā toronga homopolymer raupapatanga) me te tango i ngā taupatupatu iti ake te hiranga, ka whakarōpūhia ngā whakaurunga raupapatanga DNA N-mer kua tātarihia i mua (ngā whakaurunga) mā te ōrite. ngā whakaurunga (tae atu ki te 2 ngā turanga kore-taurite e whakaaetia ana) mā te whakamahi i tētahi raupapatanga tāruarua kua tautuhia penei: ka whakarōpūhia ngā whakaurunga i te tuatahi mā ō rātou auau (teitei ki te iti rawa), ā, ki te rite tonu, mā tō rātou whakarōpū tuarua mā te roa (roa ki te poto rawa)). Nō reira, ko ngā whakaurunga tino auau me te roa rawa e tautuhi ana i te "rōpū" tuatahi. Ka whakatakotoria te auau rōpū ki te auau matua. Kātahi, ka nganahia kia tāpirihia ia whakaurunga e toe ana i roto i te rārangi kua whakarōpūhia ki te rōpū mā te whakaritenga takirua Needleman-Wunsch. Mena kāore te maha o ngā kore taurite, ngā whakaurunga, ngā mukunga rānei i roto i tētahi whakaritenga e neke ake i te paepae o te 2, ka tāpirihia he whakaurunga ki te rōpū, ā, ka whakanuia te auau rōpū whānui mā te auau o te tāpiritanga o te whakaurunga. Ka tohua ngā whakaurunga i tāpirihia ki tētahi rōpū hei mea kua whakamahia, ā, ka tangohia mai i te tukatuka atu. Mena kāore e taea te tāpiri i te raupapa whakauru ki tētahi rōpū kua tīariari kē, ka whakamahia te raupapa whakauru hei waihanga i tētahi rōpū hou me te auau whakauru e tika ana, ā, ka tohua hei mea kua whakamahia. Ka mutu te whakahoutanga ina whakamahia ia raupapa whakauru hei hanga i tētahi rōpū hou, ka taea rānei te whakauru ki tētahi rōpū kua tīariari kē. Nā te mea, ko ngā whakauru kua whakarōpūtia kei roto ko ngā nucleotide ka whakamāoritia hei raupapa peptide (ngā whare pukapuka peptide). Ko te hua o tēnei tātaritanga he huinga whakauru me ō rātou auau e rite ana e hanga ana i te maha o ngā pānui karapīpiti (Pikitia Tāpiri 2).
Te Whakaputa Motif: I runga i te rārangi o ngā peptide ahurei, i hangaia he whare pukapuka kei roto ngā tauira waikawa amino katoa (aa) e whakaaturia ana i raro nei. I tangohia ia tauira pea o te roa 3 mai i te peptide, ā, i tāpirihia tōna tauira whakamuri me tētahi whare pukapuka motif noa kei roto ngā tauira katoa (tripeptides). I raupapahia ngā whare pukapuka o ngā motif tino auau, ā, i tangohia te taurite. Kātahi, mō ia tripeptide i roto i te whare pukapuka motif, i tirohia e mātou tōna aroaro i roto i te whare pukapuka mā te whakamahi i ngā taputapu rorohiko. I tēnei take, ka tāpirihia te auau o te peptide kei roto te tripeptide motif kua kitea, ā, ka tohaina ki te motif i roto i te whare pukapuka motif ("te maha o ngā motif"). Ko te hua o te whakaputa motif he huinga rua-ahu kei roto ngā putanga katoa o ngā tripeptide (motifs) me ō rātou uara, arā, te maha o ngā pānui raupapatanga e hua ake ai te motif e rite ana ina tātarihia, ka whakarōpūhia, ka whakamāoritia ngā pānui. Ngā inenga e whakaahuatia ana i runga ake nei.
Te whakataurite i te maha o ngā motika me ngā kauwhata marara e rite ana: I whakatauritea te maha o ngā motika mō ia tauira mā te whakamahi i
ko ni te maha o ngā pānui kei roto te kaupapa i. Nō reira, ko vi te tohu i te ōrau auau o ngā pānui (ngā peptide rānei) kei roto te motif i i roto i te tauira. I tatauhia ngā uara-P mō te maha o ngā motif kāore i te whakatauritea mā te whakamahi i te whakamātautau tika a Fisher. Mō ngā hononga o te maha o ngā motif, i tatauhia ngā hononga a Spearman mā te whakamahi i te maha o ngā motif kua whakatauritea me R.
Hei tiro i te ihirangi o ngā waikawa amino i ia tūranga i roto i te whare pukapuka peptide, i hangaia ngā tohu tukutuku 32, 33 (http://weblogo.threeplusone.com). Tuatahi, ka rongoatia te ihirangi o ngā waikawa amino i ia tūranga o te peptide 12-mer ki roto i tētahi matihiko 20×12. Kātahi ka hangaia he huinga peptide 1000 kei roto te ihirangi waikawa amino ōrite i ia tūranga i roto i te whakatakotoranga raupapa-fasta, ā, ka tukuna hei whakauru ki te tohu tukutuku 3, e whakaputa ana i tētahi whakaaturanga whakairoiro o te ihirangi waikawa amino ōrite i ia tūranga. mō tētahi whare pukapuka peptide kua hoatu. Hei tiro i ngā huinga raraunga maha-ahu, i hangaia ngā mahere wera mā te whakamahi i tētahi taputapu kua whakawhanakehia i roto i te R (biosHeatmap, he mōkihi R kāore anō kia tukuna). I tatauhia ngā dendrogram i whakaaturia i roto i ngā mahere wera mā te whakamahi i te tikanga whakarōpū hierarchical a Ward me te ine tawhiti Euclidean. Mō te tātari tatauranga o ngā raraunga whakatauira motif, i tatauhia ngā uara P mō te whakatauira kore-whakarerekētanga mā te whakamahi i te whakamātautau tika a Fisher. I tatauhia ngā uara-P mō ētahi atu huinga raraunga i roto i te R mā te whakamahi i te whakamātautau-t a te Tauira, te ANOVA rānei.
