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Uruzitiro rw'amaraso n'ubwonko hamwe n'uruzitiro rw'amaraso n'ubwonko bibuza imiti ivura indwara za kanseri kugera ku ntego zayo mu mitsi yo hagati, bityo bikabangamira uburyo bwiza bwo kuvura indwara z'imitsi. Kugira ngo tuvumbure uburyo bushya bwo gutwara ubwonko mu mubiri, twashyizeho isomero rya T7 phage peptide hamwe n'amaraso yakusanyijwe n'amazi yo mu bwonko (CSF) hakoreshejwe uburyo bwa cannulated large pool y'imbeba. Udukoko twa phage twakungahaye cyane muri CSF nyuma y'inshuro enye zo gutoranya. Igeragezwa rya peptides imwe ku yindi ryagaragaje ko CSF ​​yikubye inshuro zirenga 1000. Umurimo w'ibinyabutabire bya peptide mu bwonko byemejwe no kugabanuka kwa 40% k'urugero rwa amyloid-β mu mazi yo mu bwonko hakoreshejwe inhibitor ya BACE1 peptide ijyanye na peptide nshya yagaragaye. Ibi byavuye mu bushakashatsi bigaragaza ko peptides zagaragajwe n'uburyo bwo gutoranya phage mu mubiri zishobora kuba inzira z'ingirakamaro zo kugeza macromolecules mu bwonko mu buryo bw'umubiri hamwe n'ingaruka zo kuvura.
Ubushakashatsi ku buvuzi bushingiye ku mikorere y’imitsi yo mu bwoko bwa CNS bwibanze cyane ku kumenya imiti n’ibintu bitanga ubushobozi bwo kwibasira CNS, ariko imbaraga nke zirimo kumenyekana ku buryo butuma imiti igera mu bwonko. Ibi bitangiye guhinduka ubu kuko gutanga imiti, cyane cyane molekile nini, ari igice cy’ingenzi mu iterambere ry’imiti ikoreshwa mu mitsi yo mu bwonko. Ibidukikije by’imitsi yo mu bwoko bwa CNS birinzwe neza n’imitsi yo mu bwonko, igizwe n’imiti ihuza amaraso n’ubwonko (BBB) ​​​​n’imiti ihuza amaraso n’ubwonko (BCBB), bigatuma bigorana kugeza imiti mu bwonko1,2. Bivugwa ko hafi ya imiti yose minini n’imiti irenga 98% by’imiti mito iva mu bwonko3. Niyo mpamvu ari ingenzi cyane kumenya uburyo bushya bwo gutwara ubwonko butanga imiti ivura neza kandi yihariye kuri CNS 4,5. Ariko, BBB na BCSFB nazo zitanga amahirwe meza yo gutanga imiti kuko yinjira kandi ikinjira mu miterere yose y’ubwonko binyuze mu mitsi yabwo miremire. Bityo, imbaraga ziriho ubu zo gukoresha uburyo bwo kugeza amakuru mu bwonko zidakoreshejwe mu buryo butunguranye zishingiye ahanini ku buryo bwo gutwara amakuru hakoreshejwe uburyo bwa receptor (PMT) hakoreshejwe uburyo bwa endogenous BBB6 receptor. Nubwo hari intambwe zikomeye ziherutse guterwa hakoreshejwe uburyo bwa transferrin receptor pathway7,8, hakenewe iterambere ryinshi rya sisitemu nshya zo gutanga amakuru zifite imiterere myiza. Kugira ngo tubigereho, intego yacu yari ukumenya peptides zishobora kuyobora ubwikorezi bwa CSF, kuko muri rusange zishobora gukoreshwa mu kugeza macromolecules kuri CNS cyangwa gufungura inzira nshya za receptor. Byumwihariko, receptors na transporters za sisitemu y'imitsi yo mu bwonko (BBB na BSCFB) zishobora kuba intego zo gutanga imiti ivura indwara. Cerebrospinal fluid (CSF) ni umusaruro uva muri choroid plexus (CS) kandi ihura neza n'amazi yo mu bwonko binyuze mu mwanya wa subarachnoid n'umwanya wa ventricular4. Vuba aha byagaragaye ko amazi yo mu bwonko ya subarachnoid akwirakwira cyane mu gice cy'ubwonko9. Twifuza kugera ku mwanya wa parenchymal dukoresheje iyi nzira y’amazi ya subarachnoide cyangwa binyuze muri BBB. Kugira ngo tubigereho, twashyizeho ingamba zikomeye zo guhitamo phage muri vivo zigaragaza neza peptides zitwarwa na imwe muri izi nzira ebyiri zitandukanye.
Ubu turasobanura uburyo bwo gusuzuma mu buryo bukurikiranye bwa "in vivo phage ecran" hifashishijwe CSF sampling hamwe na "high throughput sequencing" (HTS) kugira ngo hagenzurwe ibizamini byo gutoranya bya mbere bifite ubwoko bwinshi bw'ibitabo. Isuzuma ryakozwe ku mbeba zifite ubwenge hakoreshejwe "large cisterna" (CM) cannula ihoraho kugira ngo hirindwe kwandura amaraso. Icy'ingenzi ni uko ubu buryo buhitamo "brain-target" ndetse na "peptides" zifite imikorere yo gutwara mu bwonko. Twakoresheje "T7 phages" kubera ko ari ntoya (~60 nm)10, maze tuvuga ko zikwiriye gutwara "vesicles" zemerera "transcellular cross" ku muyoboro wa "endothelial" na / cyangwa "epithelial-medulla barrier". Nyuma y'ibizamini bine byo gupima, "phages" zashyizwe ukwazo zigaragaza ko "incrementation" ikomeye ya CSF mu bwonko n'isano y'imiyoboro mito y'ubwonko. Icy'ingenzi ni uko twashoboye kwemeza ibyo twabonye twerekanye ko "peptides" nziza zatoranyijwe neza kandi zikozwe mu buryo bwa "chemical" zishobora gutwara poroteyine mu mazi y'ubwonko. Ubwa mbere, ingaruka za CNS ku bijyanye n'imiti zagaragajwe no guhuza peptide ikomeye itwara abantu n'ikintu kiyibuza gukora peptide ya BACE1. Uretse kugaragaza ko ingamba zo gusuzuma imikorere y'umubiri zishobora kugaragaza peptide nshya zitwara ubwonko nk'izitwara poroteyine neza, twiteze ko uburyo nk'ubwo bwo guhitamo imikorere buzaba ingenzi mu kumenya inzira nshya zitwara ubwonko.
Hashingiwe ku bice birema plaque (PFU), nyuma y’intambwe yo gupakira phage, hakozwe kandi hakorwa isomero rya peptides za phage za 12-mer linear T7 zifite ubwoko butandukanye bwa 109 (reba Ibikoresho n'Uburyo). Ni ngombwa kumenya ko twasesenguye neza iyi somero mbere yo gupima mu buryo bw'umubiri. Kuzamura PCR y'ibipimo bya somero za phage hakoreshejwe primers zahinduwe byatanze amplicons zari zikoreshwa neza kuri HTS (Ishusho y'inyongera ya 1a). Bitewe na a) amakosa yo gukurikirana HTS11, b) ingaruka ku bwiza bwa primers (NNK)1-12, na c) kuba hariho wild-type (wt) phages (skeleton inserts) muri isomero rya standby, hashyizweho uburyo bwo kuyungurura sequence kugira ngo hakorwe amakuru yemewe gusa y'urukurikirane (Ishusho y'inyongera ya 1b). Izi ntambwe zo kuyungurura zikoreshwa ku isomero ryose rya HTS. Ku isomero risanzwe, habonetse ibisomwa 233.868, muri byo 39% byujuje ibisabwa mu gusesengura isomero kandi byakoreshejwe mu gusesengura no gutoranya ibyiciro byakurikiyeho (Ishusho y'inyongera 1c–e). Ibisomwa byari byiganjemo ibice bitatu by'uburebure bifite uburebure bwa nucleotides 36 (Ishusho y'inyongera 1c), byemeza igishushanyo cy'isomero (NNK) 1-12. Ikigaragara ni uko hafi 11% by'abagize isomero bari bafite icyiciro cya PAGISRELVDKL cy'umugongo gifite imiterere 12 (wt), kandi hafi kimwe cya kabiri cy'ibice (49%) byari birimo ibyashyizwemo cyangwa byakuwemo. HTS y'isomero ry'isomero yemeje ko peptide nyinshi zitandukanye muri isomero: ibirenga 81% by'ibice bya peptide byabonetse rimwe gusa kandi 1.5% gusa byabonetse muri kopi ≥4 (Ishusho y'inyongera 2a). Imiterere ya aside amine (aa) mu myanya 12 yose mu gice cy’urutonde yahuye neza n’imibare yitezwe ku mubare wa codons zikorwa n’urutonde rwa NKK rwangiritse (Ishusho y’inyongera ya 2b). Imibare yagaragaye y’ibisigazwa bya aa byashyizwe muri izi ngingo yahuye neza n’umubare wabazwe (r = 0.893) (Ishusho y’inyongera ya 2c). Gutegura ibitabo bya phage kugira ngo bitererwemo harimo intambwe zo kongera no gukuraho endotoxin. Ibi byagaragaye mbere ko bishobora kugabanya ubudasa bw’ibitabo bya phage12,13. Kubwibyo, twakurikiranye igitabo cya phage cyakuweho endotoxin cya plate-amplified cyari cyarakuweho endotoxin maze tugereranya n’igitabo cy’umwimerere kugira ngo tumenye inshuro za AA. Isano ikomeye (r = 0.995) yagaragaye hagati y’ikidendezi cy’umwimerere n’ikidendezi cyakuweho kandi gisukuye (Ishusho y’inyongera ya 2d), bigaragaza ko ipiganwa hagati y’ibice byakuweho ku masahani hakoreshejwe phage ya T7 ritateye ivangura rikomeye. Iri gereranya rishingiye ku nshuro ya tripeptide muri buri isomero, kubera ko ubwinshi bw'amasomero (~109) budashobora gufatwa neza nubwo haba hari HTS. Isesengura ry'inshuro aa kuri buri mwanya ryagaragaje ko hari aho bitandukaniye n'aho biherereye mu myanya itatu ya nyuma y'urutonde rwinjiye (Ishusho y'inyongera ya 2e). Mu gusoza, twasanze ubwiza n'ubwinshi bw'isomero byari byemewe kandi ko hari impinduka nto mu budasa bwagaragaye bitewe no kongera no gutegura amasomero ya phage hagati y'ingendo nyinshi zo gutoranya.
