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Indwara ya gene yo kuvura fibrosis yo mu bihaha igomba kuba yibasiwe n'inzira ziyobora, kubera ko kwanduza ibihaha bya periferique nta ngaruka zo kuvura.Imikorere yo kwanduza virusi ifitanye isano itaziguye nigihe cyo gutura.Nyamara, amazi yo kubyara nkabatwara gene mubisanzwe akwirakwira muri alveoli mugihe cyo guhumeka, kandi uduce duto two kuvura muburyo ubwo aribwo bwose tuvanaho vuba na transport ya mucociliary.Kongera igihe cyo gutura abatwara gene mu myanya y'ubuhumekero ni ngombwa ariko biragoye kubigeraho.Imashini ya magnetiki itwara ibintu ishobora kwerekezwa hejuru yubuhumekero irashobora kunoza intego zakarere.Bitewe nibibazo bijyanye no gufata amashusho ya vivo, imyitwarire yutwo tuntu duto duto twa magnetique hejuru yumuyaga imbere yumurima wa magneti washyizweho ntabwo byumvikana neza.Icyari kigamijwe muri ubu bushakashatsi kwari ugukoresha amashusho ya synchrotron kugira ngo ugaragaze muri vivo urujya n'uruza rw'ibice bya magneti muri trachea y'imbeba zatewe aneste kugira ngo wige imbaraga n'imiterere y'imyitwarire y'ibice bimwe na byinshi muri vivo.Twasuzumye kandi niba itangwa rya lentiviral magnetique imbere yumurima wa magneti byongera imikorere ya transduction muri trachea yimbeba.Imashusho ya Synchrotron X-ray yerekana imyitwarire yibice bya magneti mumashanyarazi ihagaze kandi igenda muri vitro no muri vivo.Ibice ntibishobora gukururwa byoroshye hejuru yumuyaga uhumeka ukoresheje magnesi, ariko mugihe cyo gutwara, kubitsa byibanze murwego rwo kureba, aho imbaraga za rukuruzi zikomeye.Imikorere ya transduction nayo yariyongereye inshuro esheshatu mugihe uduce duto twa lentiviral magnetique twatanzwe imbere yumurima wa rukuruzi.Ufatiye hamwe, ibisubizo byerekana ko ibice bya magnetiki ya lentiviral hamwe na magnetique bishobora kuba inzira zingenzi zogutezimbere icyerekezo cya gene icyerekezo no kwanduza urwego rwumuyaga uyobora vivo.
Fibrosis ya Cystic (CF) iterwa no guhindagurika muri gene imwe yitwa CF transmembrane imyitwarire (CFTR).Poroteyine ya CFTR ni umuyoboro wa ion uboneka mu ngirabuzimafatizo nyinshi mu mubiri, harimo n'umuyaga uhumeka, ikibanza gikomeye mu gutera indwara ya fiboside ya cystic.Inenge ziri muri CFTR ziganisha ku gutwara amazi adasanzwe, kubura amazi hejuru yumuyaga, no kugabanuka kwamazi yo mu kirere (ASL).Irabangamira kandi ubushobozi bwubwikorezi bwa mucociliary transport (MCT) bwo gukuraho inzira zumuyaga uhumeka hamwe na virusi.Intego yacu ni uguteza imbere imiti ya Lentiviral (LV) kugirango itange kopi yukuri ya gene CFTR no guteza imbere ubuzima bwa ASL, MCT, nibihaha, no gukomeza guteza imbere tekinolojiya mishya ishobora gupima ibyo bipimo muri vivo1.
LV vectors ni umwe mu bakandida bayoboye ubuvuzi bwa cystic fibrosis, cyane cyane ko bushobora kwinjiza burundu gene ivura ingirabuzimafatizo zo mu kirere (selile stem selile).Ibi nibyingenzi kuko birashobora kugarura hydrasiyo isanzwe hamwe na mucus itandukanijwe mugutandukanya mumikorere ya selile ikora ingirabuzimafatizo yo mu kirere ifitanye isano na fibrosis ya cystic, bikavamo inyungu ubuzima bwawe bwose.LV vectors igomba kwerekezwa kumyuka ihumeka, kuko aha niho hatangirira uruhare mubihaha muri CF.Gutanga vector byimbitse mu bihaha bishobora kuviramo kwandura alveolar, ariko ibi nta ngaruka zo kuvura muri fiboside ya cystic.Nyamara, amazi nkabatwara gene mubisanzwe yimukira muri alveoli iyo ihumeka nyuma yo kubyara3,4 kandi uduce duto two kuvura birukanwa vuba mumyanya yo mu kanwa na MCTs.Imikorere ya LV transduction ifitanye isano itaziguye nuburebure bwigihe vector ikomeza kuba hafi yingirabuzimafatizo kugirango yemere gufata selile - “igihe cyo gutura” 5 igabanywa byoroshye n’umuyaga usanzwe wo mu karere kimwe no gufata hamwe na mucus na MCT.Kuri fibrosis ya cystic, ubushobozi bwo kongera igihe cyo gutura LV mumihanda ni ngombwa kugirango umuntu agere kuri transduction murwego rwo hejuru, ariko kugeza ubu biragoye.
