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Yerekana karuseli ya sisitemu eshatu icyarimwe.Koresha Utubuto na Ibikurikira kugirango unyuze mumashusho atatu icyarimwe, cyangwa ukoreshe utubuto twa slide kumpera kugirango wimuke unyuze mubice bitatu icyarimwe.
Inzitizi yubwonko bwamaraso hamwe ninzitizi yubwonko bwamaraso birinda imiti ya biotherapeutique kugera kubyo igenewe muri sisitemu yo hagati, bityo bikabuza kuvura neza indwara zifata ubwonko.Kugirango tumenye ubwikorezi bwubwonko bushya muri vivo, twashyizeho isomero rya T7 phage peptide hanyuma dukusanya amaraso hamwe nubwonko bwubwonko (CSF) dukoresheje icyitegererezo kinini cya pisine cyimbeba.Clone yihariye ya fage yakungahaye cyane muri CSF nyuma yincuro enye zo gutoranya.Ikizamini cyabakandida peptide kugiti cyabo cyerekanye ubutunzi burenga 1000 muri CSF.Bioactivite yo kwanduza peptide yunganirwa mu bwonko yemejwe no kugabanuka kwa 40% kurwego rwa amyloide-β mumazi yubwonko bwifashishije peptide ya BACE1 ifitanye isano na peptide yamenyekanye.Ibisubizo byerekana ko peptide yagaragajwe nuburyo bwo guhitamo fage ya vivo ishobora kuba ibinyabiziga byingirakamaro mugutanga sisitemu ya macromolecules mubwonko bifite ingaruka zo kuvura.
Sisitemu yo hagati yubuvuzi (CNS) yibanze kubushakashatsi bwubuvuzi bwibanze cyane cyane ku kumenya imiti n’ibikoresho byerekana imiti igamije CNS, hashyizweho ingufu nke mu kuvumbura uburyo butera ibiyobyabwenge mu bwonko.Ibi bitangiye guhinduka ubu kuko gutanga ibiyobyabwenge, cyane cyane molekile nini, nibice bigize iterambere ryibiyobyabwenge bigezweho.Ibidukikije bya sisitemu yo hagati yuburinzi bikingiwe neza na sisitemu yubwonko bwubwonko, bugizwe na bariyeri yubwonko bwamaraso (BBB) ​​na bariyeri yubwonko bwamaraso (BCBB) 1, kuburyo bigoye kugeza ibiyobyabwenge mubwonko1,2.Bigereranijwe ko imiti hafi ya yose ya molekile nini irenga 98% yimiti mito ya molekile ikurwa mubwonko3.Niyo mpamvu ari ngombwa cyane kumenya uburyo bushya bwo gutwara ubwonko butanga uburyo bunoze kandi bwihariye bwo gutanga imiti ivura CNS 4,5.Nyamara, BBB na BCSFB nazo zitanga amahirwe meza yo gutanga ibiyobyabwenge kuko byinjira kandi byinjira mubice byose byubwonko binyuze mumitsi yagutse.Niyo mpamvu, imbaraga ziriho zo gukoresha uburyo budashoboka bwo kugeza ubwonko ahanini bushingiye ku buryo bwo gutwara abantu (PMT) hakoreshejwe reseptor ya endogenous BBB6.Nubwo iterambere ryambere ryambere ukoresheje transfert ya reseptor inzira ya 7,8, birakenewe ko hajyaho iterambere rya sisitemu nshya yo gutanga ibintu hamwe nibintu byiza.Kugira ngo ibyo bishoboke, intego yacu yari iyo kumenya peptide zishobora guhuza ubwikorezi bwa CSF, kuko zishobora gukoreshwa mugutanga macromolecules kuri CNS cyangwa gufungura inzira nshya zakira.By'umwihariko, kwakira no gutwara ibintu byihariye bya sisitemu yo mu bwonko (BBB na BSCFB) birashobora kuba intego yo gutanga ku buryo bunoze kandi bwihariye bwo gutanga imiti y’ibinyabuzima.Cerebrospinal fluid (CSF) nigicuruzwa cyibanga cya choroide plexus (CS) kandi gihura neza n’amazi hagati yubwonko binyuze mumwanya wa subarachnoid n'umwanya wa ventricular4.Vuba aha, byagaragaye ko amazi ya subarachnoid cerebrospinal fluid ikwirakwira cyane muri interstitium yubwonko9.Turizera ko tuzagera kumwanya wa parenchymal dukoresheje iyi nzira ya subarachnoid yinjira cyangwa binyuze muri BBB.Kugirango tubigereho, twashyize mubikorwa ingamba zo guhitamo icyiciro cya vivo cyerekana neza peptide itwarwa nimwe murinzira zombi zitandukanye.
Ubu turasobanura urukurikirane muburyo bwa vivo fage yerekana uburyo bwo kwerekana hamwe na CSF icyitegererezo hamwe no kwinjiza ibicuruzwa byinshi (HTS) kugirango dukurikirane ibyiciro byatoranijwe hamwe nibitabo byinshi bitandukanye.Isuzumabumenyi ryakorewe ku mbeba zifite ubwenge hamwe na kanseri nini yatewe burundu (CM) kugira ngo hatabaho kwanduza amaraso.Icy'ingenzi, ubu buryo butoranya ubwonko bwibasira ubwonko na peptide hamwe nibikorwa byo gutwara abantu hejuru yubwonko bwubwonko.Twakoresheje ibyiciro bya T7 kubera ubunini bwabyo (~ 60 nm) 10 hanyuma dusaba ko bibereye gutwara transport ya vicles yemerera kwambuka ingirabuzimafatizo ya endoteliyale na / cyangwa epiteliyale-medulla.Nyuma yincuro enye zo gutekesha, abaturage ba fage bari mu bwigunge berekana imbaraga mu gutunganya vivo CSF ​​no kwishyiriraho mikorobe yubwonko.Icy'ingenzi, twashoboye kwemeza ibyo twabonye twerekana ko peptide nziza y’abakandida yatoranijwe kandi ikomatanya imiti ishobora gutwara imizigo ya poroteyine mu mazi yo mu bwonko.Ubwa mbere, ingaruka za farumasi ya CNS zashyizweho muguhuza peptide iyobora transit hamwe na inhibitor ya peptide ya BACE1.Usibye kwerekana ko muri vivo ingamba zo gusuzuma imikorere ishobora kwerekana peptide yo gutwara ubwonko bushya nk'utwara imizigo ya poroteyine nziza, turateganya ko uburyo bwo guhitamo imikorere bushobora no kuba ingenzi mu kumenya inzira zo gutwara ubwonko bushya.