Gufata ingero z'amazi yo mu bwonko mu buryo bw'uruhererekane bishobora gukorwa hakoreshejwe uburyo bwo kubaga bushyira cannula muri CM y'imbeba zifite ubwenge kugira ngo byorohere kumenya phage ya T7 yatewe mu mitsi (iv) binyuze muri BBB na/cyangwa BCSFB (Ishusho ya 1a-b). Twakoresheje amaboko abiri yigenga yo gutoranya (amaboko A na B) mu byiciro bitatu bya mbere byo gutoranya mu mubiri (Ishusho ya 1c). Twongereye buhoro buhoro imbaraga zo gutoranya tugabanya umubare wose wa phage yashyizwe mu byiciro bitatu bya mbere byo gutoranya. Ku cyiciro cya kane cyo gutoranya, twahuje ingero zo mu mashami A na B maze dukora andi mahitamo atatu yigenga. Kugira ngo twige imiterere y'uduce twa phage ya T7 muri ubu buryo, phage yo mu bwoko bwa wild-type (PAGISRELVDKL master insert) yatewe mu mbeba binyuze mu mutsi w'umurizo. Gusubirana phage mu mazi yo mu bwonko no mu maraso mu bihe bitandukanye byagaragaje ko phage nto za T7 za icosahedral zagize intera yihuse yo kuva mu gice cy'amaraso (Ishusho y'inyongera ya 3). Dushingiye ku mibare yatanzwe n'ingano y'amaraso y'imbeba, twabaze ko hafi 1% by'uburemere bw'umusemburo watanzwe ari byo byagaragaye mu maraso iminota 10 nyuma yo guterwa mu mitsi. Nyuma y'uku kugabanuka kw'umusemburo kwa mbere, hapimwe buhoro buhoro igice cy'ubuzima cy'iminota 27.7. Icy'ingenzi ni uko habonetse uduce duto cyane tw'umusemburo mu gice cya CSF, bigaragaza ko nta nkomoko y'umusemburo wo mu bwoko bwa wild-type winjira mu gice cya CSF (Ishusho y'inyongera ya 3). Ugereranyije, hafi 1 x 10-3% by'uduce twa T7 mu maraso na 4 x 10-8% by'uduce twatangiye gushyirwamo ni byo byagaragaye mu mazi yo mu bwonko mu gihe cyose cyo gupimwa (iminota 0-250). Ikigaragara ni uko igice cy'ubuzima (iminota 25.7) cy'uduce twa wild-type mu mazi yo mu bwonko cyari gisa n'icyagaragaye mu maraso. Aya makuru agaragaza ko uruzitiro rutandukanya igice cya CSF n'amaraso ruri mu mbeba za CM-cannulated, bigatuma hatoranywa ibitabo bya phage mu mubiri kugira ngo hamenyekane ubwoko bw'uturemangingo twimukanwa mu maraso tujya mu gice cya CSF.
(a) Gushyiraho uburyo bwo kongera gupima amazi yo mu bwonko (CSF) mu kidendezi kinini. (b) Ishusho igaragaza aho urusobe rw'imitsi yo mu bwonko (CNS) ruherereye n'ingamba zo guhitamo zakoreshejwe mu kumenya peptides zambuka urusobe rw'amaraso n'ubwonko (BBB) ​​​​n'urusobe rw'amaraso n'ubwonko. (c) Imbonerahamwe yo gusuzuma imiterere y'urusobe rw'amaraso mu mubiri. Muri buri cyiciro cyo gutoranya, phages (ibiranga inyamaswa biri mu myambi) byaterwaga mu mitsi. Amashami abiri yigenga (A, B) abikwa ukwayo kugeza ku cyiciro cya kane cyo gutoranya. Ku cyiciro cya 3 n'icya 4 cyo gutoranya, buri tsinda rya phages ryakuwe muri CSF ryakurikiraniwe n'intoki. (d) Isuzuma rya phages ryakuwe mu maraso (uruziga rutukura) n'amazi yo mu bwonko (impandeshatu z'icyatsi) mu cyiciro cya mbere cyo gutoranya mu mbeba ebyiri zashyizwe mu kano nyuma yo gutera isomero rya peptide ya T7 (2 x 1012 phages/inyamaswa). Udupira tw'ubururu tugaragaza impuzandengo y'ingano y'umusemburo mu maraso, tubarwa uhereye ku gipimo cya phage yatewe, hashingiwe ku bunini bw'amaraso yose. Udupira tw'umukara tugaragaza aho umurongo wa y uhurira uturutse ku gipimo cya phage mu maraso. (e, f) Garagaza inshuro n'uburyo intera y'udupira tw'inyuma twose dushoboka duhurirana twabonetse muri peptide. Umubare w'udupira twabonetse mu bipimo 1000 urerekanwa. Inyuguti z'ingenzi (p < 0.001) zirimo utudomo dutukura. (e) Ishusho y'isano igereranya inshuro igereranya intera y'ishusho ya tripeptide y'isomero ryatewe n'amaraso ya phage ikomoka ku nyamaswa #1.1 na #1.2. (f) Ishusho y'isano igereranya inshuro igereranya imbonerahamwe ya tripeptide y'inyamaswa #1.1 na #1.2 yakuwe mu maraso no mu bwonko. (g, h) Ishusho y'uruhererekane rw'ishusho ya phage ikungahaye mu maraso (g) ugereranije n'isomero ryatewe n'umusemburo na phage ikungahaye kuri CSF (h) ugereranije n'amaraso nyuma yo gutoranya mu mubiri mu nyamaswa zombi. Ingano y'inyuguti imwe yerekana inshuro iyo aside amine iboneka muri iyo myanya. Icyatsi kibisi = polar, umuhengeri = neutral, ubururu = basic, umutuku = aside na black = hydrophobic aside amine. Ishusho ya 1a, b yakozwe kandi ikorwa na Eduard Urich.