Kugira ngo dutsinde iyi nzitizi, turasaba ko LV magnetique (MPs) ishobora gufasha muburyo bubiri bwuzuzanya.Ubwa mbere, barashobora kuyoborwa na rukuruzi hejuru yumuyaga kugirango barusheho gutera imbere no gufasha ibice bitwara gene kuba ahantu heza h'umwuka;6. kuba amashanyarazi ahamye.Imirasire ya magnetiki yo kuvura kanseri 7. Ubundi buryo bwa "hyperthermic" bugamije kwica selile yibibyimba ushyushya abadepite iyo bahuye nimirima ya magneti.Ihame ryo kwanduza magnetiki, aho umurima wa rukuruzi ukoreshwa nkumukozi wo kwanduza ADN mu ngirabuzimafatizo, ukunze gukoreshwa muri vitro ukoresheje urwego rutandukanye rwa virusi na virusi kugira ngo imirongo igoye itoroshye. ..Imikorere ya LV magnetotransfection hamwe no gutanga MP MP muri vitro mumurongo ngengabuzima wa epitelium ya bronchial yumuntu imbere yumurima wa magnetiki uhagaze, byongera imikorere ya transduction inshuro 186 ugereranije na VV yonyine.LV MT yanakoreshejwe muburyo bwa vitro ya fibrosis ya cystic, aho kwanduza magnetique byongereye LV kwanduza mumico yimiterere yimiterere yimiterere yikirere ku kigero cya 20 imbere ya fibrosis spicum10.Nyamara, muri vivo organ magnetotransfection yitabiriwe cyane kandi yasuzumwe gusa mubushakashatsi buke bwinyamanswa11,12,13,14,15, cyane cyane mubihaha16,17.Nyamara, amahirwe yo kwanduza magnetique mu kuvura ibihaha muri fiboside ya cystic arasobanutse.Tan n'abandi..
Imyitwarire ya magnetique ntoya hejuru yinzira yubuhumekero imbere yumurima wa magneti washyizweho biragoye kubyiyumvisha no kwiga, kubwibyo ntibisobanutse neza.Muyindi nyigo, twateje imbere Synchrotron Kwamamaza Bishingiye ku Cyiciro Cyerekana Itandukaniro X-Ray Imashusho (PB-PCXI) uburyo bwo gufata amashusho adatera no kugereranya umunota mu mpinduka za vivo mu burebure bwa ASL18 n’imyitwarire ya MCT19, 20 kugirango bapime mu buryo butaziguye umuyoboro wa gazi hejuru. kandi ikoreshwa nkigipimo cyo kuvura hakiri kare.Mubyongeyeho, uburyo bwacu bwo gutanga amanota MCT bukoresha ibice 10-35 µm bya diametre bigizwe na alumina cyangwa ikirahure cyinshi cyerekana ibirahure nkibimenyetso bya MCT bigaragara hamwe na PB-PCXI21.Ubwo buryo bwombi burakwiriye gushushanya ubwoko butandukanye, harimo n'abadepite.
Bitewe nuburebure buri hejuru nigihe gito, ASL na MCT ishingiye kuri PB-PCXI ikwiranye neza no kwiga imbaraga nuburyo bwimyitwarire yibice bimwe na byinshi muri vivo kugirango bidufashe kumva no kunoza uburyo bwo gutanga gene ya MP.Uburyo dukoresha hano bushingiye kubushakashatsi bwacu dukoresheje urumuri rwa SPring-8 BL20B2, aho twerekanaga uburyo amazi agenda nyuma yo gutanga urugero rwa vectori ya dummy mumyuka yizuru na pulmonary yumuyaga yimbeba kugirango bidufashe gusobanura imiterere ya gene itandukanye. muri gen.ubushakashatsi bwinyamanswa hamwe nuwitwaye dose ya 3.4.
Icyari kigamijwe muri ubu bushakashatsi kwari ugukoresha synchrotron ya PB-PCXI kugirango ugaragaze amashusho ya vivo yimikorere yuruhererekane rwabadepite muri trachea yimbeba nzima.Ubu bushakashatsi bwerekana amashusho ya PB-PCXI bwateguwe kugirango bugerageze urukurikirane rwa MP, imbaraga za magnetique, hamwe n’ahantu kugirango hamenyekane ingaruka zabyo ku myigaragambyo ya MP.Twatekereje ko umurima wa magneti wo hanze uzafasha MF yatanzwe kuguma cyangwa kwimukira ahabigenewe.Ubu bushakashatsi kandi bwadushoboje kumenya ibishushanyo bya magneti byerekana umubare munini wibice bisigaye muri trachea nyuma yo kubitsa.Mu cyiciro cya kabiri cy’ubushakashatsi, twari tugamije gukoresha ubu buryo bwiza kugira ngo twerekane uburyo bwo guhererekanya ibintu biva mu gutanga vivo yo gutanga abadepite ba LV mu nzira y’imbeba, twibwira ko gutanga abadepite ba LV mu rwego rwo guhumeka inzira byavamo mu kongera LV transduction neza..