Hashingiwe ku bice bigize plaque (PFU), nyuma yintambwe yo gupakira fage, isomero rya random 12-mer umurongo wa T7 fage peptide ifite ubudasa bugera kuri 109 yarateguwe kandi irarema (reba Ibikoresho nuburyo).Ni ngombwa kumenya ko twasesenguye neza iri somero mbere muri vivo panning.Kwiyongera kwa PCR kwicyitegererezo cyibitabo byicyitegererezo ukoresheje primers yahinduwe yabyaye amplicons yakoreshwaga kuri HTS (Ishusho yinyongera. 1a).Bitewe na a) Amakosa akurikirana ya HTS11, b) ingaruka ku bwiza bwa primers (NNK) 1-12, na c) kuba hari fage yo mu bwoko bwa (wt) fage (insimburangingo ya skeleton) mu isomero ryihagararaho, hashyizweho uburyo bwo kuyungurura urutonde kugirango hakurwe gusa amakuru akurikirana (Ishusho yinyongera. 1b).Iyungurura intambwe ikoreshwa mubitabo byose bikurikirana bya HTS.Ku isomero risanzwe, habonetse ibisomwa 233.868, muri byo 39% byatsinze ibipimo byo kuyungurura kandi byakoreshejwe mu gusesengura amasomero no guhitamo icyiciro gikurikiraho (Ishusho yinyongera 1c - e).Ibisomwa byari byiganjemo inshuro 3 zifatizo zuburebure hamwe nimpinga kuri nucleotide 36 (Ishusho yinyongera 1c), yemeza igishushanyo mbonera cyibitabo (NNK) 1-12.Ikigaragara ni uko hafi 11% by'abagize isomero ryarimo ubwoko bwa 12-bwo mu bwoko bwa wt (wt) umugongo PAGISRELVDKL winjizamo, kandi hafi kimwe cya kabiri cyakurikiranye (49%) cyarimo kwinjiza cyangwa gusiba.HTS yo mu isomero ry'isomero yemeje ko peptide itandukanye cyane mu isomero: ibice birenga 81% by'urutonde rwa peptide byabonetse rimwe gusa naho 1.5% ni byo byabaye muri kopi 4 (Ishusho y'inyongera. 2a).Imirongo ya acide ya amino (aa) kumyanya 12 yose muri repertoire ifitanye isano neza numurongo uteganijwe kumubare wa code zakozwe na repertoire ya NKK yangiritse (Ishusho yinyongera 2b).Inshuro yagaragaye yibisigisigi bya aa byashizwemo nibi bisobanuro bifitanye isano neza numubare wabazwe (r = 0.893) (Ishusho yinyongera. 2c).Gutegura amasomero ya fage yo gutera inshinge harimo intambwe zo kongera no gukuraho endotoxine.Ibi byerekanwe mbere kugirango bigabanye kugabanya itandukaniro ryamasomero yicyiciro12,13.Kubwibyo, twakurikiranye isahani yongerewe isomero rya fage isomero ryakuweho endotoxine hanyuma tuyigereranya nububiko bwambere kugirango tugereranye inshuro za AA.Isano rikomeye (r = 0,995) ryagaragaye hagati ya pisine yambere na pisine yongerewe kandi isukuye (Ishusho yinyongera. 2d), byerekana ko irushanwa hagati ya clone yongerewe kumasahani ukoresheje icyiciro cya T7 ntabwo ryateje kubogama gukomeye.Iri gereranya rishingiye ku nshuro ya tripeptide motifs muri buri somero, kubera ko amasomero atandukanye (~ 109) adashobora gufatwa neza na HTS.Isesengura ryinshuro ya aa kuri buri mwanya ryerekanaga umwanya muto-ushingiye kubogama mumyanya itatu yanyuma ya repertoire yinjiye (Ishusho yinyongera. 2e).Mu gusoza, twanzuye ko ubuziranenge nubudasa bwibitabo byemewe kandi impinduka nke gusa mubitandukanye byagaragaye kubera kwongera no gutegura amasomero ya fage hagati yuburyo butandukanye bwo gutoranya.
Gutoranya ubwonko bwa cerebrospinal fluid bishobora gukorwa mugushira kubaga urumogi muri CM yimbeba zifite ubwenge kugirango byorohereze kumenya icyiciro cya T7 cyatewe mumitsi (iv) binyuze kuri BBB na / cyangwa BCSFB (Ishusho 1a-b).Twakoresheje amaboko abiri yigenga yo gutoranya (amaboko A na B) mubice bitatu byambere byo guhitamo vivo (Ishusho 1c).Twagiye buhoro buhoro twongera umurongo wo gutoranya tugabanya umubare wicyiciro cyatangijwe mubice bitatu byambere byo gutoranya.Ku cyiciro cya kane cyo gutekesha, twahujije ingero ziva mumashami A na B hanyuma dukora izindi eshatu zigenga zatoranijwe.Kugira ngo wige muri vivo imiterere ya T7 fage ibice muri ubu buryo, icyiciro cyo mu gasozi (PAGISRELVDKL master insert) cyatewe mu mbeba binyuze mumitsi yumurizo.Kugarura ibyiciro biva mu bwonko bwa cerebrospinal n'amaraso mu bihe bitandukanye byerekanaga ko icyiciro gito cya T7 icosahedral icyiciro cya mbere cyagize icyiciro cya mbere cyo gukuraho igice cyamaraso (Ishusho yinyongera 3).Dushingiye kuri titeri yatanzwe hamwe nubunini bwamaraso yimbeba, twabaze ko hafi 1% wt.icyiciro cya dose yatanzwe cyagaragaye mumaraso nyuma yiminota 10 nyuma yo gutera inshinge.Nyuma yo kugabanuka kwambere kwambere, gutinda kwibanze byapimwe hamwe nigice cyubuzima bwiminota 27.7.Icy'ingenzi, ibyiciro bike cyane byakuwe mubice bya CSF, byerekana imiterere mike yo kwimuka kwubwoko bwimiterere yimuka muri CSF (Ishusho yinyongera 3).Ugereranije, titre zigera kuri 1 x 10-3% gusa ya T7 fage mumaraso na 4 x 10-8% byicyiciro cya mbere cyinjijwe byagaragaye mumazi yubwonko mugihe cyicyitegererezo cyose (0-250 min).Ikigaragara ni uko igice cya kabiri cyubuzima (25.7 min) cyicyiciro cyubwoko bwishyamba mumazi ya cerebrospinal cyasaga nkicyagaragaye mumaraso.Aya makuru yerekana ko inzitizi itandukanya igice cya CSF n'amaraso idahwitse mu mbeba zandujwe na CM, bigatuma mu guhitamo vivo amasomero y'ibice kugirango hamenyekane clone zijyanwa mu maraso mu cyumba cya CSF.
(a) Gushiraho uburyo bwo kongera gutoranya cerebrospinal fluid (CSF) kuva muri pisine nini..(c) Muri vivo fage yerekana kwerekana ibicapo.Muri buri cyiciro cyo gutoranya, ibyiciro (ibiranga inyamaswa imbere mu myambi) byatewe inshinge.Amashami abiri yigenga yandi (A, B) abikwa ukundi kugeza icyiciro cya 4 cyo gutoranya.Kuburyo bwo gutoranya icyiciro cya 3 nicya 4, buri fage clone yakuwe muri CSF yakurikiranwe nintoki..Imirambararo yubururu yerekana impuzandengo yambere ya fage mumaraso, ubaze uhereye kumubare watewe inshinge, ukurikije ubwinshi bwamaraso.Umwanya wumukara werekana ingingo yo guhuza umurongo y umurongo utandukanijwe nubushakashatsi bwamaraso..Umubare wa motifs iboneka mubisomwa 1000 irerekanwa.Ikigaragara (p <0.001) motif ikungahaye irangwa nududomo dutukura....Ingano yinyuguti imwe yerekana inshuro iyo aside amine iboneka kuri uwo mwanya.Icyatsi = polar, umutuku = utabogamye, ubururu = shingiro, umutuku = acide n'umukara = hydrophobique amino acide.Igishushanyo 1a, b cyakozwe kandi cyakozwe na Eduard Urich.
Twashizemo isomero rya fage peptide mu mbeba ebyiri za CM (clade A na B) hanyuma fage itandukanya amazi ya cerebrospinal fluid n'amaraso (Ishusho 1d).Itangiriro ryihuse ryibitabo byibitabo ntibyagaragaye cyane ugereranije nicyiciro cyubwoko.Hagati yubuzima bwa kimwe cya kabiri cyibitabo byatewe mumatungo yombi byari iminota 24.8 mumaraso, bisa nicyiciro cyubwoko, niminota 38.5 muri CSF.Amaraso na cerebrospinal fluid fage sample ya buri nyamaswa yakorewe HTS kandi peptide zose zamenyekanye zasesenguwe kugirango habeho motif ya tripeptide.Tripeptide motifs yatoranijwe kuko itanga urufatiro ruto rwo gushiraho imiterere no guhuza peptide-protein14,15.Twabonye isano iri mu gukwirakwiza motif hagati yububiko bwibitabo bwa fage yatewe na clone yakuwe mumaraso yinyamaswa zombi (Ishusho 1e).Amakuru yerekana ko ibigize isomero bikungahaye cyane mubice byamaraso.Aminide acide ya Amino hamwe nuburyo bwumvikanyweho byongeye gusesengurwa kuri buri mwanya ukoresheje guhuza na software ya Weblogo16.Igishimishije, twabonye ubutunzi bukomeye mubisigazwa byamaraso glycine (Ishusho 1g).Iyo amaraso yagereranijwe na clon zatoranijwe muri CSF, hagaragaye guhitamo gukomeye hamwe no gutesha agaciro motif (Ishusho 1f), kandi aside amine zimwe na zimwe wasangaga biboneka ahantu hateganijwe mubanyamuryango 12 (Ishusho 1h).Ikigaragara ni uko inyamaswa ku giti cye zitandukanye cyane mu mazi yo mu bwonko, mu gihe ubutunzi bwa glycine bwagaragaye mu nyamaswa zombi (Ishusho y'inyongera 4a - j).Nyuma yo kuyungurura cyane amakuru yikurikiranya mumazi yubwonko bwinyamanswa # 1.1 na # 1.2, habonetse peptide idasanzwe ya 964 na 420 idasanzwe ya 12-mer (Ishusho yinyongera. 1d - e).Clone ya fage yihariye yongerewe kandi ikorerwa icyiciro cya kabiri cyo guhitamo vivo.Icyiciro cyakuwe mu cyiciro cya kabiri cyo gutoranya cyakorewe HTS muri buri nyamaswa kandi peptide zose zamenyekanye zakoreshejwe nk'injiza muri gahunda yo kumenyekanisha motif kugira ngo isesengure ibibaho bya tripeptide (Ishusho 2a, b, ef).Ugereranije nizunguruka ryambere ryicyiciro cyakuwe muri CSF, twabonye ubundi guhitamo no gutoranya motif nyinshi muri CSF mumashami A na B (Ishusho 2).Imiyoboro iranga algorithm yakoreshejwe kugirango hamenyekane niba bahagarariye uburyo butandukanye bwikurikiranya.Isano ryagaragaye ryagaragaye hagati yurwego rwa 12 rwagaruwe na CSF mu cyiciro cya A (Ishusho 2c, d) na B B (Ishusho 2g, h).Isesengura ryahurijwe hamwe muri buri shami ryagaragaje imyirondoro itandukanye yo guhitamo peptide 12-mer (Umugereka wongeyeho 5c, d) hamwe no kwiyongera kwa CSF / maraso ya titer yamaraso mugihe cya clone zahujwe nyuma yicyiciro cya kabiri cyo gutoranya ugereranije nicyiciro cya mbere cyo gutoranya (Ishusho yinyongera 5e).).
