Urakoze gusura Kamere.com. Verisiyo ya mushakisha ukoresha ifite inkunga nkeya kuri CSS.Kuburambe bwiza, turagusaba ko ukoresha mushakisha ivuguruye (cyangwa kuzimya uburyo bwo guhuza ibikorwa muri Internet Explorer). Hagati aho, kugirango ubone ubufasha bukomeza, tuzerekana urubuga rudafite imiterere na JavaScript.
Indwara ya gene yo kuvura indwara yibihaha ya cystic fibrosis igomba kwibasira inzira zoguhumeka kuko kwanduza ibihaha bya peripheri bidatanga inyungu zo kuvura. Uburyo bwo kwanduza virusi bifitanye isano itaziguye nigihe cyo gutura kwa vector.Nyamara, amazi yo kubyara nka gene asanzwe akwirakwira muri alveoli mugihe cyo guhumeka, kandi uduce duto two kuvura muburyo ubwo aribwo bwose bwogutwara mumashanyarazi. ibice bishobora kwerekezwa hejuru yumuyaga birashobora kunoza intego zakarere. Bitewe nibibazo byo muri vivo iyerekwa rya vivo, imyitwarire yutwo duce duto twa magnetique hejuru yumuyaga imbere yumurima wa magneti washyizweho ntisobanutse neza.Intego yubu bushakashatsi kwari ugukoresha amashusho ya synchrotron kugirango ugaragaze amashusho ya vivo yikurikiranya ryimyanya ndangagitsina muri trachea yimiterere ya vivo hamwe nudusimba twa anestheti. lentiviral magnetique imbere yumurima wa magnetiki byongera imikorere ya transduction muri trachea yimbeba.Synchrotron X-ray yerekana amashusho yerekana imyitwarire yibice bya magnetiki mumirima ihagaze kandi igenda yimuka muri vitro no muri vivo.Ibice ntibishobora gukururwa byoroshye hejuru yumuyaga muzima hamwe na magneti, ariko mugihe cyo gutwara, kubitsa byibanze cyane mubice bya magnetique byashyizwemo imbaraga. yumurima wa magneti. Hamwe na hamwe, ibisubizo byerekana ko ibice bya magnetique lentiviral nuduce twa magnetique bishobora kuba inzira zingenzi zo kunoza icyerekezo cya gene no kongera urwego rwa transduction mu kuyobora inzira zo guhumeka muri vivo.
Cystic fibrosis (CF) iterwa no guhindagurika muri gene imwe yitwa CF transmembrane conductance regulor (CFTR) .Intungamubiri ya CFTR ni umuyoboro wa ion uboneka mu ngirabuzimafatizo nyinshi za epiteliyale mu mubiri wose, harimo n'umuyoboro uhumeka, ahantu hanini h’indwara ya CF. Inenge za CFR zitera ubwikorezi bw'amazi adasanzwe kandi bikagabanya ubujyakuzimu bwa ASL. sisitemu ya transport ya mucociliary (MCT) kugirango ikureho uduce duto twahumekewe na virusi ziva mumyuka ihumeka.Intego yacu ni ugutezimbere imiti ya lentiviral (LV) kugirango itange kopi yukuri ya gene CFTR no guteza imbere ubuzima bwa ASL, MCT, nibihaha, no gukomeza guteza imbere ikoranabuhanga rishya rishobora gupima ibyo bipimo muri vivo1.
LV vectors ni umwe mu bakandida bayobora ubuvuzi bwa CF bwo mu kirere, cyane cyane ko bushobora kwinjiza burundu gene yo kuvura mu ngirabuzimafatizo zo mu kirere (ingirabuzimafatizo zo mu kirere) .Ibi ni ngombwa kuko bishobora kugarura imiyoboro isanzwe ya gene ikosorwa na CF ifitanye isano n’ingirabuzimafatizo zo mu kirere ubuzima bwabyo. birashobora kuviramo kwandura alveolar, ariko ibi nta nyungu zo kuvura muri CF.Nyamara, amazi nkabatwara gene mubisanzwe bimukira muri alveoli nyuma yo kubyara 3.4 kandi uduce duto two kuvura twahanaguwe vuba mumyanya yo mu kanwa na MCT. CF, ubushobozi bwo kongera igihe cyo gutura kwa LV mu kirere ni ngombwa kugira ngo habeho transduction nyinshi muri kano karere, ariko kugeza ubu biragoye.