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 ibyifuzo bya ARRIVE.
Amashusho yose ya x-ray yafatiwe kumurongo wa BL20XU kuri synchrotron ya SPring-8 mubuyapani ukoresheje setup isa niyasobanuwe mbere 21,22.Muri make, agasanduku k'igerageza kari m 245 uvuye kumpeta ya synchrotron.Intera-to-detector intera ya 0,6 m ikoreshwa mubushakashatsi bwerekana amashusho na 0.3 m mubushakashatsi bwa vivo bwerekana amashusho kugirango habeho ingaruka zinyuranye.Ikoreshwa rya monochromatic rifite ingufu za 25 keV ryakoreshejwe.Amashusho yabonetse hifashishijwe icyerekezo kinini cya X-ray transducer (SPring-8 BM3) ihujwe na deteri ya sCMOS.Transducer ihindura X-imirasire yumucyo ugaragara ukoresheje 10 µm yuburebure bwa scintillator (Gd3Al2Ga3O12), hanyuma ikoherezwa kuri sensor ya sCMOS ikoresheje intego ya microscope × 10 (NA 0.3).Disikete ya sCMOS yari Orca-Flash4.0 (Hamamatsu Photonics, Ubuyapani) ifite ubunini bwa 2048 × 2048 pigiseli hamwe na pigiseli mbisi ingana na 6.5 × 6.5 µm.Igenamiterere ritanga isotropic nziza ya pigiseli ingana na 0.51 µm n'umurima wo kureba hafi mm 1,1 mm × 1,1 mm.Igihe cyo kumara ms 100 cyatoranijwe kugirango hongerwe ibimenyetso byerekana urusaku-urusaku rwibice bya magnetiki imbere no hanze yumuyaga mugihe hagabanijwe ibihangano byatewe no guhumeka.Kuberako mubushakashatsi bwa vivo, icyuma cyihuta cya X-ray cyashyizwe munzira ya X-ray kugirango igabanye igipimo cyimirasire muguhagarika urumuri rwa X-hagati.
Itangazamakuru rya LV ntabwo ryakoreshejwe mubushakashatsi bwerekana amashusho ya SPring-8 PB-PCXI kuko icyumba cyerekana amashusho BL20XU ntabwo cyemewe na Biosafety Urwego rwa 2.Ahubwo, twahisemo urutonde rwabadepite barangwa neza nabacuruzi babiri bacuruza ibicuruzwa bitandukanye, ibikoresho, ibyuma bya fer, hamwe nibisabwa, - ubanza kugirango twumve uburyo imirima ya rukuruzi igira ingaruka kumyigaragambyo yabadepite muri capillaries, hanyuma hanyuma ubuzima bwo mu kirere.hejuru.Ingano yumudepite iratandukanye kuva 0.25 kugeza 18 µm kandi ikozwe mubikoresho bitandukanye (reba Imbonerahamwe 1), ariko ibigize buri cyitegererezo, harimo nubunini bwa magnetique muri MP, ntibizwi.Dushingiye ku bushakashatsi bwagutse bwa MCT 19, 20, 21, 23, 24, turateganya ko abadepite bagera kuri 5 µm bashobora kugaragara hejuru yumuhanda wa tracheal, urugero, mugukuramo ibice bikurikiranye kugirango tubone neza imikorere y’abadepite.Umudepite umwe wa 0,25 µm ni ntoya kuruta gukemura ibyuma bifata amashusho, ariko biteganijwe ko PB-PCXI izamenya itandukaniro ryayo ryinshi hamwe nigikorwa cyamazi yo hejuru bashyiramo nyuma yo kubitsa.
Ingero kuri buri Mudepite kumeza.1 yateguwe muri 20 μl capillaries (Drummond Microcaps, PA, USA) ifite diameter y'imbere ya mm 0,63.Ibice bya Corpuscular biboneka mumazi, mugihe ibice bya CombiMag biboneka mumazi nyirizina.Buri muyoboro wuzuye igice cyuzuye amazi (hafi 11 µl) ugashyirwa kuri sample (reba Ishusho 1).Ikirahuri capillaries yashyizwe mu buryo butambitse kuri stade mu cyumba cyerekana amashusho, hanyuma igashyirwa ku nkombe z’amazi.Dimetero 19 mm z'uburebure (mm 28 z'uburebure) nikel-shell magnet ikozwe mubutaka budasanzwe, neodymium, fer na boron (NdFeB) (N35, injangwe no. LM1652, Jaycar Electronics, Ositaraliya) hamwe na 1.17 T yashyizwe kuri a gutandukanya imbonerahamwe kugirango ugere kure Guhindura umwanya wawe mugihe cyo gutanga.Ishusho ya X-ray itangira iyo magnet ihagaze hafi mm 30 hejuru yicyitegererezo kandi amashusho aboneka kumurongo 4 kumasegonda.Mugihe cyo gufata amashusho, magneti yazanwe hafi yikirahure capillary (ku ntera ya mm 1) hanyuma yimukira kumuyoboro kugirango asuzume ingaruka zimbaraga zumurima nu mwanya.