Gutunganyiriza motifs na peptide mumazi ya cerebrospinal byamazi abiri akurikiranye muri vivo imikorere yicyiciro cyo gutoranya.
Ibyiciro byose byubwonko bwakuwe mubyiciro byambere bya buri nyamaswa (inyamaswa # 1.1 na # 1.2) byahurijwe hamwe, byongerwaho imbaraga, bikurikiranwa na HT hanyuma bishyirwa hamwe (2 x 1010 ibyiciro / inyamaswa) 2 SM yimbeba (# 1.1 → #).2.1 na 2.2, 1.2 → 2.3 na 2.4)..Inshuro zijyanye no gukwirakwiza motifs zerekana ibishoboka byose byuzuye tripeptide iboneka muri peptide mubyerekezo byombi.Umubare wa motifs iboneka mubisomwa 1000 irerekanwa.Motifs zagaragaye cyane (p <0.001) zatoranijwe cyangwa zitandukanijwe murimwe mumasomero yagereranijwe agaragazwa nududomo dutukura..Ingano yinyuguti imwe yerekana inshuro iyo aside amine iboneka kuri uwo mwanya.Kugirango uhagararire ikirangantego, inshuro zikurikirana za CSF zavanywe mubikoko bitandukanye hagati yizingo ebyiri zatoranijwe ziragereranywa kandi ibyiciro bikungahaye mubyiciro bya kabiri byerekanwe: (c) # 1.1– # 2.1 (d) # 1.1– # 2.2 (g) # 1.2– # 2.3 na (h) # 1.2– # 2.4.Aminide acide ikungahaye cyane kumwanya runaka muri (c, d) inyamaswa oya.2.1 na oya.2.2 cyangwa (g, h) mu nyamaswa no.2.3 na oya.2.4 irerekanwa ibara.Icyatsi = polar, umutuku = utabogamye, ubururu = shingiro, umutuku = acide n'umukara = hydrophobique amino acide.
Nyuma yicyiciro cya gatatu cyo gutoranya, twabonye urutonde 124 rwihariye rwa peptide (# 3.1 na # 3.2) kuva 332 CSF yongeye gushyirwaho clone ya fage yatandukanijwe ninyamaswa ebyiri (Ishusho yinyongera 6a).Urukurikirane LGSVS (18.7%) yari ifite igipimo kinini ugereranije, ikurikirwa no gushyiramo ubwoko bwishyamba PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), na SARGSWREIVSLS (2.2%).Mu cyiciro cya kane cyanyuma, twahujije amashami abiri yatoranijwe yigenga ku nyamaswa eshatu zitandukanye (Ishusho 1c).Muri clone 925 ikurikiranye yakuwe muri CSF, mugice cya kane twasanze urutonde rwa peptide 64 idasanzwe (Umugereka wa 6b), murirwo ugereranije ugereranije nicyiciro cyubwoko bwishyamba cyaragabanutse kugera kuri 0.8%.Clone yakunze kugaragara cyane mugice cya kane ni LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) na RLSS.%)).Uburebure buringaniye bwa peptide bwatoranijwe biterwa no kwinjiza nucleotide / gusiba cyangwa guhagarika codons imburagihe muri primaire yububiko bwibitabo mugihe ukoresheje code ya degenerate kubishushanyo mbonera byibitabo bya NNK.Guhagarika codons hakiri kare bitanga peptide ngufi kandi byatoranijwe kuko birimo aa motif nziza.Peptide ndende irashobora guterwa no gushiramo / gusiba muri primers yububiko bwibitabo.Iyi myanya yagenewe guhagarika codon hanze yikadiri ikayisoma kugeza igihe codon nshya ihagaze igaragara hepfo.Muri rusange, twabaze ibintu byo gukungahaza ibyiciro bine byatoranijwe tugereranya ibyinjijwe hamwe nicyitegererezo gisohoka.Kuburyo bwa mbere bwo kwerekana, twakoresheje ubwoko bwa fage titer nkibidasanzwe byihariye.Igishimishije, gutoranya ibyiciro bibi byari bikomeye cyane mugihe cyambere cya CSF, ariko ntabwo byari mumaraso (Igishusho 3a), ibyo bikaba bishobora guterwa no kuba bishoboka ko ikwirakwizwa rya pasiporo ryabantu benshi bagize isomero rya peptide ryinjira mubice bya CSF cyangwa ibyiciro bifitanye isano bikunda kubikwa neza cyangwa kuvanwa mumaraso kuruta bagiteri.Nyamara, mu cyiciro cya kabiri cyo gutekesha, guhitamo gukomeye mu byiciro muri CSF byagaragaye mu byiciro byombi, byerekana ko icyiciro kibanziriza iki cyakungahaye mu byiciro byerekana peptide ziteza imbere CSF (Ishusho 3a).Na none, nta gukungahaza amaraso gukomeye.Mu cyiciro cya gatatu n'icya kane, clone ya fage yakungahaye cyane muri CSF.Tugereranije inshuro zijyanye na buri peptide yihariye ikurikiranye hagati yuburyo bubiri bwanyuma bwo gutoranya, twasanze ibyakurikiranye byarushijeho gukungahazwa mugice cya kane cyo gutoranya (Ishusho 3b).Igiteranyo cya 931 tripeptide yakuwe muri 64 zose zidasanzwe za peptide ukoresheje icyerekezo cya peptide.Ibitekerezo bikungahaye cyane mu cyiciro cya kane byasuzumwe cyane kugirango imyirondoro yabo ikungahaye mu mpande zose ugereranije n’isomero ryatewe (gukata: gukungahaza 10%) (Ishusho yinyongera. 6c).Uburyo rusange bwo gutoranya bwerekanaga ko intego nyinshi zize zatunganijwe mubyiciro byose byabanjirije amashami yombi yatoranijwe.Nyamara, motif zimwe (urugero: SGL, VSG, LGS GSV) ahanini zari zivuye mubindi bisobanuro A, mugihe izindi (urugero: FGW, RTN, WGF, NTR) zikungahaye kumurongo B.
Kwemeza ubwikorezi bwa CSF bwa CSF ikungahaye kuri fagitire yerekana peptide hamwe na peptide umuyobozi wa biotinylated bihujwe na streptavidin yishyurwa.