Kugira ngo dutsinde iyi nzitizi, turasaba ko LV magnetique (MPs) zishobora gufasha muburyo bubiri bwuzuzanya. Ubwa mbere, zirashobora kuyoborwa na magnetiki hejuru yumuyaga kugirango zirusheho gutera imbere no gufasha ibice bitwara gene gutura mukarere kifuza guhumeka; na ASL) kwimukira murwego rwakagari 6.MPs yakoreshejwe cyane nkibinyabiziga bigamije gutanga ibiyobyabwenge mugihe bihujwe na antibodies, imiti ya chimiotherapeutique, cyangwa izindi molekile ntoya zifata uturemangingo twa selile cyangwa zihuza ibyakiriwe na selile kandi bikarundarunda ahantu h’ibibyimba hari amashanyarazi ahamye. Magnetic Fields yo kuvura Kanseri 7. Ubundi buryo bwa "hyperthermal" bugamije gushyushya abadepite mugihe bahuye nimirima ya magnetiki ihindagurika, bityo bakangiza ingirabuzimafatizo. Ihame ryo kwanduza magneti, aho umurima wa magneti ukoreshwa nkumukozi wo kwanduza ADN mu ngirabuzimafatizo, ukunze gukoreshwa muri vitro ukoresheje uburyo butandukanye bwo kwanduza virusi ya virusi. y'Abadepite ba LV-kumurongo wa epiteliyale ya bronchial yumuntu imbere yumurima wa magnetiki uhagaze, byongera imikorere ya transduction inshuro 186 ugereranije na LV yonyine.LV-MP nayo yakoreshejwe muburyo bwa vitro CF, aho kwanduza magnetique byongereye LV kwanduza mumico yimiterere yimiterere yikirere byikubye inshuro 20 imbere ya CF magnetotransfection. ubushakashatsi11,12,13,14,15, cyane cyane mu bihaha 16.17. Nubwo bimeze bityo ariko, amahirwe yo kwanduza magneti mu kuvura ibihaha bya CF arasobanutse.Tan n'abandi.
Imyitwarire ya uduce duto twa magnetiki hejuru yumuyaga imbere yumurima wa magneti washyizweho biragoye kubyiyumvisha no kwiga, bityo ntibisobanuke neza.Muyindi nyigo, twashyizeho uburyo bwa synchrotron-gukwirakwiza bushingiye ku cyiciro cyo gutandukanya X-ray yerekana amashusho (PB-PCXI) kugirango tuyerekane neza kandi ugereranye umunota mubihinduka bya vivo muburyo bwa tekinike ya MCT hamwe nubushakashatsi bwa MCT19,20 ikoresha ibice 10-35 µm bya diametre bigizwe na alumina cyangwa ikirahure kinini cyerekana ibirahure nkibimenyetso bya MCT bigaragara ukoresheje PB-PCXI21. Ubuhanga bwombi burakwiriye kubona amashusho yubwoko butandukanye, harimo na MP.
Bitewe nuburyo bukomeye kandi bwagateganyo, tekinoroji yacu yo gusesengura AS-na MCT ishingiye kuri PB-PCXI ikwiranye neza nogusuzuma imbaraga nuburyo bwimyitwarire yingingo imwe nini nini muri vivo kugirango bidufashe kumva no kunonosora uburyo bwo gutanga gene ya MP.Uburyo dukoresha hano bukomoka mubushakashatsi bwacu dukoresheje uburyo bwa spring-8 BL20B2 bwerekana umurongo utanga uburyo bwo gutembera kwizana rya virusi. muri gene yacu itwara dose inyamaswa 3,4.
Icyari kigamijwe muri ubu bushakashatsi kwari ugukoresha synchrotron PB-PCXI mu kwerekana amashusho yimikorere ya vivo yuruhererekane rwabadepite muri trachea yimbeba nzima. Izi nyigisho zerekana amashusho PB-PCXI zagenewe kugerageza abadepite batandukanye, imbaraga za magnetique, hamwe n’ahantu kugirango hamenyekane ingaruka zabyo ku cyerekezo cy’abadepite.Twakekaga ko umurima wa magnetiki ukoreshwa mu mahanga uzafasha abadepite batanze kuguma cyangwa kwimuka. umubare wibice byagumishijwe muri trachea nyuma yo kubitsa.Mu ruhererekane rwa kabiri rw’ubushakashatsi, twashatse gukoresha iyi miterere nziza kugirango twerekane uburyo bwo guhererekanya ibintu biva muri vivo yohereza abadepite ba LV mu nzira y’imbeba, dushingiye ku gitekerezo kivuga ko gutanga abadepite ba LV mu rwego rwo guhumeka neza byavamo uburyo bwiza bwo kohereza LV.
Ubushakashatsi bwose bw’inyamaswa bwakozwe hakurikijwe protocole yemejwe na kaminuza ya Adelayide (M-2019-060 na M-2020-022) na komite ishinzwe imyitwarire y’inyamaswa ya SPring-8 Synchrotron.Ubushakashatsi bwakozwe hakurikijwe amabwiriza ya ARRIVE.