Muri vitro yerekana amashusho arimo MP yintangarugero mubirahuri bya capillaries murwego rwo guhindura icyitegererezo cya xy.Inzira ya X-ray irangwa numurongo utukura.
Bimaze gushyirwaho muri vitro igaragara y’abadepite, igice cyabyo cyapimwe muri vivo ku mbeba zo mu bwoko bw’inyamanswa Wistar albino (~ ibyumweru 12, ~ 200 g).Medetomidine 0,24 mg / kg (Domitor®, Zenoaq, Ubuyapani), midazolam 3,2 mg / kg (Dormicum®, Astellas Pharma, Ubuyapani) na butorphanol 4 mg / kg (Vetorphale®, Meiji Seika).Imbeba zatewe aneste hamwe na Pharma (Ubuyapani) bivanze no guterwa intraperitoneal.Nyuma yo gutera anesthesia, bari biteguye gushushanya bakuramo ubwoya buzengurutse trachea, bashyiramo umuyoboro wa endotracheal (ET; 16 Ga urumogi rwitwa kanseri, Terumo BCT), hanyuma ubahagarika mumwanya wa supine kumasahani yakozwe mumashusho yabigenewe arimo umufuka wubushyuhe. kugumana ubushyuhe bwumubiri.22. Isahani yerekana amashusho noneho yometse kumurongo wicyitegererezo mumasanduku yerekana amashusho kumurongo muto kugirango uhuze trachea itambitse kumashusho ya x-ray nkuko bigaragara mumashusho 2a.
..Kuruhande rwibumoso rwishusho kuri ecran, urashobora kubona umugozi winsinga ufashe umutwe hamwe na cannula yo gutanga yashyizwe imbere muri tube ya ET.
Sisitemu ya pompe ya pompe igenzurwa na kure (UMP2, World Precision Instruments, Sarasota, FL) ikoresheje inshinge ya 100 µl ikirahuri cyahujwe no kuvoma PE10 (0,61 mm OD, 0.28 mm ID) ukoresheje urushinge rwa 30 Ga.Shyira umuyoboro kugirango umenye neza ko inama iri mumwanya ukwiye muri trachea mugihe winjizamo umuyoboro wa endotracheal.Hifashishijwe micropump, plunger ya syringe yakuweho maze isonga yigituba yibizwa mucyitegererezo cya MP kugirango gitangwe.Umuyoboro wapakiye wapakiye noneho winjijwe mumiyoboro ya endotracheal, ushyira isonga mugice gikomeye cyumuteguro wa magneti.Kugura amashusho byagenzuwe hakoreshejwe icyuma gihumeka gihujwe nagasanduku kacu gashingiye kuri Arduino, kandi ibimenyetso byose (urugero, ubushyuhe, guhumeka, gufunga / gufunga, no kubona amashusho) byanditswe hakoreshejwe Powerlab na LabChart (AD Instruments, Sydney, Ositaraliya) 22 Iyo Ishusho Iyo inzu itabonetse, kamera ebyiri za IP (Panasonic BB-SC382) zashyizwe kuri 90 ° hagati yazo kandi zikoreshwa mugucunga umwanya wa magneti ugereranije na trachea mugihe cyo gufata amashusho (Ishusho 2b, c).Kugabanya ibihangano byerekana, ishusho imwe kumyuka yabonetse mugihe cyimyanya yubuhumekero.
Magnet yometse kumurongo wa kabiri, ushobora kuba uri kure yumubiri wumubiri.Imyanya itandukanye n'ibishushanyo bya magneti byageragejwe, harimo: byashyizwe ku nguni ya 30 ° hejuru ya trachea (ibishushanyo bigaragara mu gishushanyo cya 2a na 3a);rukuruzi imwe hejuru yinyamaswa indi hepfo, hamwe ninkingi zashyizweho kugirango zikururwe (Ishusho 3b)., rukuruzi imwe hejuru yinyamaswa nimwe hepfo, hamwe ninkingi zashyizweho kugirango zange (Ishusho 3c), hamwe na rukuruzi imwe hejuru kandi itandukanijwe na trachea (Ishusho 3d).Nyuma yo gushiraho inyamaswa na magneti no gupakira umudepite uri kugeragezwa muri pompe ya syringe, tanga urugero rwa 50 µl ku gipimo cya 4 µl / sec umaze kubona amashusho.Magnet noneho yimurwa inyuma cyangwa hejuru ya trachea mugihe ikomeje kubona amashusho.