.Ibintu bikungahaye mubyiciro bitatu byanyuma (R2-R4) byabazwe ugereranije nicyiciro cyabanjirije icyiciro cya mbere (R1) hamwe namakuru yuburemere.Utubari twafunguye ni cerebrospinal fluid, utubari dufite igicucu ni plasma.(*** p <0.001, ukurikije t-test yabanyeshuri)..Ibice bitandatu bikunze kugaragara cyane byerekanwe kumurongo, kubara hamwe nibintu bikungahaza hagati yicyiciro cya 3 nicya 4 cyo guhitamo (insets)..CSF hamwe namaraso byakusanyirijwe kumwanya wagenwe..CSF ya farumasi yubuvuzi ya buri feri ya clone yatewe hamwe nububiko bwibitabo bwa peptide mugihe cyerekanwe.(d) yerekana impuzandengo ya CSF / maraso kumpande zose zagaruwe / mL mugihe cyo gutoranya..Nibura imbeba eshatu (N = 3).).Ingero za CSF zegeranijwe ku gihe cyerekanwe kandi ibipimo bya streptavidin byapimwe na CSF anti-streptavidin ELISA (nd = itamenyekanye).(* p <0.05, ** p <0.01, *** p <0.001, hashingiwe ku kizamini cya ANOVA)..Ibice bisa kandi bisa na aside amine ni ibara ryanditse.
Mu byiciro byose bikungahaye mu cyiciro cya kane (Igishusho 3b), clone esheshatu z'abakandida zatoranijwe kugirango zisesengurwe ku giti cye mu buryo bw'icyitegererezo cya CSF.Umubare uhwanye n’icyiciro cya gatandatu cy’abakandida, icyiciro cyubusa (nta gushyiramo) hamwe n’amasomero ya peptide ya peptide yatewe mu matungo atatu ya CM yanduye, kandi imiti ya farumasi yagenwe muri CSF (Ishusho 3c) n’amaraso (Ishusho yinyongera 7).Clone ya fage yose yapimwe yibasiye igice cya CSF kurwego rwikubye inshuro 10-1000 kurenza urwego rwubusa (# 1779).Kurugero, clone # 2020 na # 2077 yari ifite titereri ya CSF inshuro zigera ku 1000 kurenza icyiciro cyo kugenzura.Umwirondoro wa pharmacokinetic ya buri peptide yatoranijwe iratandukanye, ariko yose ifite ubushobozi buke bwo gutaha CSF.Twabonye kugabanuka guhoraho mugihe cya clone # 1903 na # 2011, mugihe kuri clone # 2077, # 2002 na # 2009 kwiyongera muminota 10 yambere bishobora kwerekana ubwikorezi bukora ariko bigomba kugenzurwa.Clone # 2020, # 2002, na # 2077 yahagaze neza murwego rwo hejuru, mugihe CSF yibanze ya clone # 2009 yagabanutse buhoro nyuma yo kwiyongera kwambere.Hanyuma twagereranije inshuro ugereranije na buri mukandida wa CSF hamwe nubunini bwamaraso (Ishusho 3d).Isano riri hagati ya titerite ya buri mukandida wa CSF hamwe na titre yamaraso mugihe cyose cyicyitegererezo cyerekanaga ko batatu mubakandida batandatu bakungahaye cyane mumaraso CSF.Igishimishije, clone # 2077 yerekanye umuvuduko ukabije wamaraso (Ishusho yinyongera).Kugira ngo twemeze ko peptide ubwayo ishoboye gutwara cyane imizigo itari ibice bya fage mu gice cya CSF, twahujije peptide enye z'abayobozi bakomotse kuri biotine kuri N-terminus aho peptide ifatira ku gice cya fage.Peptide ya biotinylated (nimero 2002, 2009, 2020 na 2077) yahujwe na streptavidin (SA) kugirango ibone uburyo butandukanye bwigana fage geometrie.Iyi format yatwemereye kandi gupima SA mu maraso no mu bwonko bwa cerebrospinal fluid nka peptide itwara imizigo.Icy'ingenzi, amakuru ya fage ashobora kubyara kenshi mugihe peptide ya syntetique yatanzwe muri ubu buryo bwa SA-conjugated (Ishusho 3e).Peptide yatondaguwe ntiyigeze igaragara mbere kandi byihuse CSF ifite urwego rutamenyekana mumasaha 48.Kugirango tumenye neza inzira zo gutanga ziriya peptide ya peptide mumwanya wa CSF, twasesenguye aho hitabwa kuri peptide ya fage peptide dukoresheje immunohistochemie (IHC) kugirango tumenye neza ibice bya fage nyuma yisaha 1 nyuma yo gutera inshinge muri vivo.Ikigaragara ni uko clone # 2002, # 2077, na # 2009 zishobora gutahurwa no kwanduza cyane imitsi yubwonko, mugihe icyiciro cyo kugenzura (# 1779) na clone # 2020 kitagaragaye (Ishusho yinyongera 8).Ibi birerekana ko peptide igira uruhare mukugira ingaruka mubwonko neza mukurenga BBB.Ubundi isesengura rirambuye rirasabwa kugerageza iyi hypothesis, kuko inzira ya BSCFB nayo ishobora kubigiramo uruhare.Iyo ugereranije urutonde rwa aside amine ya clone ikungahaye cyane (# 2002) hamwe nizindi peptide zatoranijwe, byagaragaye ko zimwe murizo zifite ubwiyongere bwa aside amine, bushobora kwerekana uburyo busa nubwikorezi (Ishusho 3f).
Bitewe numwirondoro wihariye wa plasma no kwiyongera cyane muri CSF mugihe, clone yerekana fage # 2077 yongeye gushakishwa mugihe cyamasaha 48 kandi yashoboye kubyara ubwiyongere bwihuse muri CSF yagaragaye ifatanije ninzego za SA zihamye (Ishusho 4a).Kubijyanye nizindi clone zamenyekanye, # 2077 yandujwe cyane na capillaries yubwonko kandi yerekanaga colocalisation ikomeye hamwe na capillary marker lectin iyo urebye neza kandi birashoboka ko bimwe byanduye mumwanya wa parenchymal (Ishusho 4b).Kugira ngo tumenye niba ingaruka za farumasi ziterwa na peptide zishobora kuboneka muri CNS, twakoze ubushakashatsi aho verisiyo ya biotinylated ya i) peptide ya # 2077 na ii) peptide ya BACE1 inhibitor peptide yavanze na SA ku mibare ibiri itandukanye.Kubufatanye bumwe twakoresheje gusa inhibitor ya BACE1 peptide naho ubundi twakoresheje igipimo cya 1: 3 cya peptide ya peptide ya BACE1 na # 2077 peptide.Izi ngero zombi zatanzwe mu maraso kandi amaraso hamwe na cerebrospinal fluid urugero rwa beta-amyloide peptide 40 (Abeta40) byapimwe mugihe.Abeta40 yapimwe muri CSF kuko igaragaza BACE1 kubuza ubwonko parenchyma.Nkuko byari byitezwe, ibice byombi byagabanije cyane urugero rwamaraso ya Abeta40 (Ishusho 4c, d).Nyamara, gusa ingero zirimo uruvange rwa peptide oya.2077 hamwe na inhibitori ya BACE1 peptide yahujwe na SA yatumye igabanuka rikabije rya Abeta40 mumazi yubwonko (Ishusho 4c).Amakuru yerekana ko peptide oya.2077 ishoboye gutwara proteine ​​60 kDa SA muri CNS kandi ikanatera ingaruka za farumasi hamwe na SA-conjugated inhibitor ya peptide ya BACE1.
..Izi fage clone zahawe imbeba 3 kandi zemererwa kuzenguruka isaha 1 mbere yo gutwikwa.Ubwonko bwaragabanijwe kandi busizwe hamwe na antibodiyite ya FITC yanditseho polyclone irwanya T7 fage capsid.Iminota icumi mbere yo kwisiga no gukosorwa nyuma, DyLight594 yanditseho disikuru yatanzwe mumitsi.Amashusho ya Fluorescent yerekana disikuru yerekana (umutuku) kuruhande rwa luminal ya microvessel na fage (icyatsi) mumitsi ya capillaries hamwe nubwonko bwubwonko bwa perivasculaire.Igipimo cyibipimo gihwanye na 10 µm..Kugabanuka kwa BACE1 peptide inhibitor-yunganirwa muri Aβ40 yapimwe na Aβ1-40 ELISA mumaraso (umutuku) na fluid cerebrospinal fluid (orange) mugihe cyagenwe.Kugirango bisobanuke neza, umurongo utudomo ushushanyije ku gishushanyo ku gipimo cya 100%...Kwibanda kwa Aβ kugenzura byari 420 pg / ml (gutandukana bisanzwe = 101 pg / ml).