Amashusho yose ya X-ray yakorewe kumurongo wa BL20XU kuri synchrotron ya SPring-8 mu Buyapani, hifashishijwe uburyo busa nubwavuzwe mbere 21,22. Muri make, agasanduku k'ubushakashatsi kari gaherereye kuri m 245 uvuye ku mpeta yo kubika synchrotron. Intera-to-detector intera ya 0,6 m ikoreshwa mubushakashatsi bwerekana amashusho hamwe na 0.3 m mu bushakashatsi bwakozwe na vivo. Ihinduramiterere ryinshi rya X-ray (SPring-8 BM3) ihujwe na deteri ya sCMOS. Ihindura ihindura X-imirasire kumucyo ugaragara ikoresheje scintillator ya 10 µm (Gd3Al2Ga3O12), hanyuma ikoherezwa kuri sensor ya sCMOS ikoresheje intego ya microscope ya NA 10 (NA 0.3). ubunini bwa 2048 × 2048 pigiseli hamwe na pigiseli mbisi ingana na 6.5 × 6.5 µm.Iyi mikorere itanga umusaruro mwiza wa isotropic pigiseli ingana na 0.51 µm hamwe n'umurima wo kureba hafi ya mm 1,1 mm × 1.1 mm.Uburebure bwa ms 100 bwatoranijwe kugirango hagabanuke igipimo cyerekana ibimenyetso-urusaku rw'ibice bya magnetiki imbere no hanze yumuyaga mu gihe hagabanywa imishwarara yatewe na moteri. Inzira ya X-yo kugabanya imishwarara ikumira imirasire ya X-hagati yerekana.
Ubwikorezi bwa LV ntabwo bwakoreshejwe mubushakashatsi bwakozwe bwerekana amashusho ya SPring-8 PB-PCXI kubera ko icyumba cyerekana amashusho cya BL20XU kitari Biosafety Urwego rwa 2 cyemejwe.Ahubwo, twahisemo abadepite barangwa neza nabashoramari babiri batanga ibicuruzwa - bikubiyemo ubunini, ibikoresho, ibyuma bya fer, hamwe nibisabwa - tubanza kumva uburyo imirima ya rukuruzi igira ingaruka kumikorere ya MP muri capillaries yikirahure, hanyuma mukirere gihumeka. hejuru.MPs ifite ubunini kuva kuri 0.25 kugeza kuri 18 mkm kandi bikozwe mubikoresho bitandukanye (reba Imbonerahamwe 1), ariko ibigize buri cyitegererezo, harimo nubunini bwa magnetique muri MP, ntibizwi. Dushingiye ku bushakashatsi bwakozwe na MCT bwagutse 19, 20, 21, 23, 24, turateganya ko abadepite bato nka 5 μm bashobora kugaragara hejuru yikurikiranya ryinzira ya MP25. MP-nini ya MP ni ntoya kuruta gukemura ibyuma bifata amashusho, ariko biteganijwe ko PB-PCXI izagaragaza itandukaniro ryabyo hamwe nigikorwa cyamazi yo hejuru bashyiramo nyuma yo kuyashyira.
Icyitegererezo kuri buri Mudepite mu mbonerahamwe ya 1 cyateguwe muri 20 μl capillaries (Drummond Microcaps, PA, USA) hamwe na diametre y'imbere ya mm 0,63. Ibice bya corpuscular biboneka mumazi, mugihe ibice bya CombiMag biboneka mumazi ya nyirubwite. Buri muyoboro wuzuyemo igice cyuzuye amazi (hafi 11 μl) hanyuma ugashyirwa kuri stade yerekana icyerekezo (reba ishusho ya 1). shyira ku nkombe z'amazi. Uburebure bwa mm 19 z'uburebure (mm 28 z'uburebure) nikel shell idasanzwe isi neodymium fer boron (NdFeB) magnet (N35, injangwe no. amakadiri kumasegonda.Mu gihe cyo gufata amashusho, magnet yazanwe hafi yikirahure capillary tube (hafi mm 1) hanyuma igahindurwa kumuyoboro kugirango isuzume ingaruka zimbaraga zumurima nu mwanya.
Muri vitro yerekana amashusho arimo MP yintangarugero muri capillaries yicyitegererezo kuri stade ya xy yo guhindura. Inzira yumurongo wa X-ray irangwa numurongo utukura.
Bimaze gushyirwaho muri vitro igaragara y’abadepite, igice cyabo muri bo cyageragejwe muri vivo mu bwoko bw’imbeba zo mu bwoko bw’imbeba zo mu bwoko bwa albino Wistar (~ ibyumweru 12, ~ 200 g). anesthete hamwe nuruvange rwa Pharma), mubuyapani) hakoreshejwe inshinge za intraperitoneal.Nyuma ya anesteziya, biteguye gushushanya bakuramo ubwoya buzengurutse trachea, binjiza umuyoboro wa endotracheal (ET; 16 Ga iv cannula, Terumo BCT) hanyuma ubabuza supine kumasahani yakozwe mumashusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana ishusho yerekana isahani yerekana isahani. trachea itambitse mu ishusho ya X-ray, nkuko bigaragara ku gishushanyo cya 2a.
.