Ibikoresho bya magneti kubishusho ya vivo (a) rukuruzi imwe hejuru ya trachea kumpande ya 30 °, (b) magnesi ebyiri zagenewe gukurura, (c) magnesi ebyiri zagenewe kwangwa, (d) rukuruzi imwe hejuru na perpendicular kuri trachea.Indorerezi yarebye hasi kuva mu kanwa yerekeza mu bihaha inyuze muri trachea maze urumuri rwa X-runyura mu ruhande rw'ibumoso rw'imbeba maze rusohoka iburyo.Magnet yaba yimuwe muburebure bwumuhanda cyangwa ibumoso niburyo hejuru ya trachea yerekeza kumurongo wa X-ray.
Twashatse kandi kumenya neza imyitwarire yimyitwarire yumuyaga mugihe hatabayeho kuvanga guhumeka nigipimo cyumutima.Kubwibyo, igihe cyo gufata amashusho kirangiye, inyamanswa zarahawe abantu kubera kunywa inzoga nyinshi za pentobarbital (Somnopentyl, Pitman-Moore, Washington Crossing, Amerika; ~ 65 mg / kg ip).Inyamaswa zimwe zasigaye kurubuga rwo gufata amashusho, hanyuma nyuma yo guhagarika guhumeka no gutera umutima, inzira yo gufata amashusho yarasubiwemo, yongeraho urugero rwumudepite niba nta mudepite wagaragaye hejuru yumuyaga.
Amashusho yavuyemo yakosowe kumurima uringaniye kandi wijimye hanyuma akoranyirizwa muri firime (amakaramu 20 kumasegonda; 15-25 × umuvuduko usanzwe bitewe nigipimo cyo guhumeka) ukoresheje inyandiko yihariye yanditse muri MATLAB (R2020a, Imibare).
Ubushakashatsi bwose bwakozwe ku itangwa rya virusi ya LV bwakorewe muri kaminuza ya Adelaide Laboratory Laboratwari y’inyamaswa kandi bugamije gukoresha ibyavuye mu bushakashatsi bwa SPring-8 kugira ngo harebwe niba itangwa rya LV-MP imbere y’umuriro wa rukuruzi rishobora kuzamura ihererekanyabubasha muri vivo. .Kugirango hamenyekane ingaruka za MF na magnetique, havuwe amatsinda abiri yinyamaswa: itsinda rimwe ryatewe LV MF hamwe na magnet, naho irindi tsinda ryatewe nitsinda rishinzwe kugenzura hamwe na LV MF nta rukuruzi.
LV gene vectors yakozwe hakoreshejwe uburyo bwasobanuwe mbere 25, 26.Indwara ya LacZ igaragaza gene ya beta-galactosidase ya kirimbuzi itwarwa na porotokoro ya MPSV (LV-LacZ), itanga umusaruro wubururu mu ngirabuzimafatizo, bigaragara ku bice no mu bice bigize ibihaha.Titration yakorewe mumico ya selile mukubara intoki umubare wa selile nziza ya LacZ ukoresheje hemocytometero kugirango ubare titer muri TU / ml.Abatwara ibintu bibikwa kuri -80 ° C, bikonjeshwa mbere yo kubikoresha, kandi bigahuzwa na CombiMag bivanga 1: 1 hanyuma bigashyirwa ku rubura byibuze iminota 30 mbere yo kubyara.
Imbeba zisanzwe za Dawley (n = 3 / itsinda, ~ 2-3 anesthetised ip hamwe nuruvange rwa 0.4mg / kg medetomidine (Domitor, Ilium, Ositaraliya) na 60mg / kg ketamine (Ilium, Ositaraliya) ukwezi kumwe) ip ) gutera inshinge no kubaga umunwa urumogi hamwe na 16 Ga ya kanseri.Kugirango tumenye neza ko inzira yo mu kirere ya tracheal yakira LV transduction, byasabwaga gukoresha protocole yacu yasobanuwe mbere aho uburyo bwo guhumeka bwa tracheal bwakoreshwaga mu cyuma hamwe nigitebo cyinsinga (N-Circle, ikuramo amabuye ya nitinol idafite inama NTSE-022115) -UDH, Teka Ubuvuzi, Amerika) 30 p28.Noneho, nyuma yiminota 10 nyuma yo kwangirika muri minisiteri y’ibinyabuzima, hakozwe ubuyobozi bwa tracheal ya LV-MP.