Twateye isomero rya phage peptide mu mbeba ebyiri za CM (clades A na B) hamwe na phage yakuwe mu mazi yo mu bwonko n'amaraso (Ishusho ya 1d). Gusohoka vuba kw'isomero byabanje byari bike ugereranije na phage yo mu bwoko bwa wild-type. Impuzandengo y'ubuzima bw'isomero ryatewe mu nyamaswa zombi yari iminota 24.8 mu maraso, kimwe na phage yo mu bwoko bwa wild-type, n'iminota 38.5 muri CSF. Ingero z'amaraso na phage yo mu bwonko yavuye kuri buri nyamaswa zakorewe HTS kandi peptide zose zagaragaye zarasesenguwe kugira ngo harebwe niba hari ishusho ngufi ya tripeptide. Inyuguti za tripeptide zatoranyijwe kuko zitanga ishingiro rito ryo gukora imiterere n'imikoranire ya peptide na poroteyine14,15. Twabonye isano nziza mu ikwirakwizwa ry'inyuguti hagati y'isomero rya phage ryatewe n'uturemangingo twakuwe mu maraso y'inyamaswa zombi (Ishusho ya 1e). Amakuru agaragaza ko imiterere y'isomero ikungahaye gato mu gice cy'amaraso. Imiterere ya aside amino n'uruhererekane rw'ibitekerezo byasesenguwe kuri buri mwanya hakoreshejwe porogaramu ya Weblogo16. Igishimishije ni uko twabonye ubwiyongere bukomeye mu bisigazwa bya glycine mu maraso (Ishusho ya 1g). Ubwo amaraso yagereranywaga n'ama-clone yatoranijwe muri CSF, hagaragaye guhitamo gukomeye no gukuraho inyuguti zimwe na zimwe (Ishusho ya 1f), kandi aside amine zimwe na zimwe zari zihari cyane mu myanya yagenwe mu bice 12 (Ishusho ya 1h). Igitangaje ni uko inyamaswa zitandukanye cyane mu mazi yo mu bwonko, mu gihe ubwiyongere bwa glycine mu maraso bwagaragaye mu nyamaswa zombi (Ishusho y'inyongera ya 4a-j). Nyuma yo gusesengura neza amakuru y'uruhererekane mu mazi yo mu bwonko y'inyamaswa #1.1 na #1.2, habonetse peptide 964 na 420 zidasanzwe za 12-mer (Ishusho y'inyongera ya 1d-e). Ama-clone ya phage yihariye yongerewe kandi akorerwa icyiciro cya kabiri cyo guhitamo mu mubiri. Phage yakuwe mu cyiciro cya kabiri cy’itoranywa yashyizwemo HTS muri buri nyamaswa kandi peptide zose zagaragaye zakoreshejwe nk'inyongera muri gahunda yo kumenya motif kugira ngo hasesengurwe igaragara rya motif za tripeptide (Ishusho ya 2a, b, ef). Ugereranije n'izunguruka rya mbere rya phage yakuwe muri CSF, twabonye andi mahitamo n'itandukaniro ry'inyuguti nyinshi muri CSF mu mashami A na B (Ishusho ya 2). Hakoreshejwe uburyo bwo kumenya umuyoboro kugira ngo hamenyekane niba zihagarariye imiterere itandukanye y'urukurikirane ruhoraho. Hagaragaye isano igaragara hagati y'urukurikirane rw'ibice 12 rwakuwemo na CSF mu bundi buryo bwa clade A (Ishusho ya 2c, d) na clade B (Ishusho ya 2g, h). Isesengura ryashyizwe hamwe muri buri shami ryagaragaje imiterere itandukanye y'amahitamo ya peptide 12-mer (Ishusho y'inyongera ya 5c, d) n'ubwiyongere bw'igipimo cya CSF/amaraso mu gihe runaka ku ma clones yashyizwe hamwe nyuma y'izunguruka rya kabiri ry'itoranywa ugereranije n'izunguruka rya mbere ry'itoranywa (Ishusho y'inyongera ya 5e).).
Kongera isura ya peptides na peptides mu mazi yo mu bwonko binyuze mu byiciro bibiri bikurikirana by’itoranywa ry’ishusho ya phage ikora mu mubiri.
Inyamaswa zose zo mu bwonko zavuye mu cyiciro cya mbere cya buri nyamaswa (inyamaswa #1.1 na #1.2) zashyizwe hamwe, zongerwamo imbaraga, zishyirwa mu byiciro bya HT hanyuma zigashyirwa hamwe (2 x 1010 phages/inyamaswa) Imbeba 2 za SM zashyizwe mu kano (#1.1 → #). 2.1 na 2.2, 1.2 → 2.3 na 2.4). (a,b,e,f) Imiterere y’isano igereranya inshuro igereranya igereranya ry’inyuguti za tripeptide z’inyuguti zose zikomoka kuri CSF mu cyiciro cya mbere n’icya kabiri cyo guhitamo. Inshuro igereranya n’ikwirakwizwa ry’inyuguti zigaragaza inyuguti zose zishobora guhurirana ziboneka muri peptides mu cyerekezo cyombi. Umubare w’inyuguti zabonetse mu bipimo 1000 uragaragazwa. Inyuguti zatoranijwe cyane (p < 0.001) cyangwa zitashyizwe muri imwe mu isomero ryagereranyijwe zigaragazwa n’utudomo dutukura. (c, d, g, h) Ikimenyetso cy'urutonde rw'urutonde rw'urutonde rw'urutonde rw'aside amine 12 zikungahaye kuri CSF hashingiwe ku mpande za 2 na 1 z'ihitamo ryakozwe mu mubiri. Ingano y'inyuguti imwe igaragaza inshuro iyo aside amine iboneka kuri uwo mwanya. Kugira ngo hagaragazwe ikimenyetso, hagereranywa inshuro urutonde rwa CSF rukurwa mu nyamaswa imwe hagati y'urutonde rw ...
Nyuma y'icyiciro cya gatatu cyo gutoranya, twabonye urukurikirane rw'amapeptidi 124 yihariye (#3.1 na #3.2) muri 332 CSF-reconstituted phage clones zakuwe mu nyamaswa ebyiri (Ishusho y'inyongera ya 6a). Urukurikirane rwa LGSVS (18.7%) rwari rufite igipimo cyo hejuru cyane, hagakurikiraho inyito za wild-type PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), na SARGSWREIVSLS (2.2%). Mu cyiciro cya kane cya nyuma, twakusanyije amashami abiri yatoranijwe ku giti cyayo mu nyamaswa eshatu zitandukanye (Ishusho ya 1c). Mu 925 clones za phage zakuwe muri CSF, mu cyiciro cya kane twabonye 64 peptide sequences zidasanzwe (Ishusho y'inyongera ya 6b), muri zo igipimo cya wild-type phage cyagabanutse kigera kuri 0.8%. Ingero za CSF zikunze kugaragara mu cyiciro cya kane zari LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) na RLSSVDSDLSGC (3, 2%). %). Uburebure bwa peptide zatoranijwe buterwa no gushyiramo/gusiba nucleotide cyangwa codons zihagarara mbere y'igihe mu bibanza bya library iyo hakoreshejwe codons zangiritse mu gishushanyo cya library ya NNK. Codons zihagarara mbere y'igihe zikora peptides ngufi kandi zitoranywa kuko zirimo aa motif nziza. Peptides ndende zishobora guterwa no gushyiramo/gusiba mu bibanza bya library by'ibihangano. Ibi bishyira codon yashyizweho hanze y'ishusho kandi bikayisoma kugeza igihe codon nshya ihagaze igaragara inyuma. Muri rusange, twabaze ibintu by'inyongera ku byiciro bine byose byo guhitamo tugereranya amakuru yinjiye n'amakuru y'ingero. Ku cyiciro cya mbere cyo gusuzuma, twakoresheje uduce twa phage twitwa wild-type nk'aho ari ikigereranyo kitari cyo cy’inyuma. Igishimishije ni uko guhitamo phage mbi byari bikomeye cyane mu cyiciro cya mbere cya CSF, ariko ntabwo byari mu maraso (Ishusho ya 3a), bishobora guterwa n’amahirwe make yo gukwirakwira kw’abagize isomero rya peptide mu gice cya CSF cyangwa phage zifitanye isano zikunze kubikwa neza cyangwa gukurwa mu maraso kurusha bacteriophages. Ariko, mu cyiciro cya kabiri cyo gupima, guhitamo gukomeye kwa phage muri CSF byagaragaye mu bice byombi, bigaragaza ko igice cyabanje cyari gikungahaye kuri phage zigaragaza peptides zitera CSF kwinjira (Ishusho ya 3a). Nanone, nta kongera amaraso cyane. Nanone mu cyiciro cya gatatu n’icya kane, phage clones zari zikungahaye cyane muri CSF. Tugereranije inshuro zihariye za buri cyiciro cyihariye cya peptide hagati y’ibihe bibiri bya nyuma byo guhitamo, twasanze ibihe byarushijeho kwiyongera mu cyiciro cya kane cyo guhitamo (Ishusho ya 3b). Imiterere ya tripeptide 931 yose yakuwe muri peptide zose uko ari 64 zihariye hakoreshejwe imiterere ya peptide zombi. Imiterere yagutse cyane mu cyiciro cya kane yasuzumwe neza kugira ngo harebwe imiterere yayo mu byiciro byose ugereranije n'isomero ryashyizwemo (incamake: 10% by'ubuhanga) (Ishusho y'inyongera ya 6c). Imiterere rusange y'uburyo bwo guhitamo yagaragaje ko impamvu nyinshi zasuzumwe zakungahaye mu byiciro byose byabanje by'amashami yombi yo guhitamo. Ariko, zimwe mu ngingo (urugero: SGL, VSG, LGS GSV) zari zishingiye cyane ku cyiciro cya A, mu gihe izindi (urugero: FGW, RTN, WGF, NTR) zari zikungahaye ku cyiciro cya B.
Kwemeza uburyo CSF ​​itwara peptides zigaragaza phage zikungahaye kuri CSF hamwe na peptides zigize biotinylated leader zihujwe na streptavidin payloads.