Sisitemu ya pompe ya siringe igenzurwa na kure (UMP2, World Precision Instruments, Sarasota, FL) ikoresheje siringi ya 100 μl ikirahuri cyahujwe no kuvoma PE10 (OD 0,61 mm, ID 0.28 mm) ukoresheje urushinge rwa 30 Ga. Shyira umuyoboro kugirango umenye neza ko igitekerezo kiri mumwanya mwiza muri trachea mugihe washyizemo igituba cya microp. murugero rwabadepite rugomba gutangwa. Umuyoboro wapakiwe wapakiye noneho winjizwa mumiyoboro ya endotracheal, ushyira igitekerezo mugice kinini cyumwanya wa magneti uteganijwe gukoreshwa. Kugura amashusho byagenzuwe hakoreshejwe icyuma gihumeka gihuza agasanduku kacu ka Arduino, kandi ibimenyetso byose (urugero: ubushyuhe, guhumeka, gufungura gufungura / gufunga no kugura amashusho) byanditswe hakoreshejwe Powerlab na LabChart (AD Instruments). .
Magneti ifatanye ku cyiciro cya kabiri gishobora kuba kure y’amazu yerekana amashusho. Imyanya itandukanye ya magneti hamwe n’ibishushanyo byageragejwe, harimo: Yashyizwe ku nguni ya 30 ° hejuru ya trachea (ibishushanyo bigaragara ku gishushanyo cya 2a na 3a); rukuruzi imwe hejuru yinyamaswa nindi hepfo, hamwe ninkingi zashyizweho kugirango zikurure (Ishusho 3b); rukuruzi imwe hejuru yinyamaswa indi hepfo, hamwe ninkingi zashyizweho kugirango zisubire inyuma (Ishusho 3c); na rukuruzi imwe hejuru na perpendicular kuri trachea (Igicapo 3d) .Iyo inyamanswa na magneti bimaze gushyirwaho hanyuma umudepite agomba gupimwa yapakiwe muri pompe ya syringe, tanga urugero rwa 50 μl ku gipimo cya 4 μl / sek mugihe ubonye amashusho. Magneti ihita yimurwa inyuma cyangwa ikanyura kuri trachea mugihe ikomeje kubona amashusho.
Ibikoresho bya magneti kuri mashusho ya vivo (a) rukuruzi imwe hejuru ya trachea kumpande zigera kuri 30 °, (b) magnesi ebyiri zashyizweho kugirango zikurure, (c) magnesi ebyiri zashyizweho kugirango zisubire inyuma, (d) rukuruzi imwe hejuru na perpendicular muri trachea. Indorerezi yarebye hasi kumunwa yerekeza mubihaha inyuze muri trachea. inzira yo guhumeka cyangwa ibumoso n'iburyo hejuru ya trachea mu cyerekezo cya X-ray.
Twashatse kandi kumenya neza n’imyitwarire y’uduce duto two mu kirere mu gihe hatabayeho guhumeka neza no kugenda umutima. Kubera iyo mpamvu, igihe cyo gufata amashusho kirangiye, inyamaswa zishwe n’abantu bazira kunywa inzoga nyinshi za pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, Amerika; ~ 65 mg / kg ip) Depite yagaragaye hejuru yumuyaga.
Amashusho yabonetse yari umurima-mwijima-mwijima-warakosowe hanyuma ukusanyirizwa muri firime (amakaramu 20 kumasegonda; 15-25 × umuvuduko usanzwe bitewe nigipimo cyubuhumekero) ukoresheje inyandiko yihariye yanditse muri MATLAB (R2020a, Imibare).
Ubushakashatsi bwose bwo gutanga virusi ya LV bwakorewe mu kigo cy’ubushakashatsi bw’amatungo muri Laboratwari muri kaminuza ya Adelaide kandi bugamije gukoresha ibisubizo by’igeragezwa rya SPring-8 kugira ngo harebwe niba itangwa rya LV-MP imbere y’umurima wa rukuruzi rishobora kuzamura ihererekanyabubasha muri vivo. Kugira ngo hamenyekane ingaruka z’umudugudu wa MP na magnetique, amatsinda abiri y’inyamaswa yaravuwe: itsinda rimwe ryahawe LV-MP hamwe na magneti yashyizwe hamwe na magneti.
LV gene vectors yakozwe hakoreshejwe uburyo bwasobanuwe mbere 25, 26 .Icyerekezo cya LacZ cyerekana gene ya beta-galactosidase ya kirimbuzi ikwirakwizwa na porotokoro ya MPSV (LV-LacZ), itanga umusaruro wubururu mu ngirabuzimafatizo zanduye, zigaragara mu bice by’ibihaha no mu bice bya tissue. TU / ml. Inzitizi zibikwa kuri -80 ° C, zikonjeshwa mbere yo kuzikoresha, hanyuma zigahuzwa na CombiMag zivanga ku kigereranyo cya 1: 1 hanyuma zigashyirwa ku rubura byibuze iminota 30 mbere yo kubyara.
Imbeba zisanzwe za Sprague Dawley (n = 3 / itsinda, ~ 2-3 zatewe anesthetite intraperitoneally hamwe nuruvange rwa 0.4 mg / kg medetomidine (Domitor, Ilium, Ositaraliya) na 60 mg / kg ketamine (Ilium, Ositaraliya) ukwezi) ip) inshinge hamwe na kanseri yo mu kanwa itabanje kubagwa hakoreshejwe uburyo bwo kwanduza LV. poroturbation protocole, aho inzira yumuyaga ya tracheal yakubiswe mu buryo bwikurikiranya hamwe nigitebo cyinsinga (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Ubuyobozi bwa tracheal bwa LV-MP bwakorewe mumabanga yumutekano wibinyabuzima nyuma yiminota 10 nyuma yo kwangirika.