Umwanya wa magneti wakoreshejwe muri ubu bushakashatsi washyizweho kimwe nubushakashatsi bwakozwe na vivo x-ray, hamwe na magnesi zimwe zifatiye kuri trachea hamwe na stent clamps (Ifoto 4).Ingano ya 50 µl (2 x 25 µl aliquots) ya LV-MP yagejejwe kuri trachea (n = inyamaswa 3) ikoresheje umuyoboro wa geli nkuko byasobanuwe mbere.Itsinda rishinzwe kugenzura (n = inyamaswa 3) ryakiriye LV-MP imwe idakoresheje magneti.Nyuma yo kurangiza gushiramo, urumogi ruvanwa mu muyoboro wa endotracheal hanyuma inyamaswa ikongerwa.Magnet iguma muminota 10 mbere yo gukurwaho.Imbeba zafashwe mu buryo bwihuse hamwe na meloxicam (1 ml / kg) (Ilium, Ositaraliya) hanyuma hakurikiraho gukuramo anesthesia no gutera intraperitoneal inshinge 1 mg / kg atipamazole hydrochloride (Antisedan, Zoetis, Ositaraliya).Imbeba zabitswe neza kandi zirubahirizwa kugeza zatewe neza na anesthesia.
Igikoresho cyo gutanga LV-MP muri guverenema yumutekano wibinyabuzima.Urashobora kubona ko urumuri rwerurutse rwa Luer-gufunga urumuri rwa ET ruva mu kanwa, kandi inama ya gel pipette yerekanwe ku gishushanyo yinjizwa mu muyoboro wa ET kugera ku bujyakuzimu bwifuzwa muri trachea.
Icyumweru kimwe nyuma yubuyobozi bwa LV-MP, inyamaswa zatambwe abantu guterwa no guhumeka 100% CO2 kandi imvugo ya LacZ yasuzumwe dukoresheje uburyo busanzwe bwa X-gal.Impeta eshatu za caudal karitsiye zavanyweho kugirango harebwe niba ibyangiritse byangiritse cyangwa kubika amazi bitewe no gushyira imiyoboro ya endotracheal bitazashyirwa mubisesengura.Buri trachea yaciwe mu burebure kugirango ibone ibice bibiri byo gusesengura hanyuma ishyirwa mu gikombe kirimo reberi ya silicone (Sylgard, Dow Inc) ukoresheje urushinge rwa Minutien (ibikoresho byiza bya siyansi) kugirango ubone ishusho yumucyo.Ikwirakwizwa n'imiterere ya selile yahinduwe byemejwe no gufotora imbere ukoresheje microscope ya Nikon (SMZ1500) hamwe na kamera ya DigiLite na software ya TCapture (Tucsen Photonics, Ubushinwa).Amashusho yabonetse kuri 20x yo gukuza (harimo igenamigambi ntarengwa ry'ubugari bwuzuye bwa trachea), hamwe n'uburebure bwose bwa trachea bwerekanwe intambwe ku yindi, butanga guhuzagurika bihagije hagati ya buri shusho kugirango amashusho "adoda".Amashusho yo muri buri trachea yahise ahuzwa mumashusho imwe akomatanya ukoresheje Composite Image Editor verisiyo ya 2.0.3 (Ubushakashatsi bwa Microsoft) ukoresheje algorithm ya planar. Agace kerekana imvugo ya LacZ mumashusho ya tracheal igizwe na buri nyamaswa yagereranijwe hifashishijwe inyandiko ya MATLAB yikora (R2020a, MathWorks) nkuko byasobanuwe mbere28, ukoresheje igenamiterere rya 0.35 <Hue <0.58, Saturation> 0.15, nagaciro <0.7. Agace kerekana imvugo ya LacZ mumashusho ya tracheal igizwe na buri nyamaswa yagereranijwe hifashishijwe inyandiko ya MATLAB yikora (R2020a, MathWorks) nkuko byasobanuwe mbere28, ukoresheje igenamiterere rya 0.35 <Hue <0.58, Saturation> 0.15, nagaciro <0.7. Площадь ларесЗии LacZ в совирных изображениях трахеи от kaждого животного б бар количественно определена с использованием аататизированного черакат MATLAB (R2020a, MathWorks), как описов , насщенность> 0,15 и значение <0 , 7. Agace kerekana imvugo ya LacZ mumashusho ya tracheal igizwe na buri nyamaswa yagereranijwe hifashishijwe inyandiko ya MATLAB ikora (R2020a, MathWorks) nkuko byasobanuwe mbere 28 ukoresheje igenamiterere rya 0.350.15 n'agaciro <0 .7.如前所述 ， 使用 自动 MATLAB 脚本 （R2020a ， MathWorks） 对 来自 每只 动物 的 气管 复合 图像 的 ac LacZ 表达 区域 进行 量化 ， 使用 0.35 <色调 <0.58 、 饱和 度> 0.15 和 值 <0.7 的 设置。。如 前所 述 ， 自动 自动 Matlab 脚本 （（r2020a work Imibare） 来自 每 只 的 气管 图像 图像 的 的 的 使用 35 35 0.35 <色调 <0.58 、> 0.15 和 值 <0.7 的 。。。。。 HIP Области кресрессии LacZ на соверных изображениях трахеи kaждого животного количественно определяли с использованием автоматизированного черака MATLAB (R2020a, MathWorks), как описано роте асыщенность> 0,15 и значение <0,7 . Ibice byerekana imvugo ya LacZ kumashusho yibice bya trachea ya buri nyamaswa byagereranijwe hifashishijwe inyandiko ya MATLAB yikora (R2020a, MathWorks) nkuko byasobanuwe mbere ukoresheje igenamiterere rya 0.35 <hue <0.58, kwiyuzuza> 0.15 nagaciro <0.7.Mugukurikirana ibice bigize tissue muri GIMP v2.10.24, hakozwe mask yakozwe nintoki kuri buri shusho igizwe kugirango igaragaze agace kama tissue kandi ikumire ikintu icyo ari cyo cyose kitagaragara hanze yumubiri wa tracheal.Ibice byanditseho amashusho yose yibumbiye muri buri nyamaswa byegeranijwe kugirango bitange ubuso bwuzuye kuri iyo nyamaswa.Ahantu hasize irangi noneho hagabanijwe nubuso bwuzuye bwa mask kugirango ubone ahantu hasanzwe.