(a) Ibipimo by’ubwiyongere bw’amaraso byabazwe mu byiciro bine byose (R1-R4) hashingiwe ku bipimo byatewe (input = I) phage (PFU) n’ibipimo byagenwe bya phage ya CSF (output = O). Ibipimo by’ubwiyongere bw’amaraso mu byiciro bitatu bya nyuma (R2-R4) byabazwe hagereranyijwe n’icyiciro cyabanje n’icyiciro cya mbere (R1) hamwe n’ibipimo by’uburemere. Uduce dufunguye ni amazi yo mu bwonko, utundi duce dufite ibara ni plasma. (***p<0.001, hashingiwe ku ikizamini cya t cy’umunyeshuri). (b) Urutonde rwa peptide za phage nyinshi cyane, zitondekwa hakurikijwe ingano yazo n’uduce twose twakusanyijwe muri CSF nyuma y’icyiciro cya 4 cy’itoranywa. Uduce dutandatu twa phage dukunze kugaragara cyane tugaragara mu ibara, imibare n’ibipimo by’ubwiyongere bwazo hagati y’icyiciro cya 3 n’icya 4 cy’itoranywa (insets). (c,d) Uduce dutandatu twa phage twungutse cyane, uduce duto twa phage na peptide y’ababyeyi ya phage two mu cyiciro cya 4 twasesenguwe ukwatwo mu buryo bwo gupima CSF. Uduce twa CSF n’ibipimo by’amaraso byakusanyijwe mu bihe byagenwe. (c) Ingano zingana ya 6 candidate phage clones (2 x 1010 phages/inyamaswa), empty phages (#1779) (2 x 1010 phages/inyamaswa) na stock phage peptide libraries (2 x 1012 phages/inyamaswa) Shyira nibura 3 CM ku nyamaswa yashyizwe mu kanombe ukwayo binyuze mu mutsi w'umurizo. Ibyerekeye pharmacokinetics ya CSF ya buri kanombe ya phage na phage peptide library uko igihe kigenda gihita biragaragazwa. (d) igaragaza impuzandengo ya CSF/amaraso kuri phage zose zasubiwemo/mL mu gihe cyo gufata ingero. (e) Peptides enye za synthetic leader na imwe yakoreshejwe byahujwe na biotin na streptavidin binyuze muri N-terminus yazo (tetramer display) hagakurikiraho inshinge (umutsi w'umurizo iv, 10 mg streptavidin/kg). Nibura imbeba eshatu zashyizwe mu cyuma (N = 3).). Ingero za CSF zakusanyijwe mu gihe cyagenwe, kandi igipimo cya streptavidin cyapimwe hakoreshejwe CSF anti-streptavidin ELISA (nd = ntabwo byagaragaye). (*p<0.05, **p<0.01, ***p<0.001, hashingiwe ku isuzuma rya ANOVA). (f) Kugereranya urukurikirane rw'aside amine ya clone ya phage peptide ikungahaye cyane #2002 (umuhengeri) n'izindi clone za phage peptide zatoranijwe zo mu cyiciro cya kane cy'itoranywa. Uduce twa aside amine zisa n'izisa nazo twashyizwe mu mabara atandukanye.
Mu matsinda yose ya phage yuzuye mu cyiciro cya kane (Ishusho ya 3b), hatoranijwe amoko atandatu ya clone kugira ngo akorerwe isesengura ryimbitse mu buryo bwa CSF. Ingano zingana za phage esheshatu, phage y'ubusa (nta insert) na peptide ya prophage zatewe mu nyamaswa eshatu za CM zashyizwe mu kato, kandi pharmacokinetics zagenwe muri CSF (Ishusho ya 3c) no mu maraso (Ishusho y'inyongera ya 7). Amatsinda yose ya phage yageragejwe yerekeje ku gice cya CSF ku rwego ruri hejuru y'inshuro 10-1000 ugereranyije n'ay'agace ka empty control phage (#1779). Urugero, amoko ya clone #2020 na #2077 yari afite titers za CSF zikubye inshuro 1000 ugereranyije n'agace ka empty control phage. Imiterere ya pharmacokinetic ya buri peptide yatoranijwe iratandukanye, ariko yose afite ubushobozi bwo kongera kwinjira muri CSF. Twabonye igabanuka rihoraho uko igihe cyagiye gihita kuri clones #1903 na #2011, mu gihe kuri clones #2077, #2002 na #2009 kwiyongera mu minota 10 ya mbere bishobora kugaragaza ko hari ubwikorezi bukora ariko bigomba kwemezwa. Clones #2020, #2002, na #2077 zahagaze ku rugero rwo hejuru, mu gihe ingano ya CSF ya clone #2009 yagabanutse buhoro buhoro nyuma y’ubwiyongere bwa mbere. Hanyuma twagereranije inshuro za buri mvange ya CSF n’ingano yayo mu maraso (Ishusho ya 3d). Isano y’ikigereranyo cya buri mvange ya CSF n’ingano yayo mu maraso mu bihe byose byo gupima yagaragaje ko bitatu muri bitandatu byakungahaye cyane kuri CSF mu maraso. Igishimishije ni uko mvange #2077 yagaragaje ko amaraso ahamye cyane (Ishusho y’inyongera ya 7). Kugira ngo twemeze ko peptides ubwazo zishobora gutwara imizigo itari uduce twa phage mu gice cya CSF, twakoze peptides enye z’ingenzi zikomoka kuri biotin kuri N-terminus aho peptides zifatanye n’agace ka phage. Peptide za Biotinylated (nos. 2002, 2009, 2020 na 2077) zahujwe na streptavidin (SA) kugira ngo haboneke imiterere myinshi isa n’imiterere ya phage. Ubu buryo bwanatumye dushobora gupima uburyo SA yagaragaye mu maraso no mu mazi yo mu bwonko nk'amapeptide ya poroteyine yinjizwamo imizigo. Icy'ingenzi ni uko amakuru ya phage yashoboraga gukosorwa kenshi igihe peptide za sintetike zatanzwe muri ubu buryo bwa SA-conjugated (Ishusho ya 3e). Peptide zasuguwe zagize ibyago bike byo kwangirika mbere kandi zigahita zishyirwa mu buryo bwa CSF hamwe n'urugero rutagaragara mu masaha 48. Kugira ngo tumenye neza inzira izi peptide phage clones zijya mu mwanya wa CSF, twasesenguye aho peptide za phage zihurira hakoreshejwe immunohistochemistry (IHC) kugira ngo tumenye neza uduce twa phage isaha 1 nyuma yo guterwa mu mitsi. Ikigaragara ni uko clones #2002, #2077, na #2009 zashoboraga kugaragara hakoreshejwe ibara rikomeye mu mitsi yo mu bwonko, mu gihe control phage (#1779) na clone #2020 bitabonetse (Ishusho y'inyongera ya 8). Ibi bigaragaza ko izi peptide zigira uruhare mu ngaruka ku bwonko binyuze mu kwambuka BBB. Hakenewe isesengura ryimbitse kugira ngo hagenzurwe iki gitekerezo, kuko inzira ya BSCFB ishobora no kuba irimo. Mu kugereranya urukurikirane rwa aside amine ya clone ikungahaye cyane (#2002) n'izindi peptide zatoranijwe, byagaragaye ko zimwe muri zo zifite aside amine zisa, zishobora kugaragaza uburyo bumwe bwo gutwara (Ishusho ya 3f).
Bitewe n’imiterere yayo yihariye ya plasma n’ubwiyongere bugaragara bwa CSF uko igihe cyagiye gihita, phage display clone #2077 yakomeje gukorerwa ubushakashatsi mu gihe cy’amasaha 48 kandi yashoboye kongera kwiyongera kwa CSF kwagaragaye hamwe n’urugero rwa SA ruhoraho (Ishusho ya 4a). Ku bijyanye n’izindi phage clones zagaragaye, #2077 yahinduye cyane imitsi yo mu bwonko kandi yagaragaje kolocalization ikomeye hamwe na lectine y’imitsi iyo irebewe ku rwego rwo hejuru ndetse wenda n’aho ihinduka mu mwanya wa parenchymal (Ishusho ya 4b). Kugira ngo dusuzume niba ingaruka za peptide zishingiye ku buvuzi zishobora kuboneka muri CNS, twakoze igerageza aho verisiyo za biotinylated za i) peptide ya #2077 transit na ii) peptide ya BACE1 inhibitor zavanzwe na SA ku bipimo bibiri bitandukanye. Ku ruvange rumwe twakoresheje BACE1 peptide inhibitor gusa naho ku rundi twakoresheje 1:3 ratio ya BACE1 peptide inhibitor na peptide ya #2077. Ingero zombi zatanzwe mu mitsi, hanyuma urugero rw'amaraso n'amazi yo mu bwonko ya beta-amyloid peptide 40 (Abeta40) bipimwa uko igihe cyagiye gihita. Abeta40 yapimwe muri CSF kuko igaragaza uburyo BACE1 ibuza mu bwonko. Nkuko byari byitezwe, izo complexes zombi zagabanyije cyane urugero rwa Abeta40 mu maraso (Ishusho ya 4c, d). Ariko, ingero gusa zirimo uruvange rwa peptide no. 2077 n'umuti ubuza peptide ya BACE1 ihujwe na SA byateje igabanuka rikomeye rya Abeta40 mu mazi yo mu bwonko (Ishusho ya 4c). Amakuru agaragaza ko peptide no. 2077 ishobora gutwara poroteyine ya 60 kDa SA muri CNS kandi ikanatera ingaruka mbi ku miti hamwe n'imiti ibuza peptide ya BACE1 ihujwe na SA.