I werohia ngā clone phage me ngā phage kāore he whakaurunga mā te uaua hiku (2×1010 phages/kararehe i roto i te 300 μl PBS). Tekau meneti i mua i te whakaheke toto me te whakapiringa i muri mai, i werohia ngā kararehe kotahi ki te 100 μl o te lectin DyLight594-tohu (Vector Laboratories Inc., DL-1177). 60 meneti i muri i te werohanga phage, i ringihia ngā kiore ki te 50 ml PBS mā te ngākau, ā, i whai muri ko te 50 ml 4% PFA/PBS. I whakapirihia anō ngā tauira roro i roto i te 4% PFA/PBS i te pō, ā, i rumakina ki te 30% sucrose i te pō i te 4°C. Ka whakatio wawetia ngā tauira ki roto i te ranunga OCT. I whakahaerehia te tātaritanga ā-ira-matū o ngā tauira tio i te pāmahana rūma i runga i ngā wehenga kiri 30 µm kua āraia ki te 1% BSA, ā, i whaowhia ki ngā paturopi polyclonal FITC-tohu ki te T7 phage (Novus NB 600-376A) i te 4 °C. Whakawhaowhia i te pō. Hei whakamutunga, i horoia ngā wāhanga e 3 ngā wā ki te PBS, ā, i tirohia ki te karu hiko confocal laser (Leica TCS SP5).
I hangaia ngā peptide katoa me te iti rawa o te parakore o te 98% e GenScript USA, i whakakoioratia, ā, i whakakoioratia. Ka herea te Biotin mā te whakamahi i tētahi atu āputa glycine toru i te pito-N. Tirohia ngā peptide katoa mā te whakamahi i te ine papatipu.
I konatunatua a Streptavidin (Sigma S0677) ki te taikaha e 5-ngā wā e rite ana te nui o te peptide biotinylated, te peptide aukati BACE1 biotinylated, te huinga rānei (ōwehenga 3:1) o te peptide aukati BACE1 biotinylated me te peptide aukati BACE1 i roto i te 5–10% DMSO/i whakatōkia ki roto i te PBS. 1 hāora i te pāmahana rūma i mua i te werohanga. I werohia ngā peptide honohono-streptavidin mā roto i te uaua i te horopeta o te 10 mg/kg ki roto i tētahi o ngā uaua hiku o ngā kiore he kōhao roro.
I aromatawaihia te kukū o ngā matatini streptavidin-peptide mā te ELISA. I pania ngā pereti Nunc Maxisorp microtiter (Sigma) i te pō i te 4°C ki te 1.5 μg/ml paturopi anti-streptavidin kiore (Thermo, MA1-20011). I muri i te aukati (pūnaha aukati: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) i te pāmahana rūma mō ngā hāora e 2, horoia te pereti ki te 0.05% Tween-20/PBS (pūnaha horoi) mō ngā hēkona e 3, i tāpirihia ngā tauira CSF me te plasma ki ngā puna i whakaranua ki te pūnaha aukati (plasma 1:10,000, CSF 1:115). Kātahi ka whakatōkia te pereti i te pō i te 4°C ki te paturopi kite (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). I muri i ngā horoinga e toru, i kitea te streptavidin mā te whaowhia ki roto i te otinga TMB (Roche) mō te 20 meneti. I muri i te whakamutu i te whanaketanga tae ki te 1M H2SO4, inehia te mimiti i te 450 nm.
I aromatawaihia te mahi a te matatini aukati streptavidin-peptide-BACE1 mā te Aβ(1-40) ELISA e ai ki te kawa a te kaihanga (Wako, 294-64701). I roto i te poto, i whakaranua ngā tauira CSF ki roto i te wai whakaranu paerewa (1:23) ā, i whaowhia mō te pō i te 4°C i roto i ngā pereti 96-pū kua pania ki te paturopi hopu BNT77. I muri i ngā taahiraa horoi e rima, i tāpirihia te paturopi BA27 honohono-HRP ā, i whaowhia mō ngā haora e 2 i te 4°C, ā, i muri mai ko ngā taahiraa horoi e rima. I kitea te Aβ(1–40) mā te whaowhia i roto i te otinga TMB mō ngā meneti 30 i te pāmahana rūma. I muri i te whakamutua o te whanaketanga tae ki te otinga aukati, inehia te mimiti i te 450 nm. I tukuna ngā tauira plasma ki te tangohanga wāhanga totoka i mua i te Aβ(1–40) ELISA. I tāpirihia te plasma ki te 0.2% DEA (Sigma) i roto i ngā pereti 96-pū, ā, i whaowhia i te pāmahana rūma mō ngā meneti 30. I muri i te horoinga o ngā pereti SPE (Oasis, 186000679) ki te wai me te 100% methanol, ka tāpirihia ngā tauira plasma ki ngā pereti SPE, ā, ka tangohia katoatia te wai. I horoia ngā tauira (tuatahi ki te 5% methanol, kātahi ki te 30% methanol) ā, ka whakamāoritia ki te 2% NH4OH/90% methanol. I muri i te whakamaroketanga o te eluate i te 55°C mō te 99 meneti i te iahiko N2 pumau, ka whakaitihia ngā tauira ki roto i ngā wai whakamāori paerewa, ā, ka inehia te Aβ(1–40) e ai ki te whakaahuatanga i runga ake nei.
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