Buri trachea yashyizwe muri paraffin igabanywa 5 µm z'ubugari.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 yo gufata (verisiyo 5.20.00).
Isesengura ryibarurishamibare ryose ryakozwe muri GraphPad Prism v9 (GraphPad Software, Inc.).Ubusobanuro bwibarurishamibare bwashyizwe kuri p ≤ 0.05.Ubusanzwe bwageragejwe hakoreshejwe ikizamini cya Shapiro-Wilk kandi itandukaniro rya LacZ ryasuzumwe hifashishijwe t-test idakorewe.
Abadepite batandatu basobanuwe mu mbonerahamwe ya 1 basuzumwe na PCXI, kandi ibiboneka bisobanurwa mu mbonerahamwe ya 2. Abadepite babiri ba polystirene (MP1 na MP2; 18 µm na 0,25 µm) ntibagaragaye na PCXI, ariko ingero zisigaye zishobora kumenyekana. (ingero zerekanwa mu gishushanyo cya 5).MP3 na MP4 biragaragara cyane (10-15% Fe3O4; 0,25 µm na 0,9 µm).Nubwo MP5 (98% Fe3O4; 0,25 µm) yarimo uduce duto duto twapimwe, niyo yagaragaye cyane.Ibicuruzwa bya CombiMag MP6 biragoye kubitandukanya.Mubibazo byose, ubushobozi bwacu bwo kumenya MF bwarushijeho kuba bwiza twimura magnet imbere ninyuma ihwanye na capillary.Mugihe magnesi yimukiye kure ya capillary, ibice byakuwe muminyururu miremire, ariko mugihe magnesi yegeraga kandi imbaraga za magnetique zikagenda ziyongera, ingoyi zingingo zagabanutse uko uduce twimukiye hejuru yubuso bwa capillary (reba Video yinyongera S1 : MP4), kongera ubwinshi bwibice hejuru.Ku rundi ruhande, iyo magneti akuwe muri capillary, imbaraga zumurima ziragabanuka kandi abadepite bongera guhinduranya iminyururu ndende iva hejuru ya capillary (reba Video yinyongera S2: MP4).Nyuma ya rukuruzi ihagaritse kugenda, ibice bikomeza kugenda mugihe runaka nyuma yo kugera kumwanya uringaniye.Mugihe umudepite agenda yerekeza kure yubuso bwa capillary, ibice bya magneti bikunda gukuramo imyanda mumazi.
Kugaragara kwa MP munsi ya PCXI biratandukanye cyane hagati yintangarugero.(a) MP3, (b) MP4, (c) MP5 na (d) MP6.Amashusho yose yerekanwe hano yafashwe hamwe na rukuruzi ihagaze hafi mm 10 hejuru ya capillary.Inziga nini zigaragara ni umwuka mwinshi wafashwe muri capillaries, werekana neza ibiranga umukara n'umweru biranga ishusho itandukanye.Agasanduku gatukura kerekana ubunini bwongera itandukaniro.Menya ko diameter yumuzunguruko wa magneti mumibare yose itagomba gupimwa kandi irikubye inshuro 100 kurenza iyerekanwe.
Nkuko rukuruzi igenda ibumoso n'iburyo hejuru ya capillary, inguni yumugozi wa MP ihinduka kugirango ihuze na rukuruzi (reba Ishusho ya 6), bityo igasobanura imirongo yumurongo wa rukuruzi.Kuri MP3-5, nyuma ya chord igeze ku mbibi zinjira, ibice bikurura hejuru ya capillary.Ibi akenshi bivamo abadepite bahurira mumatsinda manini hafi yumurima wa magneti ukomeye (reba Video yinyongera S3: MP5).Ibi kandi bigaragarira cyane cyane mugihe amashusho yegereye iherezo rya capillary, bigatuma umudepite yegeranya kandi akibanda kumazi-mwuka.Ibice biri muri MP6, byari bigoye gutandukanya kurusha ibyo muri MP3-5, ntibyigeze bikurura igihe magneti yagendaga hejuru ya capillary, ariko imirongo ya MP yatandukanijwe, hasigara ibice (reba Video y'inyongera S4: MP6).Rimwe na rimwe, iyo umurima wa magnetiki washyizweho wagabanutse mukwimura magneti intera ndende kurubuga rwa mashusho, abadepite bose basigaye bamanuka buhoro buhoro hejuru yubutaka bwumuvuduko ukabije, basigara mumurongo (reba Video yinyongera S5: MP3) .