(a) Gutera clonal (2 × 10 phages/inyamaswa) bya T7 phage bigaragaza imiterere y’igihe kirekire ya pharmacokinetic ya CSF peptide #2077 (RLSSVDSDLSGC) na uninjected control phage (#1779) mu mbeba eshatu za CM-intubated. (b) Ishusho ya microscopic ya confocal y’imitsi mito y’umubiri ihagarariye cortical mu mbeba zatewe plage (2 × 10 10 phages/inyamaswa) igaragaza counterdating ya peptide #2077 n’imitsi (lectin). Izi clones za phage zahawe imbeba 3 maze zirekurwa zigatembera isaha 1 mbere yo guterwa. Ubwonko bwagabanyijwemo ibice maze bushyirwamo irangi rya polyclonal FITC-label antibodies kuri T7 phage capsid. Iminota icumi mbere yo guterwa no guterwa nyuma, DyLight594-label lectin yatanzwe mu mitsi. Amashusho y'urumuri agaragaza ibara rya lectin (umutuku) ku ruhande rw'urumuri rw'imitsi mito na phages (icyatsi kibisi) mu rumuri rw'imitsi mito n'imitsi yo mu bwonko. Umwanya w'igipimo uhuye na µm 10. (c, d) Peptide ibuza BACE1 ya Biotinylated yonyine cyangwa ivanze na peptide ya biotinylated transit #2077 yahujwe na streptavidin hagakurikiraho gutera nibura imbeba eshatu za CM za cannulated (10 mg streptavidin / kg). Kugabanuka kwa peptide ya BACE1 ibuza Aβ40 kwapimwe na Aβ1-40 ELISA mu maraso (umutuku) no mu mazi yo mu bwonko (icunga) mu bihe byagenwe. Kugira ngo birusheho kuba byiza, umurongo ushushanyijeho ushushanyije ku gipimo cya 100%. (c) Kugabanuka kw'ijanisha rya Aβ40 mu maraso (inyabutatu zitukura) n'amazi yo mu bwonko (inyabutatu z'icunga) mu mbeba zavuwe na streptavidin ihujwe na peptide ya transit #2077 na peptide ibuza BACE1 ku gipimo cya 3:1. (d) Kugabanuka kw'ijanisha mu maraso ya Aβ40 (inyabutatu zitukura) n'amazi yo mu bwonko (inyabutatu z'icunga) by'imbeba zavuwe na streptavidin zihujwe na peptide ibuza BACE1 gusa. Igipimo cya Aβ mu igenzura cyari 420 pg/ml (itandukaniro risanzwe = 101 pg/ml).
Ishusho ya phage yakoreshejwe neza mu nzego zitandukanye z'ubushakashatsi ku buvuzi17. Ubu buryo bwakoreshejwe mu bushakashatsi ku mitsi y'amaraso mu mubiri18,19 ndetse no mu bushakashatsi ku mitsi y'ubwonko20,21,22,23,24,25,26. Muri ubu bushakashatsi, twongereye ikoreshwa ry'ubu buryo bwo guhitamo atari mu kumenya gusa peptides zireba imitsi y'ubwonko, ahubwo no mu kuvumbura abagomba kuba bafite imiterere ikora yo gutwara abantu kugira ngo bambuke umugozi w'amaraso n'ubwonko. Ubu turasobanura iterambere ry'uburyo bwo gutoranya CM mu mbeba zifashishijwe CM kandi tugaragaza ubushobozi bwazo bwo kumenya peptides zifite imiterere ya CSF homing. Dukoresheje phage ya T7 igaragaza isomero rya peptides 12-mer random, twashoboye kwerekana ko phage ya T7 ari nto bihagije (hafi 60 nm mu murambararo)10 kugira ngo ihuzwe n'umugozi w'amaraso n'ubwonko, bityo ikanyura mu mugozi w'amaraso n'ubwonko cyangwa choroid plexus. Twabonye ko gusarura CSF mu mbeba za CM zashyizwe mu kato ari uburyo bwo gusuzuma imikorere y’umubiri bugenzurwa neza, kandi ko phage yakuwemo idafata gusa ku mitsi y’amaraso ahubwo yanakoraga nk'umuyoboro unyura mu ruzitiro rw’amaraso n’ubwonko. Byongeye kandi, mu gukusanya amaraso icyarimwe no gushyira HTS kuri CSF n’udukoko dukomoka mu maraso, twemeje ko guhitamo CSF ​​kwacu kutagizweho ingaruka no kongera amaraso cyangwa ubushobozi bwo kwaguka hagati y’ingendo zo guhitamo. Ariko, igice cy’amaraso ni kimwe mu bigize uburyo bwo guhitamo, kubera ko udukoko dushobora kugera mu gice cya CSF tugomba kubaho no gutembera mu maraso igihe gihagije kugira ngo twikungahaze mu bwonko. Kugira ngo dukure amakuru yizewe y’uruhererekane mu makuru ya HTS, twashyizeho filters zahinduwe ku makosa yihariye yo gukurikirana mu buryo bw’isesengura. Dukoresheje ibipimo bya kinetic mu buryo bwo gusuzuma, twemeje pharmacokinetics yihuta ya phage za T7 zo mu bwoko bwa wild-type (t½ ~ 28 min) mu maraso24, 27, 28 ndetse tunamenya igice cyazo cy’ubuzima mu mazi yo mu bwonko (t½ ~ 26 min) ku munota). Nubwo hari imiterere isa ya farumasi mu maraso na CSF, 0.001% by'umubare w'amaraso ya phage ni byo byashoboraga kugaragara muri CSF, bigaragaza ko phage yo mu bwoko bwa T7 yo mu bwoko bwa wild-type igenda gahoro mu gice cy'amaraso n'ubwonko. Uyu murimo ugaragaza akamaro k'icyiciro cya mbere cyo guhitamo iyo hakoreshejwe ingamba zo gupima mu mubiri, cyane cyane ku buryo bwa phage zisohoka vuba mu nzira z'amaraso, kuko clones nke zishobora kugera mu gice cya CNS. Bityo, mu cyiciro cya mbere, kugabanuka k'ubusumbane mu bubiko byari byinshi cyane, kuko umubare muto w'clones ni wo waje gukusanywa muri ubu buryo bukomeye cyane bwa CSF. Iyi ngamba yo gupima mu mubiri yari ikubiyemo intambwe nyinshi zo guhitamo nko gukusanya mu gice cya CSF, kubaho kwa clone mu gice cy'amaraso, no gukuraho vuba kwa clones za phage za T7 mu maraso mu minota 10 ya mbere (Ishusho ya 1d n'Ishusho y'inyongera ya 4M). Bityo, nyuma y'icyiciro cya mbere, clones zitandukanye za phage zabonetse muri CSF, nubwo ikigega kimwe cya mbere cyakoreshejwe ku nyamaswa imwe. Ibi bigaragaza ko intambwe nyinshi zikomeye zo guhitamo amasomero y’isoko afite umubare munini w’abanyamuryango b’isomero zigabanya cyane ubudasa. Kubwibyo, ibintu bitunguranye bizaba igice cyingenzi cy’uburyo bwo guhitamo bwa mbere, bigira ingaruka zikomeye ku musaruro. Birashoboka ko clones nyinshi mu isomero rya mbere zari zifite ubushake bwo kongera CSF. Ariko, nubwo haba hari imiterere imwe y’igerageza, ibisubizo byo guhitamo bishobora gutandukana bitewe n’umubare muto wa buri clone runaka mu itsinda rya mbere.
Imiterere y’uturemangingo twinshi muri CSF itandukanye n’iyo mu maraso. Igishimishije ni uko twabonye impinduka ya mbere igana kuri peptides zikungahaye kuri glycine mu maraso y’inyamaswa ku giti cyazo. (Ishusho ya 1g, Ishusho y’inyongera. 4e, 4f). Peptides za glycine zirimo Phage zishobora kuba zihamye kandi zidakunze gukurwa mu miyoboro y’amaraso. Ariko, izi peptides zikungahaye kuri glycine ntizabonetse mu bipimo by’amazi yo mu bwonko, bigaragaza ko amasomero yatoranyijwe yanyuze mu ntambwe ebyiri zitandukanye zo guhitamo: imwe mu maraso indi yemerewe kwirundanya mu mazi yo mu bwonko. Clone zikungahaye kuri CSF zavuye mu cyiciro cya kane cy’itoranywa zageragejwe cyane. Clone hafi ya zose zapimwe ku giti cyazo zemejwe ko zikungahaye kuri CSF ugereranije na blank control phage. Peptide imwe yakubiswe (#2077) yasuzumwe birambuye. Yagaragaje ko igice cy’ubuzima kirekire cya plasma ugereranije n’izindi hits (Ishusho ya 3d n’Ishusho y’inyongera ya 7), kandi igishimishije ni uko iyi peptide yari irimo ibisigazwa bya cysteine ​​​​kuri C-terminus. Biherutse kugaragara ko kongeramo cysteine ​​​​muri peptides bishobora kunoza imiterere yabyo ya pharmacokinetic binyuze mu gufatanya na albumin 29. Ibi ntibizwi kuri peptide #2077 kandi bisaba ubushakashatsi bwimbitse. Peptides zimwe zagaragaje ko zishingiye kuri valence mu kongera CSF (amakuru ntabwo yagaragajwe), bishobora kuba bifitanye isano n'imiterere y'ubuso bwa T7 capsid. Sisitemu ya T7 twakoresheje yagaragaje kopi 5-15 za buri peptide kuri buri gace ka phage. IHC yakozwe ku ma clones ya lead phage yashyizwe mu mitsi mu bwonko bw'imbeba (Ishusho y'inyongera ya 8). Amakuru yagaragaje ko nibura clones eshatu (No. 2002, No. 2009 na No. 2077) zakoranaga na BBB. Haracyari ngombwa kumenya niba iyi mikoranire ya BBB ituma CSF yiyongera cyangwa ko izi clones zijya muri BCSFB. Icy'ingenzi ni uko twerekanye ko peptides zatoranijwe zigumana ubushobozi bwazo bwo gutwara CSF iyo zikozwe kandi zifatanye na poroteyine. Guhuza peptide za N-terminal zikomoka kuri biotinyle kuri SA bisubiramo ibisubizo byabonetse hamwe na clones zazo za phage mu maraso no mu mazi yo mu bwonko (Ishusho ya 3e). Hanyuma, twerekanye ko peptide y'icyitegererezo #2077 ishobora guteza imbere imikorere y'ubwonko bw'ingufu ya peptide ya BACE1 ihujwe na SA, bigatera ingaruka zikomeye za pharmacodynamic muri CNS mu kugabanya cyane urugero rwa Abeta40 muri CSF (Ishusho ya 4). Ntitwashoboye kumenya homologues iyo ari yo yose muri database dukora ubushakashatsi ku buryo bwa peptide zikomoka kuri homologue ku buryo bwose bwa homologue. Ni ngombwa kumenya ko ingano y'isomero rya T7 ari hafi 109, mu gihe ingano y'isomero rya teoriyori rya 12-mers ari 4 x 1015. Kubwibyo, twahisemo igice gito cy'ahantu hatandukanye h'isomero rya peptide ya 12-mer, bishobora gusobanura ko peptides nziza zishobora kumenyekana hakoreshejwe gusuzuma umwanya w'aho homologue iri hafi y'aho homologues yabonetse. Mu buryo bw'ibitekerezo, imwe mu mpamvu tutabonye ubwoko bw'ibi binyabuzima bisanzwe bishobora kuba ari uguhitamo mu gihe cy'ubwihindurize kugira ngo hirindwe ko ibyiciro bimwe na bimwe bya peptide byinjira mu bwonko mu buryo butagenzurwa.