Icyiciro cyerekanwe cyakoreshejwe neza mubice byinshi byubushakashatsi bwibinyabuzima17.Ubu buryo bwakoreshejwe mubushakashatsi bwubwoko butandukanye bwa vivo 18,19 kimwe nubushakashatsi bwibasira imiyoboro yubwonko20,21,22,23,24,25,26.Muri ubu bushakashatsi, twongereye uburyo bwo guhitamo ubu buryo bwo gutoranya gusa mu kumenya mu buryo butaziguye peptide yibasira imiyoboro y’ubwonko, ariko tunavumbura abakandida bafite imitungo itwara abantu kugira ngo barenze inzitizi y’amaraso n'ubwonko.Ubu turasobanura iterambere ryuburyo bwo gutoranya vivo muri CM intubated imbeba kandi twerekana ubushobozi bwayo bwo kumenya peptide ifite imitungo ya CSF.Twifashishije icyiciro cya T7 cyerekana isomero rya peptide ya 12-mer idasanzwe, twashoboye kwerekana ko fage ya T7 ari nto bihagije (hafi 60 nm ya diametre) 10 kugirango ihuze na bariyeri yubwonko bwamaraso, bityo turenga imbogamizi yubwonko bwamaraso cyangwa poroque ya choroide.Twabonye ko gusarura CSF bivuye ku mbeba za CM zanduye byagenzuwe neza muburyo bwa vivo bwo gusuzuma imikorere, kandi ko fage yakuweho itajyanye gusa na vasulature gusa ahubwo yanakoraga nk'ubwikorezi bwambukiranya inzitizi y'amaraso n'ubwonko.Byongeye kandi, mugihe kimwe cyo gukusanya amaraso no gukoresha HTS kuri CSF hamwe nicyiciro gikomoka kumaraso, twemeje ko guhitamo kwa CSF kutatewe no gukungahaza amaraso cyangwa ubuziranenge bwo kwaguka hagati yo gutoranya.Nyamara, igice cyamaraso kiri muburyo bwo gutoranya, kubera ko ibyiciro bishobora kugera muri CSF bigomba kubaho kandi bikazenguruka mumaraso igihe kirekire bihagije kugirango bikungahaze mubwonko.Kugirango dukuremo amakuru yizewe yamakuru avuye mumibare mbisi ya HTS, twashyize mubikorwa muyunguruzi yahujwe namakosa yihariye akurikirana mugikorwa cyo gusesengura.Mugushyiramo ibipimo bya kinetic muburyo bwo gusuzuma, twemeje farumasi yihuta yibice byo mu bwoko bwa T7 (t½ ~ 28 min) mumaraso24, 27, 28 kandi tunagena ubuzima bwabo bwa kabiri mumazi ya cerebrospinal fluid (t½ ~ 26 min) kumunota).Nubwo imiti isa na farumasi ikoreshwa mumaraso na CSF, 0.001% gusa byamaraso yibice bya fage byashoboraga kugaragara muri CSF, byerekana ko umuvuduko muke wubwoko bwa T7 bwishyamba bwambukiranya inzitizi yubwonko bwamaraso.Aka kazi kagaragaza akamaro k'icyiciro cya mbere cyo gutoranya mugihe ukoresheje ingamba zo gutondekanya vivo, cyane cyane kuri sisitemu ya fage ikurwaho vuba vuba, kuko clone nke zishobora kugera muri CNS.Rero, mu cyiciro cya mbere, kugabanya itandukaniro ryibitabo byari binini cyane, kuko umubare muto gusa wa clone waje gukusanywa murubu buryo bukomeye bwa CSF.Ibi muri vivo panning stratégie harimo intambwe nyinshi zo gutoranya nko kwegeranya cyane mu gice cya CSF, kubaho kwa clone mu gice cyamaraso, no kuvana vuba T7 fage clone mumaraso muminota 10 yambere (Igicapo 1d nigishushanyo cya 4M).).Rero, nyuma yicyiciro cya mbere, clone zitandukanye zamenyekanye muri CSF, nubwo pisine imwe yambere yakoreshejwe kubinyamaswa.Ibi birerekana ko intambwe nyinshi zo gutoranya amasomero yinkomoko hamwe numubare munini wabanyamuryango b'isomero bivamo kugabanuka gukomeye muburyo butandukanye.Kubwibyo, ibintu bitunguranye bizahinduka igice cyibikorwa byambere byo gutoranya, bigira ingaruka cyane kubisubizo.Birashoboka ko clone nyinshi mubitabo byumwimerere byari bifite CSF itunganijwe cyane.Nubwo bimeze bityo ariko, no mubihe bimwe byubushakashatsi, ibisubizo byatoranijwe birashobora gutandukana bitewe numubare muto wa buri clone yihariye muri pisine ibanza.
Motifs ikungahaye muri CSF itandukanye niyiri mumaraso.Igishimishije, twabonye ihinduka ryambere ryerekeza kuri peptide ikungahaye kuri glycine mumaraso yinyamaswa.(Ishusho 1g, Igishushanyo cy'inyongera. 4e, 4f).Icyiciro kirimo peptide ya glycine kirashobora kuba gihamye kandi ntigishobora gukurwa mubitembera.Nyamara, izo peptide zikungahaye kuri glycine ntizigeze zigaragara mu byitegererezo by’amazi yo mu bwonko, byerekana ko amasomero yatunganijwe yanyuze mu nzira ebyiri zitandukanye zo gutoranya: imwe mu maraso indi yemerewe kwirundanyiriza mu mazi yo mu bwonko.Clone ikungahaye kuri CSF ituruka kumurongo wa kane wo gutoranya yageragejwe cyane.Hafi ya clone zose zapimwe kugiti cye zemejwe ko zikungahaye muri CSF ugereranije nicyiciro cyo kugenzura ubusa.Peptide imwe yakubiswe (# 2077) yasuzumwe muburyo burambuye.Yerekanye plasma ndende igice cyubuzima ugereranije nizindi hit (Igishusho 3d ninyongera Ishusho 7), kandi igishimishije, iyi peptide yarimo ibisigazwa bya sisitemu kuri C-terminus.Vuba aha byerekanwe ko kongeramo sisitemu kuri peptide bishobora guteza imbere imiti ya farumasi ikomatanya na albumin 29.Kugeza ubu ntabwo bizwi kuri peptide # 2077 kandi bisaba ubushakashatsi bwimbitse.Peptide zimwe zerekanaga agaciro-biterwa no gukungahaza CSF (amakuru aterekanwa), bishobora kuba bifitanye isano na geometrike yerekanwe hejuru ya T7 capsid.Sisitemu ya T7 twakoresheje yerekanaga kopi 5-15 za buri peptide kuri buri cyiciro.IHC yakorewe ku bakandida bayobora fage clone yatewe mu bwonko bwubwonko bwimbeba (Ishusho yinyongera 8).Amakuru yerekanaga ko byibura clone eshatu (No 2002, No 2009 na No 2077) zakoranye na BBB.Hasigaye kumenya niba iyi mikoranire ya BBB itera gukusanya CSF cyangwa kwimuka kwi clone kuri BCSFB.Icyangombwa, twerekana ko peptide yatoranijwe igumana ubushobozi bwubwikorezi bwa CSF mugihe ikomatanyirijwe hamwe nu mutwaro wa protein.Guhambira peptide ya N-terminal biotinylated peptide isubiramo cyane cyane ibisubizo byabonetse hamwe na clone ya fage yabyo mumaraso hamwe nubwonko bwubwonko (Ishusho 3e).Hanyuma, twerekana ko peptide ya piside # 2077 ishoboye guteza imbere ubwonko bwimikorere ya biotinylated peptide inhibitor ya BACE1 ihujwe na SA, itera ingaruka za farumakodinike muri CNS igabanya cyane urwego Abeta40 muri CSF (Ishusho 4).Ntabwo twashoboye kumenya homologues iyo ari yo yose muri data base dukora peptide ikurikirana homology ishakisha hits zose.Ni ngombwa kumenya ko ingano yububiko bwibitabo bwa T7 bugera kuri 109, mugihe ingano yububiko bwibitabo bwa 12-mers ari 4 x 1015. Kubwibyo, twahisemo gusa agace gato k’ahantu hatandukanye h’isomero rya peptide ya 12-mer, rishobora gusobanura ko peptide nziza cyane ishobora kumenyekana mugusuzuma umwanya ukurikiranye wibi byamenyekanye.Hypothetically, imwe mumpamvu zituma tutabona homologues naturel ya peptide irashobora gutorwa mugihe cyubwihindurize kugirango hirindwe kwinjizwa mu buryo butagengwa na peptide zimwe na zimwe mu bwonko.