Umwanya wa magneti wakoreshejwe murubu bushakashatsi washyizweho muburyo busa nubushakashatsi bwakozwe na vivo X-ray, hamwe na magnesi imwe yafashwe hejuru ya trachea ikoresheje clip stent distillation (Igicapo 4) .Ubunini bwa 50 μl (2 × 25 μl aliquots) za LV-MP bwagejejwe muri trachea (n = 3 inyamaswa) hakoreshejwe umuyoboro wa geli nkuko byavuzwe haruguru. magnetic kugeza gukira byuzuye kuva anesthesia.
Igikoresho cyo gutanga LV-MP muri guverenema yumutekano wibinyabuzima.Icyatsi cyoroshye cya Luer hub yumuyoboro wa ET kirashobora kugaragara kiva mumunwa kandi isonga ya gel ya pipeti yerekanwe kumashusho yinjizwa mumiyoboro ya ET kugeza mubwimbuto bwifuzwa muri trachea.
Icyumweru kimwe nyuma yuburyo bwa LV-MP, inyamaswa zishwe nubumuntu n’umwuka wa CO2 100% kandi imvugo ya LacZ yasuzumwe hifashishijwe uburyo busanzwe bwo kuvura X-gal.Impeta eshatu za caudal impeta nyinshi za karitsiye zavanyweho kugira ngo harebwe niba ibyangiritse cyangwa imashini zitwara amazi zashyizwe mu miyoboro ya endotracheal zitashyizwe mu isesengura. Dow Inc. intambwe ku yindi, kwemeza guhuzagurika bihagije hagati ya buri shusho kugirango yemere ishusho "kudoda" .Ibishusho biva muri buri trachea byahise bikusanyirizwa hamwe mu ishusho imwe ihuriweho hifashishijwe Ishusho ya Composite Editor v2.0.3 (Ubushakashatsi bwa Microsoft) ukoresheje algorithm ya planar. 0.58.
Buri trachea yashyizwe muri paraffine hanyuma ibice 5 μm biracibwa.Ibice byabujijwe gutukura bitagira aho bibogamiye mu minota 5 kandi amashusho yabonetse hakoreshejwe microscope ya Nikon Eclipse E400, kamera ya DS-Fi3 na software ya NIS (verisiyo 5.20.00).
Isesengura ry’ibarurishamibare ryose ryakorewe muri GraphPad Prism v9 (Porogaramu ya GraphPad, Inc.
Abadepite batandatu basobanuwe mu mbonerahamwe ya 1 basuzumwe bakoresheje PCXI, kandi ibiboneka bisobanurwa mu mbonerahamwe ya 2. Abadepite babiri ba polystirene (MP1 na MP2; 18 mkm na 0,25 mm), ntibagaragaye kuri PCXI, ariko izindi ngero zasigaye zaramenyekanye (ingero zerekanwa ku gishushanyo cya 5) .MP3 na MP4 (10-15% Fe3O4 zigaragara kuri 0.25 μm na 0.4); uduce duto duto twageragejwe, MP5 (98% Fe3O4; 0,25 mm) niyo yagaragaye cyane.Ibicuruzwa bya CombiMag MP6 biragoye kubibona.Mu bihe byose, ubushobozi bwacu bwo gutahura MP bwongerewe imbaraga muguhindura magnet imbere n'inyuma bigereranywa na capillary.Iyo magneti yimuka kuva mumirongo miremire, ariko uko magneti yagendaga yiyongera mumirongo miremire, ariko uko magneti yagendaga yiyongera mumirongo miremire, ariko uko magneti yagendaga yiyongera mumirongo miremire. hejuru hejuru ya capillary (reba Video yinyongera S1: MP4), kwiyongera Ubwinshi bwibice byubuso.Ibinyuranye, iyo magneti akuwe muri capillary, imbaraga zumurima ziragabanuka hanyuma abadepite bongera guhinduranya imirongo miremire iva hejuru yubuso bwa capillary (reba Video yinyongera S2: MP4) kure yubuso bwo hejuru bwa capillary, ibice bya magneti mubisanzwe bikurura imyanda mumazi.
Kugaragara kwa MP munsi ya PCXI biratandukanye cyane hagati yintangarugero. ibishushanyo mubishushanyo byose ntabwo bigomba gupimwa kandi bigera hafi inshuro 100 kurenza uko byerekanwe.