Ibyo twagezeho byose hamwe bitanga ishingiro ry'ibikorwa byo mu gihe kizaza byo kumenya no kugena uburyo bwo gutwara imiyoboro y'ubwonko mu mubiri mu buryo burambuye. Uburyo bw'ibanze bw'ubu buryo bushingiye ku ngamba zo guhitamo imikorere idasobanura gusa clones zifite ubushobozi bwo gufatanya imiyoboro y'ubwonko, ahubwo inakubiyemo intambwe ikomeye aho clones nziza zigira imikorere yo kwambuka inzitizi za biyoloji mu mubiri mu mubiri zijya mu gice cya CNS. Ni ukugaragaza uburyo bwo gutwara izi peptides n'ibyo zikunda gufatana na microvasculature yihariye mu gace k'ubwonko. Ibi bishobora gutuma havumburwa inzira nshya zo gutwara BBB na receptors. Twiteze ko peptides zagaragaye zishobora gufatana neza na receptors zo mu bwonko cyangwa ligands zizenguruka zinyuzwa muri BBB cyangwa BCSFB. Vectors za peptides zifite imikorere ya CSF transportation yavumbuwe muri ubu bushakashatsi zizasuzumwa cyane. Ubu turimo gukora iperereza ku buryo ubwonko bw'izi peptides bufite ubushobozi bwo kwambuka BBB na/cyangwa BCSFB. Izi peptide nshya zizaba ibikoresho by'ingirakamaro cyane mu kuvumbura uburyo bushya bwo kwakira cyangwa inzira no guteza imbere uburyo bushya bwo kugeza ku bwonko ibintu bishya, nka biologique.
Gucukura imbeba nini (CM) hakoreshejwe uburyo bwahinduwe mbere. Imbeba za Wistar zatewe ikinya (200-350 g) zashyizwe ku gikoresho cya stereotaxic hanyuma hakorwa icyuho cyo hagati ku mutwe wogoshwe kandi wateguwe neza kugira ngo agahanga kagaragare. Fukura imyobo ibiri mu gice cyo hejuru cy'agasashi hanyuma uhambire vis zo kuyishyiraho mu myobo. Hatobowe undi mwobo mu gice cyo ku ruhande rw'ijisho kugira ngo hayobore visyo ya cannula y'icyuma kitagira umugese muri CM. Shyira sima y'amenyo hafi y'agasatsi hanyuma ushyireho vis. Nyuma yo gufotora no gukomera kwa sima, igikomere cy'uruhu cyafunzwe n'umugozi wa 4/0 supramid. Gushyira cannula neza byemezwa no kuva amaraso mu bwonko (CSF) mu buryo butunguranye. Kura imbeba mu gikoresho cya stereotaxic, witezweho ubuvuzi bukwiye nyuma yo kubagwa no kugabanya ububabare, kandi ureke ikire nibura icyumweru kimwe kugeza igihe ibimenyetso by'amaraso bigaragaye mu mazi yo mu bwonko. Imbeba za Wistar (Crl:WI/Han) zavuye muri Charles River (France). Imbeba zose zorojwe mu buryo bwihariye butagira indwara. Igerageza ryose ry’inyamaswa ryemejwe n’Ibiro by’Ubuvuzi bw’Amatungo byo mu Mujyi wa Basel, mu Busuwisi, kandi ryakozwe hakurikijwe uruhushya rw’inyamaswa No. 2474 (Isuzuma ry’uburyo ubwonko bukora hifashishijwe gupima urwego rw’abakandida bavura mu mazi yo mu bwonko n’ubwonko bw’imbeba).
Komeza witonze imbeba ufite CM cannula mu ntoki. Kura Datura muri cannula hanyuma utore µl 10 z'amazi yo mu bwonko atemba mu buryo bwikora. Kubera ko ubushobozi bwa cannula bwaje kwangirika, muri ubu bushakashatsi hashyizwemo gusa ingero z'amazi yo mu bwonko zidafite ibimenyetso by'uko amaraso yanduye cyangwa yahindutse. Muri ubwo bushakashatsi, hafi µl 10-20 z'amaraso zakuwe mu gace gato ku mutwe w'umurizo mu miyoboro irimo heparin (Sigma-Aldrich). CSF n'amaraso byakusanyijwe mu bihe bitandukanye nyuma yo guterwa T7 phage mu mitsi. Hafi µl 5-10 z'amazi zajugunywe mbere yuko buri gipimo cya CSF gikusanywa, bihuye n'ingano ya catheter yapfuye.
Amasomero yakozwe hakoreshejwe vekteri ya T7Select 10-3b nk'uko byasobanuwe mu gitabo cy'amabwiriza ya sisitemu ya T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). Muri make, inkingo ya ADN ya 12-mer yakozwe mu buryo bukurikira:
Kodoni ya NNK yakoreshejwe mu kwirinda kodoni ebyiri zihagarara no kwiyongera cyane kwa aside amine mu gice cyo hejuru. N ni igipimo cya equimolar cya buri nucleotide givanze n'intoki, naho K ni igipimo cya equimolar cya adenine na cytosine nucleotides givanze n'intoki. Uduce duto twashyizwemo ADN ebyiri zashyizwemo binyuze mu kongera gukwirakwiza dNTP (Novagen) na enzyme ya Klenow (New England Biolabs) muri Klenow buffer (New England Biolabs) mu gihe cy'amasaha 3 kuri 37°C. Nyuma yo gukora, ADN ebyiri zabonetse hakoreshejwe imvura ya EtOH. ADN yavuyemo yahinduwe hifashishijwe enzymes zo kugabanya EcoRI na HindIII (zombi zivuye muri Roche). Insert yaciwe kandi isukurwa (QIAquick, Qiagen) (T4 ligase, New England Biolabs) yashyizwe mu gice cyo hejuru cya T7 vector nyuma ya aside amine 348 ya gene ya capsid ya 10B. Inzira zo gufunga zashyizwe mu bushyuhe bwa 16°C. mu gihe cy'amasaha 18 mbere yo gupakira muri vitro. Gupakira mu buryo bwa "Phage" byakozwe hakurikijwe amabwiriza yatanzwe na T7Select 10-3b cloning kit (Novagen) kandi umuti wo gupakira wongerewe inshuro imwe kugira ngo uhinduke hakoreshejwe Escherichia coli (BLT5615, Novagen). Lysates zashyizwe muri centrifuge, zishyirwamo tit hanyuma zikonjeshwa kuri -80°C. nk'umuti wa glycerol.