Dufatiye hamwe, ibisubizo byacu bitanga urufatiro rwakazi kazaza kugirango tumenye kandi turanga sisitemu yo gutwara inzitizi yubwonko bwamaraso muri vivo muburyo burambuye.Ishingiro ryibanze ryubu buryo rishingiye ku ngamba zo gutoranya imikorere itagaragaza gusa clone ifite imiterere yubwonko bwubwonko, ariko kandi ikubiyemo intambwe ikomeye aho clone igenda neza ifite ibikorwa byimbere kugirango yambure inzitizi zibinyabuzima muri vivo mubice bya CNS.ni ugusobanura uburyo bwo gutwara izo peptide nibyifuzo byabo byo guhuza microcasculature yihariye ubwonko.Ibi birashobora kuganisha ku kuvumbura inzira nshya zo gutwara BBB hamwe na reseptors.Turateganya ko peptide yamenyekanye ishobora guhuza byimazeyo na reseptor ya cerebrovasculaire cyangwa ligande zizenguruka zinyuzwa muri BBB cyangwa BCSFB.Peptide vectors hamwe nibikorwa byo gutwara CSF byavumbuwe muriki gikorwa bizakomeza gukorwaho iperereza.Muri iki gihe turimo gukora iperereza ku bwonko bwihariye bwa peptide kubushobozi bwabo bwo kurenga BBB na / cyangwa BCSFB.Iyi peptide nshya izaba ibikoresho byingirakamaro cyane kubishobora kuvumburwa kwakirwa cyangwa inzira nshya no guteza imbere urubuga rushya rukora neza rwo kugeza macromolecules, nka biologiya, mubwonko.
Kuraho cisterna nini (CM) ukoresheje guhindura uburyo bwasobanuwe mbere.Imbeba za Wistar zatewe anestheti (200-350 g) zashyizwe ku cyuma cya stereotaxique hanyuma hakorwa intambwe yo hagati yo kogosha kandi yateguwe neza kugirango yerekane igihanga.Siba ibyobo bibiri mumwanya wigishishwa cyo hejuru hanyuma uhambire imigozi ikosora mumyobo.Hacukuwe umwobo wongeyeho mu cyerekezo cya octipital kugira ngo uyobore stereotactique yo kuyobora urumogi rudafite ingese muri CM.Koresha sima y'amenyo ukikije urumogi kandi ushireho imigozi.Nyuma yo gukiza ifoto na sima gukomera, igikomere cyuruhu cyafunzwe hamwe na suture ya 4/0.Gushyira urumogi neza byemezwa no gutembera kwamazi ya cerebrospinal fluid (CSF).Kuraho imbeba mubikoresho bya stereotaxic, wakire ubuvuzi bukwiye nyuma yo kubagwa no kubabara, hanyuma ubemerera gukira byibuze icyumweru kimwe kugeza ibimenyetso byamaraso bibonetse mumazi yubwonko.Imbeba za Wistar (Crl: WI / Han) zabonetse mu ruzi rwa Charles (mu Bufaransa).Imbeba zose zabitswe mugihe cyihariye kidafite virusi.Ubushakashatsi bwose bw’inyamaswa bwemejwe n’ibiro by’amatungo by’Umujyi wa Basel, mu Busuwisi, kandi bwakozwe hakurikijwe uruhushya rw’inyamanswa No 2474 (Isuzuma ry’ubwikorezi bw’ubwonko bukora hifashishijwe gupima urwego rw’abakandida bavura muri Cerebrospinal Fluid na Ubwonko bw’imbeba).
Witonze komeza imbeba hamwe na CM cannula mu ntoki.Kuraho Datura muri cannula hanyuma ukusanyirize 10 µl y'amazi ya cerebrospinal itemba.Kubera ko amaherezo urumogi rwa cannula rwahungabanijwe, gusa ubushakashatsi bwerekana ko amazi yo mu bwonko asobanutse adafite ibimenyetso byerekana ko yanduye cyangwa afite ibara ryashyizwe muri ubu bushakashatsi.Mu buryo bubangikanye, hafi 10-20 μl z'amaraso zavanywe mu gutemagura gato ku mutwe wumurizo mu tubari hamwe na heparin (Sigma-Aldrich).CSF n'amaraso byakusanyirijwe ahantu hatandukanye nyuma yo gutera inshinge T7 fage.Hafi ya 5-10 μl y'amazi yajugunywe mbere yuko buri cyitegererezo cya CSF cyegeranywa, ibyo bikaba bihuye nubunini bwapfuye bwa catheter.
Amasomero yakozwe hifashishijwe icyerekezo cya T7Select 10-3b nkuko byasobanuwe mu gitabo cya T7Select (Novagen, Rosenberg n'abandi., InNovations 6, 1-6, 1996).Muri make, insimburangingo ya 12-mer ya ADN yashizwemo muburyo bukurikira:
Codon ya NNK yakoreshejwe kugirango yirinde kode ebyiri zihagarara hamwe na aside amine ikabije kurenza urugero.N ni intoki ivanze nintoki ya buri nucleotide, na K ni intoki ivanze nintoki ya adenine na nucleotide ya cytosine.Uturere tumwe na tumwe twahinduwe duhinduka ADN ikubye kabiri hifashishijwe ubundi buryo hamwe na DNTP (Novagen) na Klenow enzyme (New England Biolabs) muri buffer ya Klenow (New England Biolabs) amasaha 3 kuri 37 ° C.Nyuma yo kubyitwaramo, ADN ifite imirongo ibiri yagaruwe nubushyuhe bwa EtOH.ADN yavuyemo yashizwemo imisemburo ibuza EcoRI na HindIII (byombi kuva Roche).Kwinjizamo no kwezwa (QIAquick, Qiagen) winjizamo (T4 ligase, Biolabs yo mu Bwongereza Nshya) noneho washyizwe mu murongo mu cyerekezo cya T7 cyabanjirije icyuma nyuma ya aside amine 348 ya 10B ya capsid.Imyitwarire yimanza yashizwe kuri 16 ° C. mumasaha 18 mbere yo gupakira vitro.Gupakira fage muri vitro byakozwe hakurikijwe amabwiriza yatanzwe na T7Select 10-3b ya cloni yo mu bwoko bwa Novagen) kandi igisubizo cyo gupakira cyongerewe inshuro imwe kuri lysis ukoresheje coli ya Escherichia (BLT5615, Novagen).Lysates yashizwemo, yitirirwa kandi ikonjeshwa kuri -80 ° C. nkumuti wa glycerol.
Kwiyongera kwa PCR kwakarere ka fage ihindagurika yongerewe mumyanda cyangwa isahani ukoresheje nyirubwite 454 / Roche-amplicon fusion primers.Imbere ya fusion primer ikubiyemo urutonde ruhuza akarere gahinduka (NNK) 12 (inyandikorugero yihariye), GS FLX Titanium Adapter A, hamwe nurufunguzo rwibitabo bine rwibanze (TCAG) (Ishusho yinyongera 1a):
Primer reversion primer nayo irimo biotine ifatanye no gufata amasaro hamwe na GS FLX Titanium Adapter B isabwa kugirango amplifisione ya clone mugihe cya emulion PCR:
Amplicons yahise ikorerwa 454 / Roche pyrosequencing ukurikije 454 GS-FLX Titanium protocole.Kuburyo bwa Sanger bukurikirana (Gukoresha Biosystems Hitachi 3730 xl ADN isesengura), T7 fage ADN yongerewe imbaraga na PCR hanyuma ikurikiranwa na primer ebyiri zikurikira:
Kwinjiza mu byapa byihariye byakorewe PCR ikoresheje Roche Byihuta Gutangira ADN Polymerase Kit (ukurikije amabwiriza yabakozwe).Kora intangiriro ishyushye (iminota 10 kuri 95 ° C) na 35 bizunguruka (50 s kuri 95 ° C, min 1 kuri 50 ° C, na min 1 kuri 72 ° C).