Nkuko rukuruzi isobanurwa ibumoso n'iburyo hejuru ya capillary, inguni yumugozi wa MP irahinduka kugirango ihuze na magneti (reba Ishusho ya 6), bityo igasobanura imirongo yumurongo wa magneti. Kuri MP3-5, nyuma ya chord igeze kumpande zingana, ibice bikururwa hejuru yubuso bwa capillary. Ibi bikunze kuvamo abadepite bahurira mumatsinda manini hafi ya S5: Reba imbaraga za Magnetiki. cyane cyane iyo ishusho yegereye impera ya capillary, itera abadepite guteranya no kwibanda kumurongo wa fluid-air.Ibice byo muri MP6, byari bigoye gutahura kuruta MP3-5, ntibyakururutswe kuko magneti yagendaga hejuru ya capillary, ariko imirongo yabadepite yatandukanijwe, hasigara ibice bitandukanijwe numwanya wa magneti washyizwemo na magneti mugihe washyizwe mubikorwa na magnetique. abadepite basigaye bamanuka gahoro gahoro hejuru yigitereko nuburemere mugihe bagumye kumurongo (reba Video yinyongera S5: MP3).
Inguni yumurongo wa MP irahinduka nkuko magnet yahinduwe iburyo hejuru ya capillary.
Ibizamini byacu byerekanaga ko kwimura magneti gahoro gahoro imbere na trachea byorohereza iyerekwa rya MP murwego rwo kugenda rugoye muri vivo.Mu igeragezwa rya vivo ntabwo ryakozwe kuko amasaro ya polystirene (MP1 na MP2) atagaragaye muri capillary. Buri mu badepite bane basigaye bapimwe muri vivo hamwe na axe ndende ya magneti yagereranijwe hejuru ya trachea kuri metero zigera kuri 30). gukora neza kuruta iboneza rya magneti ryarangiye.MP3, MP4 na MP6 ntabwo byagaragaye muri trachea yinyamaswa nzima.Iyo inzira yumuyaga yimbeba yashushanijwe nyuma yinyamaswa ziciwe abantu, ibice byaragumye bitagaragara nubwo hongeweho ingano yinyongera hifashishijwe pompe ya siringi.MP5 yari ifite ibice byinshi bya okiside yicyuma kandi niyo yari ikoreshwa muburyo bwo gusuzuma no kuranga MP muri vivo.
Gushyira magneti hejuru ya trachea mugihe cyo gutanga abadepite byaviriyemo benshi, ariko siko bose, abadepite bibanze murwego rwo kureba.Ibice byinjira muri trachea bigaragarira cyane mu nyamaswa zatambwe abantu.Figure 7 na Video yinyongera S6: MP5 yerekana gufata magnetiki byihuse no guhuza ibice hejuru yubutaka bwa trachea, byerekana ko abadepite bashobora kwerekezwa kuri trachea. Abadepite basanze hafi ya karina, bavuga ko imbaraga za magneti zidahagije mu gukusanya no kugumana abadepite bose, kubera ko zatanzwe binyuze mu karere k’ingufu nyinshi za magnetiki mu gihe cy’amazi. Nubwo bimeze bityo ariko, abadepite nyuma yo kubyara bari hejuru y’ahantu hafashwe amashusho, byerekana ko abadepite benshi bagumye mu turere tw’umuyaga aho imbaraga za rukuruzi zikoreshwa cyane.
Amashusho kuva (a) mbere na (b) nyuma yo gutanga MP5 muri trachea yimbeba iherutse gutangwa hamwe na magneti ihagaze neza hejuru y’ahantu hagaragara amashusho. Agace kashushanijwe kari hagati yimpeta zombi.
Guhindura magneti kuruhande rwa trachea muri vivo byatumye urunigi rwabadepite ruhindura inguni hejuru yumuyaga muburyo busa nubwa capillaries (reba Ishusho ya 8 na Video yinyongera S7: MP5) .Nyamara, mubushakashatsi bwacu, abadepite ntibashobora gukururwa hejuru yumuyaga muzima nkuko bashoboye hamwe na capillaries.Mu bihe bimwe na bimwe, urunigi rwabadepite ruzagenda rurerure kandi rukomeye. ubujyakuzimu bw'amazi yo hejuru hejuru iyo magnet yimuwe igihe kirekire kuri trachea, kandi ikaguka iyo magneti yimuwe hejuru hanyuma umugozi wuduce ukazenguruka kumwanya uhagaze (reba Video yinyongera S7). . bihuye nimyitwarire yabadepite twarebye munsi yumurongo wa magneti ukoreshwa mubirahuri bya capillary.
Urugero rwerekana amashusho yerekana MP5 muri trachea yimbeba nzima.