Gutera imbaraga za PCR mu buryo butaziguye mu bice bihinduka bya phage byazamuwe mu muto cyangwa mu isahani hakoreshejwe primers za 454/Roche-amplicon fusion primers. Primers za forward fusion primers zirimo ibice biri ku ruhande rw'akarere gahinduka (NNK) 12 (template-specific), GS FLX Titanium Adapter A, hamwe n'urukurikirane rw'urufunguzo rw'isomero rufite base enye (TCAG) (Ishusho y'inyongera ya 1a):
Iyi primer yo guhuza ikoresheje reverse fusion irimo kandi biotin ifatanye n'udupira two gufata hamwe na GS FLX Titanium Adapter B ikenewe kugira ngo clonal amplification irusheho kwiyongera mu gihe cya emulsion PCR:
Hanyuma amplicons zakorewe 454/Roche pyrosequencing hakurikijwe protocole ya 454 GS-FLX Titanium. Ku bijyanye no gukora Sanger sequencing n'intoki (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage ADN yongerewe ubushobozi na PCR hanyuma ikurikiranywa hamwe n'ibi bikurikira:
Ibyuma byakuwe mu bipimo byashyizwemo PCR amplification hakoreshejwe Roche Fast Start DNA Polymerase Kit (dukurikije amabwiriza y'uwabikoze). Kora hot start (iminota 10 kuri 95 °C) na boost cycles 35 (50 s kuri 95 °C, 1 s kuri 50 °C, n'umunota 1 kuri 72 °C).
Phage yakuwe mu bubiko bw'ibitabo, phage yo mu bwoko bwa wild-type, phage yakuwe muri CSF n'amaraso, cyangwa clones ku giti cyayo byongerewe muri Escherichia coli BL5615 mu musupu wa TB (Sigma Aldrich) cyangwa mu masahani ya cm2 500 (Thermo Scientific) mu gihe cy'amasaha 4 kuri 37°C. Phage yakuwe mu masahani yoza amasahani hakoreshejwe Tris-EDTA buffer (Fluka Analytical) cyangwa ikusanyirizwa hamwe na plaque zirimo pipette. Phage yakuwe mu gipimo cya culture supernatant cyangwa extraction buffer hakoreshejwe round imwe ya polyethylene glycol (PEG 8000) precipitation (Promega) hanyuma yongera gushyirwa muri Tris-EDTA buffer.
Ifu yakuweho endotoxin inshuro 2-3 hakoreshejwe uduce twa endotoxin (Miltenyi Biotec) mbere yo guterwa inshinge (500 μl/inyamaswa). Mu cyiciro cya mbere, hashyizweho phage 2 × 1012; mu cyiciro cya kabiri, phage 2 × 1010; mu cyiciro cya gatatu n'icya kane cyo guhitamo, phage 2 × 109 kuri buri nyamaswa. Ingano ya phage muri CSF no mu maraso yakusanyijwe ku gihe cyagenwe yagenwe hakoreshejwe kubara plaque hakurikijwe amabwiriza y'uwakoze (T7Select system manual). Gutoranya phage byakozwe hakoreshejwe gutera isomero ryasukuwe mu mitsi cyangwa kongera gutera phage yakuwe muri CSF mu cyiciro cyabanje cyo guhitamo, hanyuma umusaruro wakurikiyeho ukorwa mu minota 10, iminota 30, iminota 60, iminota 90, iminota 120, iminota 180, n'iminota 240 muri rusange CSF n'amaraso. Hakozwe intera enye zose zo gupima mu mubiri aho amashami abiri yatoranijwe yabikwaga ukwayo kandi agasesengurwa mu byiciro bitatu bya mbere byo gutoranya. Ingirangingo zose za phage zakuwe muri CSF mu byiciro bibiri bya mbere byo gutoranya zashyizwe muri 454/Roche pyrosequencing, mu gihe clones zose zakuwe muri CSF mu byiciro bibiri bya nyuma byo gutoranya zashyizwe mu byiciro n'intoki. Ingirangingo zose z'amaraso zo mu cyiciro cya mbere cyo gutoranya nazo zashyizwe muri 454/Roche pyrosequencing. Mu gutera clones za phage, phage zatoranijwe zongerewe muri E. coli (BL5615) ku masahani ya cm2 500 kuri 37°C mu gihe cy'amasaha 4. Ingirangingo zatoranijwe ku giti cyazo kandi zashyizwe mu byiciro n'intoki zakwirakwijwe mu buryo bwa TB. Nyuma yo gukuramo phage, gusukura no gukuraho endotoxin (nk'uko byasobanuwe haruguru), phage 2 × 1010 / inyamaswa muri 300 μl zashyizwe mu mitsi mu mutsi umwe w'umurizo.
Gutegura no gusesengura amakuru y’uruhererekane. Amakuru ya 454/Roche yahinduwe ava kuri format ya binary standard stream map (sff) ajya kuri format ya Pearson human can belocable (fasta) hakoreshejwe porogaramu ya vendor. Gutunganya uruhererekane rwa nucleotide byakozwe hakoreshejwe porogaramu za C na scripts (software package itarasohoka) nk'uko byasobanuwe hano hepfo. Isesengura ry'amakuru y'ibanze rikubiyemo uburyo bukomeye bwo gusesengura ibyiciro byinshi. Kugira ngo hasukurwe isomwa ritari ririmo 12mer insert ADN sequence yemewe, isomwa ryahujwe ku buryo bukurikiranye n'ikirango cyo gutangira (GTGATGTCGGGGATCCGAATTCT), ikirango cyo guhagarika (TAAGCTTGCGGGCGCACTCGAGTA) n'ikirango cyo gutangira (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC) hakoreshejwe ikizamini cya Needleman-Wunsch global. guhuza kwemerera kugera ku bintu 2 bivuguruzanya kuri buri shusho31. Kubwibyo, isomwa ridafite tagi zo gutangira na guhagarika n'isomwa ririmo inyuguti zo inyuma, ni ukuvuga imbonerahamwe zirenze umubare wemewe w'ibidahuye, byakuwe muri bibliote. Ku bijyanye n'ibisigaye, urukurikirane rwa ADN rwa N-mer ruhera ku kimenyetso cyo gutangira kugeza rurangiye mbere yuko ikimenyetso cyo guhagarika gikurwa mu rukurikirane rw'ibanze rw'isomwa hanyuma gikomeza gutunganywa (hanyuma byitwa "gushyira"). Nyuma yo guhindura urukurikirane rw'inyuguti, igice cya nyuma ya kodoni ya mbere yo guhagarika ku mpera ya 5′ y'icyitegererezo gikurwa mu rukurikirane rw'inyuguti. Byongeye kandi, nucleotides zijyana kuri kodoni zidahagije ku mpera ya 3′ y'icyitegererezo nazo zakuweho. Kugira ngo hatangwemo urukurikirane rw'inyuguti zirimo urukurikirane rw'inyuma gusa, urukurikirane rw'inyuguti zahinduwe rutangirana n'icyitegererezo cya aside amine "PAG" narwo rwakuweho. Peptides zifite uburebure bwa nyuma yo guhindura imiterere y'inyuguti butarenze 3 zakuwe mu bubiko bw'ibitabo. Amaherezo, gukuraho urukurikirane rw'inyuguti mu rukurikirane rw'inyuguti hanyuma umenye inshuro za buri rukurikirane rw'inyuguti. Ibisubizo by'iri sesengura byakubiyemo urutonde rw'urukurikirane rw'inyuguti za nucleotide (inyuguti) n'inshuro zarwo (inyuguti z'inyongera 1c na 2).
Itsinda rya N-mer ADN rishyira hamwe hakurikijwe imiterere isa: Kugira ngo hakurweho amakosa ya 454/Roche-specific sequencing (nk'ibibazo byo kongera homopolymer mu guhuza) no gukuraho redundancies zidakomeye cyane, ibyinjizwa bya N-mer ADN byabanje gushungurwa (ibyinjizwa) bitondekwa hakurikijwe imiterere isa. (kugeza ku mizingo 2 idahuye byemerewe) hakoreshejwe algorithm isubiramo isobanurwa gutya: ibyinjizwa bibanza gutondekwa hakurikijwe imiterere yabyo (hejuru kugeza hasi), kandi niba ari bimwe, hakurikijwe imiterere yabyo ya kabiri hakurikijwe uburebure (bure kugeza bugufi)). Bityo, ibyinjizwa kenshi kandi birebire bigena "itsinda" rya mbere. Inshuro y'itsinda ishyirwa ku nshuro y'urufunguzo. Hanyuma, buri nshuro isigaye ku rutonde rwatunganijwe yageragejwe kongerwa mu itsinda hakoreshejwe uburyo bwa Needleman-Wunsch. Niba umubare w'ibitajyanye, ibyinjizwa, cyangwa ibyasibwe mu buryo butarenze urugero rwa 2, ibyinjizwa byongerwa mu itsinda, kandi frequency rusange y'itsinda yiyongera bitewe n'inshuro iyo nshuro yongewemo. Ibyinjizwa byongewe mu itsinda bishyirwa mu majwi nk'ibyakoreshejwe kandi ntibikorwe mu buryo burambuye. Iyo urukurikirane rw'inyito rudashobora kongerwa ku itsinda ryari risanzweho, urukurikirane rw'inyito rukoreshwa mu gukora itsinda rishya rifite inshuro zikwiye zo gushyiramo kandi rigashyirwamo ikimenyetso nk'uko ryakoreshejwe. Isubiramo rirangira iyo buri rukurikirane rw'inyito rwakoreshejwe mu gukora itsinda rishya cyangwa rushobora gushyirwa mu itsinda ryari risanzweho. N'ubundi kandi, inyito zigizwe n'uduce twa nucleotide zihinduka urukurikirane rwa peptide (amasomero ya peptide). Umusaruro w'iri sesengura ni urukurikirane rw'inyito n'inshuro zijyanye nazo zigize umubare w'inyito zikurikiranye (Ishusho y'inyongera ya 2).