Icyiciro kiva mumasomero, icyiciro cyubwoko bwishyamba, fage yakijijwe muri CSF namaraso, cyangwa clone kugiti cye byongerewe imbaraga muri Escherichia coli BL5615 mumyanya yigituntu (Sigma Aldrich) cyangwa mumasahani 500 cm2 (Thermo Scientific) kuri 4 h kuri 37 ° C.Icyiciro cyakuwe mubisahani kwoza amasahani hamwe na buffer ya Tris-EDTA (Fluka Analytical) cyangwa mugukusanya ibyapa hamwe ninama za pipile sterile.Icyiciro cyatandukanijwe n’umuco ndengakamere cyangwa kuvoma hamwe n’umuzingi umwe w’imvura ya polyethylene glycol (PEG 8000) (Promega) hanyuma uhagarikwa muri buffer ya Tris-EDTA.
Icyiciro cyongerewe imbaraga cyakuweho inshuro 2-3 zo gukuraho endotoxine ukoresheje amasaro yo gukuraho endotoxine (Miltenyi Biotec) mbere yo gutera inshinge (IV) (500 μl / inyamaswa).Mu cyiciro cya mbere, hatangijwe ibyiciro 2 × 1012;mu cya kabiri, ibyiciro 2 × 1010;mu cyiciro cya gatatu n'icya kane cyo gutoranya, ibyiciro 2 × 109 kuri buri nyamaswa.Ibyiciro biri muri CSF hamwe nicyitegererezo cyamaraso yakusanyirijwe kumwanya wagenwe byagenwe no kubara icyapa ukurikije amabwiriza yabakozwe (T7Select system manual).Guhitamo icyiciro byakozwe no gutera inshinge zatewe mumasomero yatunganijwe mumurongo wumurizo cyangwa no kongera gutera inshinge zavanywe muri CSF mugice cyambere cyatoranijwe, hanyuma ibisarurwa byakurikiyeho byakozwe muminota 10, 30 min, 60 min, 90 min, 120 min, 180 min, na 240 min hamwe na CSF hamwe namaraso.Hafi yincuro enye za vivo panning zakozwe aho amashami abiri yatoranijwe yabitswe ukwe kandi asesengurwa mugihe cyicyiciro cya mbere cyo gutoranya.Ibyiciro byose byashyizwe muri CSF mubyiciro bibiri byambere byatoranijwe byakorewe 454 / Roche pyrosequensiya, mugihe clone zose zakuwe muri CSF mubyiciro bibiri byanyuma byatoranijwe zikurikiranwa nintoki.Ibyiciro byose byamaraso kuva murwego rwa mbere rwo gutoranya nabyo byakorewe 454 / Roche pyrosequencing.Kugirango utere inshinge za fage, ibyiciro byatoranijwe byongerewe muri E. coli (BL5615) kumasahani ya cm2 kuri 37 ° C mumasaha 4.Umuntu ku giti cye yatoranijwe kandi akurikirana intoki yakwirakwijwe mu gituntu.Nyuma yo gukuramo fage, kweza no gukuraho endotoxine (nkuko byasobanuwe haruguru), ibyiciro 2 × 1010 / inyamaswa muri 300 μl zatewe inshinge mumitsi imwe.
Gutunganya kandi byujuje ubuziranenge muyunguruzi yamakuru.Raw 454 / Roche data yahinduwe kuva muburyo bubiri bwikarita yerekana ikarita (sff) ihinduka Pearson yumuntu usomeka (fasta) ukoresheje software y'abacuruzi.Ibindi gutunganya urutonde rwa nucleotide byakozwe hakoreshejwe porogaramu yihariye ya C hamwe ninyandiko (porogaramu ya software idasohotse) nkuko byasobanuwe hano hepfo.Isesengura ryibanze ryibanze ririmo uburyo bukomeye bwo gushungura.Kugirango ushungure ibyasomwe bitarimo 12mer yemewe yo gushyiramo urutonde rwa ADN, ibyasomwe byahujwe bikurikirana kugirango utangire label (GTGATGTCGGGGATCCGAATTCT), guhagarika label (TAAGCTTGCGGCCGCACTCGAGTA) hamwe no gushyiramo inyuma (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC)guhuza byemerera kugera kuri 2 bidahuye kuri alignement31.Kubwibyo, soma udatangiye kandi uhagarike ibirango kandi usome urimo ibyinjijwe inyuma, ni ukuvuga, guhuza birenze umubare wemewe wo kudahuza, byakuwe mubitabo.Kubijyanye nibisigaye bisomwa, urutonde rwa N-mer rwa ADN rwatangiye kuva kuntangiriro no kurangira mbere yuko ikimenyetso cyo guhagarara cyacukuwe uhereye kumurongo wambere wasomwe hanyuma bigakomeza gutunganywa (nyuma bita "shyiramo").Nyuma yo guhinduranya kwinjiza, igice nyuma yo guhagarika codon yambere kuri 5 ′ impera ya primer ikurwa mubyinjijwe.Mubyongeyeho, nucleotide iganisha kuri codons ituzuye kuri 3 ′ impera ya primer nayo yavanyweho.Kugira ngo usibye gushiramo ibintu bikurikiranye gusa, ibisobanuro byahinduwe bitangirana na aside amine “PAG” nabyo byavanyweho.Peptide ifite uburebure bwa nyuma yubusobanuro bwa acide ya amino 3 munsi yakuwe mubitabo.Hanyuma, kura ubudahangarwa muri pisine yinjizamo hanyuma umenye inshuro ya buri kintu cyihariye.Ibisubizo by'iri sesengura birimo urutonde rwa nucleotide ikurikirana (insert) hamwe ninshuro zabo (soma) (Ishusho yinyongera 1c na 2).
Itsinda N-mer ADN yinjiza muburyo bukurikiranye: Kurandura amakosa akurikirana 454 / Roche yihariye (nkibibazo byo gukurikiranya kwaguka kwa homopolymer) no kuvanaho ibintu bitagabanije cyane, mbere byayungurujwe N-mer ADN ikurikirana (inserts) itondekanya kubisa.kwinjiza (kugeza kuri 2 bidahuye shingiro byemewe) ukoresheje itera itera algorithm isobanuwe gutya: ibyinjijwe bitondekanya mbere numurongo wabyo (hejuru kugeza hasi), kandi niba ari bimwe, kubwoko bwabo bwa kabiri kuburebure (birebire kugeza bigufi)).Rero, ibyinjijwe cyane kandi birebire bisobanura "itsinda" rya mbere.Itsinda ryinshyi ryashyizwe kumurongo wingenzi.Hanyuma, buri kwinjiza kwasigaye kurutonde rwatoranijwe byageragejwe kongerwaho mumatsinda hamwe na Needleman-Wunsch guhuza.Niba umubare udahuye, winjizamo, cyangwa gusiba mukuringaniza utarenze igipimo cya 2, iyinjizwamo ryongewe mumatsinda, kandi muri rusange itsinda ryinshyi ryiyongera ninshuro zongeweho.Kwinjiza byongewe mumatsinda birangwa nkibikoreshwa kandi usibye kubitunganya.Niba urutonde rwinjizwamo rudashobora kongerwaho mumatsinda asanzweho, iyinjizamo ryakoreshejwe mukurema itsinda rishya hamwe ninshuro zinjizwamo zikwiye kandi zikoreshwa nkuko byakoreshejwe.Itera irangira iyo buri kwinjiza ibintu byakoreshejwe mugushinga itsinda rishya cyangwa birashobora gushirwa mumatsinda asanzweho.Nyuma ya byose, gushyiramo amatsinda agizwe na nucleotide amaherezo bihindurwa muburyo bwa peptide (amasomero ya peptide).Ibisubizo by'iri sesengura ni urutonde rwinjizwamo hamwe ninshuro zijyanye nazo zigize umubare wibisomwa bikurikiranye (Ishusho yinyongera 2).