Iyo inkingi zombi zashyizwe mu cyerekezo cy’amajyaruguru y’amajyepfo hejuru no munsi ya trachea (ni ukuvuga gukurura; Igishushanyo cya 3b), chord ya MP yagaragaye igihe kirekire kandi yari iherereye ku kayira kegereye trachea aho kuba hejuru ya tracheal (reba Video yinyongera S9: MP5) .Nyamara, ubushakashatsi bwinshi bwibintu byari ahantu hatandukanye (ni ukuvuga, icyerekezo cya dcheque). ikoreshwa, ubusanzwe ibaho mugihe hakoreshejwe igikoresho kimwe rukuruzi.Noneho mugihe rukuruzi imwe yashizweho kugirango isubize inkingi ihindutse (Igishusho 3c), umubare wibice bigaragara mumwanya wo kureba ntiwagaragaye ko wiyongereye nyuma yo kubyara. Gushiraho ibice byombi bya magneti biragoye kubera imbaraga zumurima wa magneti ukurura cyangwa ugasunika kuri magneti, bikagenda bihinduka kuri moteri imwe. imirongo yambukije urukuta rwa tracheal orthogonally (Igishusho 3d), icyerekezo cyateguwe kugirango hamenyekane niba igiteranyo cyo guteranya ibice kurukuta rwuruhande rushobora kugaragara .Nyamara, muri iyi miterere, nta cyerekezo cyamenyekanye cyo gukusanya abadepite cyangwa urujya n'uruza rwa magneti. Hashingiwe kuri ibyo bisubizo byose, rukuruzi imwe rukumbi, ibyerekezo bya dogere 30 (Ishusho 3a) byatoranijwe mubushakashatsi bwa vivo gene.
Iyo inyamanswa yashushanijwe inshuro nyinshi nyuma yo kwica abantu, kutagira urujya n'uruza rw'imitsi byasobanuraga ko imirongo yoroheje kandi ngufi ishobora gutahurwa mu murima usobanutse, “wobbly” ijyanye no guhinduranya kwa rukuruzi. Nubwo bimeze bityo, ntushobora kubona neza ko MP6 ihari kandi igenda.
Titer ya LV-LacZ yari 1.8 × 108 TU / ml, hanyuma nyuma ya 1: 1 ivanze na MP CombiMag MP (MP6), inyamaswa zabonye urugero rwa 50 μl tracheal ya 9 × 107 TU / ml LV (ni ukuvuga 4.5 × 106 TU / imbeba). ) .Muri ubu bushakashatsi, aho guhindura magneti mugihe cyakazi, twashyizeho magneti mumwanya umwe kugirango tumenye niba transduction ya LV (a) ishobora kunozwa ugereranije no gutanga za vector mugihe hatabayeho umurima wa rukuruzi, kandi (b) hashobora kwibandwaho ingirabuzimafatizo zo mu kirere zihererekanwa mukarere ka magnetiki kagenewe inzira yo hejuru yumuyaga wo hejuru.
Kubaho kwa magnesi no gukoresha CombiMag bifatanije na LV vectors ntabwo byagaragaye ko bigira ingaruka mbi kubuzima bwinyamaswa, kimwe na protocole yacu isanzwe ya LV vector itanga amashusho. Amashusho yimbere yakarere ka tracheal yatewe no gukomeretsa imashini (Ishusho yinyongera. itsinda rishinzwe kugenzura (Igicapo 9b) .Kwerekana ahantu hasanzwe X-Gal hasize irangi ryerekanaga ko ubuyobozi bwa LV-MP imbere yumurima wa rukuruzi bwatanze umusaruro wikubye inshuro 6 (Ishusho 9c).
Urugero rwerekana amashusho yerekana transduction ya tracheal yakozwe na LV-MP (a) imbere yumurima wa magneti na (b) mugihe habuze magneti.
Ibice bitagira aho bibogamiye bitukura (urugero rwerekanwe ku gishushanyo cya 2) byerekanaga ingirabuzimafatizo za LacZ ziboneka muburyo busa naho biherereye nkuko byavuzwe mbere.
Ikibazo cy'ingutu cyo kuvura gene yo mu kirere gikomeje kuba ahantu nyaburanga mu turere tw’inyungu no kugera ku ntera yo hejuru yo kwanduza mu bihaha byimuka imbere y’imyuka ihumeka ndetse no mu mucyo ukabije. Ku batwara LV bagenewe kuvura indwara zo mu kirere cya CF, kongera igihe cyo gutura mu bice bitwara abagenzi mu nzira zo mu kirere byabaye intego zoroshye kugeza ubu ku buryo bwo gukoresha za magneti ku zindi ngendo zakozwe na Castellani n'ibindi. amashanyarazi, kuko ashobora guhuza ubworoherane, gukora neza-kugiciro, gutanga aho biherereye, kongera imikorere, hamwe nigihe gito cyo gukuramo, kandi birashoboka ko umutwaro muto wa dose10.Nyamara, muri vivo gushira hamwe nimyitwarire yibice bya magnetique mumyuka yumuyaga byatewe ningufu za rukuruzi zo hanze ntabwo byigeze bisobanurwa, cyangwa mubyukuri uburyo bushoboka bwubu buryo bwerekanwe muri vivo kugirango hazamurwe urwego rwimyuka mibi.