Gutunganya Motif: Hashingiwe ku rutonde rwa peptides zidasanzwe, hakozwe isomero ririmo imiterere yose ishoboka ya aside amine (aa) nk'uko bigaragara hepfo. Buri shusho ishoboka y'uburebure bwa 3 yakuwe muri peptide maze imiterere yayo inyuranyije yongerwa hamwe n'isomero risanzwe rya motif ririmo imiterere yose (tripeptides). Amasomero y'inyuguti zisubiramo cyane yarakurikiranywe kandi hakurwaho ubusumbane. Hanyuma, kuri buri tripeptide iri mu isomero rya motif, twagenzuye niba ihari muri isomero dukoresheje ibikoresho byo kubara. Muri iki gihe, inshuro ya peptide irimo motif tripeptide yabonetse yongerwaho kandi igashyirwa kuri motif iri mu isomero rya motif ("umubare w'inyuguti"). Umusaruro wo gukora motif ni urutonde rw'ibice bibiri rurimo ibintu byose bya tripeptides (motifs) n'agaciro kabyo, ari byo mubare w'inyuguti zikurikirana zitanga motif ijyanye iyo abasomyi bayunguruwe, bashyizwe mu matsinda, kandi bagahindurwa. Ibipimo nk'uko byasobanuwe mu buryo burambuye haruguru.
Gushyira mu buryo busanzwe umubare w'ibishushanyo n'amabara ajyanye nabyo: Umubare w'ibishushanyo kuri buri rugero washyizwe mu buryo busanzwe hakoreshejwe
aho ni ari umubare w'ibisomwa birimo ingingo i. Bityo, vi ihagarariye ijanisha ry'ibisomwa (cyangwa peptides) birimo inyuguti i mu rugero. Agaciro ka P ku mubare utari usanzwe w'inyuguti kabazwe hakoreshejwe ikizamini nyacyo cya Fisher. Ku bijyanye n'igereranya ry'umubare w'inyuguti, isano ya Spearman yabazwe hakoreshejwe umubare usanzwe w'inyuguti na R.
Kugira ngo hamenyekane ingano ya aside amine kuri buri mwanya mu bubiko bwa peptide, hakozwe logogram za web 32, 33 (http://weblogo.threeplusone.com). Ubwa mbere, ingano ya aside amine kuri buri mwanya wa peptide ya 12-mer ibikwa muri matrix ya 20 × 12. Hanyuma, itsinda rya peptide 1000 zirimo aside amine imwe kuri buri mwanya zikorwa mu buryo bwa fasta-sequence kandi zigatangwa nk'inyongera kuri web-logo 3, itanga ishusho y'ingano ya aside amine kuri buri mwanya. kuri bibliote ya peptide runaka. Kugira ngo hamenyekane amakuru menshi, hakozwe amakarita y'ubushyuhe hakoreshejwe igikoresho cyakozwe imbere muri R (biosHeatmap, paki ya R itarasohoka). Dendrogram zagaragajwe mu makarita y'ubushyuhe zabazwe hakoreshejwe uburyo bwa Ward bwo gukusanya hierarchical hamwe na Euclidean interaction metric. Mu gusesengura imibare y'amakuru yo gupima motif, agaciro ka P ku manota adasanzwe kabazwe hakoreshejwe ikizamini nyacyo cya Fisher. Agaciro ka P ku yandi makuru kabazwe muri R hakoreshejwe ikizamini cya T cy'Umunyeshuri cyangwa ANOVA.
Udukoko twatoranijwe twa phage na phage zitarimo inshinge twatewe mu mitsi binyuze mu mutsi w'umurizo (2 × 1010 phage/inyamaswa muri 300 μl PBS). Iminota icumi mbere yo guterwa no gusimburwa, inyamaswa zimwe zatewe mu mitsi 100 μl ya DyLight594-labeled lectin (Vector Laboratories Inc., DL-1177). Iminota 60 nyuma yo guterwa phage, imbeba zatewe mu mutima 50 ml PBS hagakurikiraho 50 ml 4% PFA/PBS. Ingero z'ubwonko nazo zarafashwe mu ijoro ryose muri 4% PFA/PBS kandi zinjizwa muri 30% sucrose mu ijoro ryose kuri 4°C. Ingero zirakonjeshwa mu mvange ya OCT. Isesengura ry’ibipimo byakonjeshejwe ryakozwe ku bushyuhe bwa µm 30, rigashyirwamo ibice bya BSA 1%, rigashyirwamo antibodies za polyclonal FITC zirwanya T7 phage (Novus NB 600-376A) kuri 4 °C. Bika mu ijoro ryose. Amaherezo, ibice byogejwe inshuro 3 na PBS kandi bisuzumwa hakoreshejwe mikorosikopi ya laser ya confocal (Leica TCS SP5).
Peptide zose zifite ubuziranenge bwa nibura 98% zakozwe na GenScript USA, zigashyirwamo biotinyle hanyuma zigashyirwamo lyophilized. Biotin ihuzwa binyuze mu gice cy’inyongera cya glycine spacer kuri N-terminus. Reba peptides zose ukoresheje mass spectrometry.
Streptavidin (Sigma S0677) yavanzwe n'inshuro 5 z'ubunini bwa peptide ya biotinylated, peptide ibuza BACE1 ya biotinylated, cyangwa uruvange (igipimo cya 3:1) rwa peptide ibuza BACE1 ya biotinylated na peptide ibuza BACE1 muri 5–10% DMSO/incubated muri PBS. Isaha 1 ku bushyuhe bw'icyumba mbere yo guterwa inshinge. Peptide za Streptavidin zashyizwe mu mitsi ku gipimo cya 10 mg/kg muri imwe mu mitsi y'inyuma y'imbeba zifite ubwonko.
Ubunini bwa streptavidin-peptide complexes bwapimwe na ELISA. Ibice bya Nunc Maxisorp microtiter (Sigma) byasizweho ubushyuhe bwa 1.5 μg/ml bya anti-streptavidin antibody (Thermo, MA1-20011) nijoro kuri 4°C hakoreshejwe anti-streptavidin ya mbeba ya 1.5 μg/ml (Thermo, MA1-20011). Nyuma yo gufunga (buffer yo gufunga: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) ku bushyuhe bw'icyumba mu gihe cy'amasaha 2, oza isahani na 0.05% Tween-20/PBS (buffer yo gufunga) mu gihe cy'amasaha 3, CSF na plasma ingero zongewe mu byobo byavanzwe na buffer yo gufunga (plasma 1:10,000, CSF 1:115). Hanyuma isahani yashyizwe mu bushyuhe bwa 4°C hakoreshejwe anti-detection antibody (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Nyuma yo gukaraba intambwe eshatu, streptavidin yagaragaye mu gukaraba mu buryo bwa TMB substrate (Roche) mu gihe cy'iminota 20. Nyuma yo guhagarika ibara ukoresheje 1M H2SO4, pima ubushobozi bwo kwinjiza ibara kuri 450 nm.
Imikorere ya streptavidin-peptide-BACE1 inhibitor complex yagenzuwe na Aβ (1-40) ELISA hakurikijwe protocole y'uwakoze (Wako, 294-64701). Muri make, ingero za CSF zavanzwe mu buryo busanzwe (1:23) maze zishyirwa mu buryo busanzwe (1:23) ijoro ryose kuri 4°C mu masahani 96 yasizweho antibody ya BNT77. Nyuma y'intambwe eshanu zo gukaraba, antibody ya BA27 ya HRP yashyizwemo hanyuma ishyirwa mu buryo busanzwe amasaha 2 kuri 4°C, hagakurikiraho intambwe eshanu zo gukaraba. Aβ (1–40) yabonetse hakoreshejwe uburyo bwo gukaraba mu buryo bwa TMB mu gihe cy'iminota 30 ku bushyuhe bw'icyumba. Nyuma yo guhagarara kw'ibara hakoreshejwe uburyo bwo gukaraba, pima ubushobozi bwo gukaraba kuri 450 nm. Ingero za plasma zashyizwe mu buryo buhamye mbere yo gukaraba Aβ (1–40) ELISA. Plasma yongewe kuri 0.2% DEA (Sigma) mu masahani 96 yashyizwe mu buryo busanzwe kandi ishyirwa mu bushyuhe bw'icyumba mu gihe cy'iminota 30. Nyuma yo koza amasahani ya SPE (Oasis, 186000679) n'amazi na methanoli 100%, ingero za plasma zongewe ku masahani ya SPE maze amazi yose arakurwaho. Ingero zogejwe (habanje na methanoli 5% hanyuma 30% methanoli) hanyuma zigashyirwamo 2% NH4OH/90% methanoli. Nyuma yo kumisha eluate kuri dogere selisiyusi 55 mu gihe cy'iminota 99 ku muyoboro wa N2 uhoraho, ingero zagabanijwe mu bipimo bisanzwe maze Aβ(1–40) ipimwa nk'uko byasobanuwe haruguru.
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