Motif Generation: Ukurikije urutonde rwa peptide idasanzwe, hashyizweho isomero ririmo uburyo bwose bushoboka bwa aside amine (aa) nkuko bigaragara hano hepfo.Buri gishushanyo gishoboka cyuburebure bwa 3 cyakuwe muri peptide kandi igishushanyo cyacyo cyongeweho hiyongereyeho isomero rusange rya motif ririmo imiterere yose (tripeptide).Amasomero ya motif yisubiramo cyane yarakurikiranye kandi arengerwa.Hanyuma, kuri buri tripeptide mumasomero ya motif, twagenzuye niba ihari mubitabo dukoresheje ibikoresho byo kubara.Muri iki kibazo, inshuro ya peptide irimo motif tripeptide yabonetse yongeweho kandi ihabwa motif mu isomero rya motif (“umubare wa motifs”).Igisubizo cyibisekuru bya motif ni ibice bibiri-bingana umurongo urimo ibintu byose bibaho bya tripeptide (motifs) hamwe nagaciro kabo, aribwo mubare wikurikiranya usoma bivamo motif ihuye mugihe ibyasomwe bishungura, bishyizwe hamwe, kandi bihindurwa.Ibipimo nkuko byasobanuwe muburyo burambuye hejuru.
Ubusanzwe umubare wa motifs hamwe na scatterplots ihuye: Umubare wa motif kuri buri sample wari usanzwe ukoresheje
aho ni numubare wabasomyi urimo ingingo i.Rero, vi yerekana ijanisha inshuro yo gusoma (cyangwa peptide) irimo motif i murugero.P-indangagaciro kumubare utari usanzwe wa motif yabazwe ukoresheje ikizamini nyacyo cya Fisher.Kubijyanye na korrelogramu yumubare wimpamvu, Isano rya Spearman ryabazwe ukoresheje umubare usanzwe wimpamvu hamwe na R.
Kugirango ugaragaze ibiri muri acide ya amino kuri buri mwanya mububiko bwibitabo bwa peptide, logogramu y'urubuga 32, 33 (http://weblogo.threeplusone.com) yarakozwe.Ubwa mbere, ibirimo aside amine kuri buri mwanya wa peptide ya 12-mer ibikwa muri matrike 20 × 12.Noneho, urutonde rwa peptide 1000 irimo aside amine igereranije kuri buri mwanya ikorwa muburyo bwa fasta-ikurikiranye kandi itangwa nkinjiza kurubuga-logo 3, itanga ishusho yerekana ishusho ya aside amine igereranije kuri buri mwanya.kububiko bwibitabo bwa peptide.Kugirango ubone amashusho yamakuru menshi, amakarita yubushyuhe yarakozwe hifashishijwe igikoresho cyatejwe imbere muri R (biosHeatmap, paketi R itarasohoka).Dendrogramu zerekanwe ku ikarita yubushyuhe zabazwe hifashishijwe uburyo bwa Ward's hierarchical clustering hamwe na metero ya Euclidean.Kubisesengura mibare yamakuru yatanzwe na motif, P agaciro kumanota adasanzwe yabazwe ukoresheje ikizamini nyacyo cya Fisher.P-indangagaciro zindi mibare yabazwe muri R ukoresheje t-test yabanyeshuri cyangwa ANOVA.
Ibyiciro byatoranijwe byatoranijwe hamwe nibice bitashyizwemo byatewe mumitsi ikoresheje umurizo (2 × 1010 fage / inyamaswa muri 300 μl PBS).Iminota icumi mbere yo gutunganirwa no gukosorwa nyuma, inyamaswa zimwe zatewe inshinge 100 μl za DyLight594 yanditseho (Vector Laboratories Inc., DL-1177).Nyuma yiminota 60 nyuma yo guterwa fage, imbeba zashizwe mumutima hamwe na ml 50 PBS ikurikirwa na ml 50 4% PFA / PBS.Ingero zubwonko zongeweho ijoro ryose muri 4% PFA / PBS hanyuma zishiramo 30% sucrose ijoro ryose kuri 4 ° C.Ingero ni flash yahagaritswe muruvange rwa OCT.Isesengura rya Immunohistochemicals yubushakashatsi bwakonjeshejwe ryakozwe mubushyuhe bwicyumba kuri 30 µm cryosections yahagaritswe na 1% BSA kandi yinjizwamo antibodiyite ya FITC yanditswemo na T7 (Novus NB 600-376A) kuri 4 ° C.Incubate ijoro ryose.Hanyuma, ibice byogejwe inshuro 3 hamwe na PBS hanyuma bisuzumwa hamwe na microscope ya lazeri (Leica TCS SP5).
Peptide zose zifite ubuziranenge bwa 98% zahujwe na GenScript yo muri Amerika, biotinylated na lyophilize.Biotin ihambiriwe hifashishijwe inyongera ya gatatu ya glycine kuri N-terminus.Reba peptide zose ukoresheje mass spectrometrie.
Streptavidin (Sigma S0677) yavanze ninshuro 5 zingana na peptide ya biotinylated, biotinylated BACE1 inhibitor peptide, cyangwa guhuza (3: 1 igereranyo) ya biotinylated BACE1 inhibitor peptide na peptide ya BACE1 muri 5-10% DMSO / yashyizwe muri PBS.Isaha 1 mubushyuhe bwicyumba mbere yo gutera inshinge.Peptide ya Streptavidin-conjugated yatewe mu maraso ku gipimo cya mg / kg 10 muri rumwe mu mitsi umurizo w’imbeba zifite ubwonko bw’ubwonko.
ELISA yibanze kuri streptavidin-peptide yibigo.Isahani ya microtiter ya Nunc Maxisorp (Sigma) yashizwemo ijoro ryose kuri 4 ° C hamwe na 1.5 μg / ml imbeba anti-streptavidin antibody (Thermo, MA1-20011).Nyuma yo guhagarika (guhagarika buffer: 140 nM NaCL, 5 mM EDTA, 0,05% NP40, 0,25% gelatine, 1% BSA) ku bushyuhe bwicyumba cyamasaha 2, oza isahani hamwe na 0.05% Hagati ya 20 / PBS (gukaraba buffer) kumasegonda 3 Isegonda, CSF na plasma byongewe kumariba yavanze na bffer (plasma 1: 10,000, CSF 1: 115).Isahani noneho yashizwemo ijoro ryose kuri 4 ° C hamwe na antibody yo gutahura (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239).Nyuma yo gukaraba intambwe eshatu, streptavidin yatahuwe na incubation muri TMB substrate igisubizo (Roche) muminota 20.Nyuma yo guhagarika iterambere ryamabara hamwe na 1M H2SO4, bapima kwinjiza kuri 450 nm.
Imikorere ya streptavidin-peptide-BACE1 inhibitor yagenzuwe na Aβ (1-40) ELISA ukurikije protocole yuwabikoze (Wako, 294-64701).Muri make, ingero za CSF zavomwe mu ndimi zisanzwe (1:23) hanyuma zishyirwa ijoro ryose kuri 4 ° C mu masahani 96 yuzuye hamwe na BNT77 ifata antibody.Nyuma yintambwe eshanu zo gukaraba, HRP-conjugated BA27 antibody yongewemo kandi yongerwaho amasaha 2 kuri 4 ° C., ikurikirwa nintambwe eshanu zo gukaraba.Aβ (1-40) yatahuwe nubushakashatsi muri TMB muminota 30 mubushyuhe bwicyumba.Nyuma yiterambere ryamabara ryahagaritswe hamwe no guhagarika igisubizo, bapima kwinjiza kuri 450 nm.Icyitegererezo cya plasma cyakuweho icyiciro gikomeye mbere ya Aβ (1-40) ELISA.Plasma yongerewe kuri 0.2% DEA (Sigma) mumasahani 96-yuzuye kandi yashyizwe mubushyuhe bwicyumba muminota 30.Nyuma yo gukaraba neza ibyapa bya SPE (Oasis, 186000679) ukoresheje amazi na methanol 100%, icyitegererezo cya plasma cyongewe kumasahani ya SPE hanyuma amazi yose arakurwaho.Ingero zogejwe (ubanza hamwe na 5% methanol hanyuma 30% methanol) hanyuma zisukurwa na 2% NH4OH / 90% methanol.Nyuma yo kumisha eluate kuri 55 ° C kuminota 99 kuri N2 ihoraho, ingero zagabanutse mumazi asanzwe kandi Aβ (1-40) yapimwe nkuko byasobanuwe haruguru.
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