Ubushakashatsi bwakozwe na vitro synchrotron PCXI bwerekanye ko ibice byose twagerageje, usibye umudepite wa polystirene, byagaragaye mugushiraho amashusho twakoresheje.Muhari umurima wa magneti, abadepite bakora imirongo ifite uburebure bujyanye nubwoko bwimiterere nimbaraga za magnetique (ni ukuvuga hafi no kugenda kwa magneti) .Nkuko bigaragara mumashusho ya 10, imirongo ya magneti iterwa na buri gice cya magneti. ibice byo guteranya no guhuza, hamwe nitsinda ryumugozi-nkimigendere bitewe nimbaraga zaho ziva mukarere kaho gakurura kandi kwanga kwizindi ngingo.
Igishushanyo cyerekana (a, b) gari ya moshi zakozwe imbere muri capillaries zuzuye amazi na (c, d) trachea yuzuye umwuka. Menya ko capillaries na trachea bidakururwa mubipimo.Panel (a) ikubiyemo kandi ibisobanuro byumudepite, bikubiyemo uduce twa Fe3O4 twateguwe mumirongo.
Iyo magnet yimuwe hejuru ya capillary, inguni yumugozi wibice yageze kumupaka ukomeye kuri MP3-5 irimo Fe3O4, nyuma yumugozi wigice ntukigume kumwanya wambere, ariko wimukiye hejuru yumwanya mushya.magnet.Iyi ngaruka birashoboka ko ishobora kubaho kuko hejuru yikirahure cya capillary cyoroshye kuburyo butemewe kugirango ibice bigende neza. MP6 (CombiMag) kwishyurwa hejuru, cyangwa ibintu bitwara ibintu byigenga byagize ingaruka kubushobozi bwabo bwo kwimuka. Itandukaniro ryishusho ryibice bya CombiMag naryo rifite intege nke, byerekana ko ayo mazi nuduce bishobora kuba bifite ubucucike busa bityo ntibishobora kugenda byoroshye. tekereza ko, mugihe magnesi zishobora gufata abadepite benshi banyura mumwanya wabigenewe, ntibishoboka ko magnesi zishobora gushingirwaho kugirango zimure ibice bya CombiMag hejuru ya trachea.Niyo mpamvu, twanzuye ko muri vivo LV-MP ubushakashatsi bugomba gukoresha imirima ya magnetiki ihagaze kugirango yibasire uturere tumwe na tumwe twigiti cyumuyaga.
Iyo ibice bitangiwe mumubiri, biragoye kubimenya murwego rwimiterere yumubiri wimibiri igenda, ariko ubushobozi bwo kubimenya bwongerewe imbaraga muguhindura magneti mu buryo butambitse hejuru ya trachea kugirango "wiggle" imirongo yabadepite.Nubwo amashusho yerekana ubuzima bushoboka, biroroshye gutahura urujya n'uruza mugihe inyamaswa zimaze kwicwa mubantu. kugeza mubushakashatsi bwa vitro, ibice ntibishobora gukururwa kuruhande rwa trachea muguhindura magnet.Iyi myanzuro ijyanye nuburyo urusenda rutwikiriye ubuso bwa trachea rusanzwe rutunganya uduce duto twahumetse, tukabifata mumitsi hanyuma bigakurwaho nuburyo bwo gukuraho mucociliary.
Twakekereje ko gukoresha magnesi kugirango bikururwe hejuru no munsi ya trachea (Igishusho 3b) bishobora kuvamo umurima umwe rukuruzi wa magneti, aho kuba umurego wa rukuruzi wibanze cyane kumwanya umwe, bikaba bishoboka ko habaho gukwirakwiza ibice bimwe.Nyamara, ubushakashatsi bwacu bwibanze ntabwo bwabonye ibimenyetso bifatika bishyigikira iki gitekerezo. ko ibice bibiri-magnet bidatezimbere cyane kugenzura kwibanze kwintego za MP, kandi ko imbaraga zikomeye za magnetique zavuyemo zigoye kubishyiraho, bigatuma ubu buryo budakorwa neza. Kimwe nuko, kwerekera magneti hejuru no kunyura muri trachea (Igishusho 3d) nayo ntiyigeze yongera umubare wibice byagumishijwe mukarere kashushanijwe. Bimwe muribi bipimo bya magnetique. 3a) ifatwa nkuburyo bworoshye kandi bunoze bwo gupima vivo.
Ubushakashatsi bwa LV-MP bwerekanye ko iyo LV vectors zahujwe na CombiMag hanyuma zigatangwa nyuma yo kwangirika kwumubiri imbere yumurima wa magneti, urwego rwa transduction rwiyongereye cyane muri trachea ugereranije nubugenzuzi.Bishingiye ku bushakashatsi bwerekana amashusho ya synchrotron hamwe n ibisubizo bya LacZ, bigaragara ko umurima wa magneti washoboye kurinda LV muri trachea kandi bikagabanya umubare wibice bya vectori bigahita byinjira mubitereko byinjira mu mitsi. trans-transduction, ingaruka ziterwa no gukingira indwara, hamwe nigiciro cyabatwara gene.Ni ngombwa, nkuko uwabikoze abivuga, CombiMag irashobora gukoreshwa ifatanije nubundi buryo bwo kohereza gene, harimo nizindi virusi (nka AAV) na acide nucleic.