Inguni yumugozi wa MP ihinduka nkuko magnet yimuka iburyo hejuru ya capillary.(a) MP3, (b) MP4, (c) MP5 na (d) MP6.Agasanduku gatukura kerekana ubunini bwongera itandukaniro.Nyamuneka menya ko videwo yinyongera igamije gutanga amakuru nkuko igaragaza imiterere yingirakamaro namakuru yingirakamaro adashobora kugaragara muri aya mashusho ahamye.
Ibizamini byacu byerekanye ko kwimura magneti inyuma no kugenda buhoro buhoro kuri trachea byoroha kubona amashusho ya MF murwego rwo kugenda muri vivo.Oya muri vivo ibizamini byakozwe kuko amasaro ya polystirene (MP1 na MP2) ntabwo yagaragaye muri capillary.Buri kimwe muri bine MF isigaye cyapimwe muri vivo hamwe na axe ndende ya magneti ihagaze hejuru ya trachea ku nguni ya 30 ° kugera kuri vertical (reba Ishusho 2b na 3a), kuko ibyo byaviriyemo iminyururu ndende ya MF kandi ikora neza kuruta rukuruzi..iboneza ryarangiye.MP3, MP4 na MP6 ntabwo byabonetse muri trachea yinyamaswa nzima.Iyo wiyumvishaga inzira y'ubuhumekero y'imbeba nyuma yo kwica inyamaswa abantu, ibice byakomeje kutagaragara kabone niyo byongerwagaho amajwi hakoreshejwe pompe ya syringe.MP5 yari ifite ibice byinshi bya okiside ya fer kandi niyo yonyine yagaragaye, bityo yakoreshejwe mugusuzuma no kuranga imyitwarire yabadepite muri vivo.
Gushyira magnet hejuru ya trachea mugihe cyo kwinjiza MF byavuyemo benshi, ariko sibyose, MF yibanze murwego rwo kureba.Inzira ya tracheal yinjira mubice bigaragara neza mubikoko byabantu.Igicapo 7 hamwe na Video yinyongera S6: MP5 yerekana gufata magneti byihuse no guhuza ibice hejuru yumusemburo wa ventrale, byerekana ko abadepite bashobora kwibasirwa ahantu bifuza trachea.Iyo ushakishije kure cyane ya trachea nyuma yo gutanga MF, wasangaga MF zimwe zisanga hafi ya karina, ibyo bikaba byerekana imbaraga zumurima wa magneti zidahagije zo gukusanya no gufata MF zose, kubera ko zanyujijwe mukarere k’imbaraga nini za magneti mugihe cyo kuyobora amazi.inzira.Icyakora, abadepite nyuma yo kubyara bari hejuru y’ishusho, byerekana ko abadepite benshi bagumye mu turere tw’umuyaga aho imbaraga za rukuruzi zikoreshwa zari nyinshi.
Amashusho ya (a) mbere na (b) nyuma yo gutanga MP5 muri trachea yimbeba iherutse guterwa hamwe na rukuruzi yashyizwe hejuru yishusho.Agace kerekanwe kari hagati yimpeta ebyiri.Hano hari amazi mu kirere mbere yuko umudepite atangwa.Agasanduku gatukura kerekana ubunini bwongera itandukaniro.Aya mashusho yakuwe kuri videwo igaragara muri S6: MP5 Yinyongera.
Kwimura magnet kuri trachea muri vivo byaviriyemo impinduka kumpande zumunyururu wa MP hejuru yumuyaga, bisa nkibigaragara muri capillaries (reba Ishusho ya 8 na Video yinyongera S7: MP5).Ariko, mubushakashatsi bwacu, abadepite ntibashoboraga gukururwa hejuru yinzira zubuhumekero nzima, nkuko capillaries yabikora.Rimwe na rimwe, urunigi rw'abadepite rurambura uko rukuruzi igenda ibumoso n'iburyo.Igishimishije, twasanze kandi urunigi rw'uduce duhindura ubujyakuzimu bw'ubuso bw'amazi iyo rukuruzi yimuwe igihe kirekire kuri trachea, kandi ikaguka iyo rukuruzi yimuwe hejuru kandi urunigi rukaba ruzunguruka rukaba ruhagaze (reba Video y'inyongera S7).: MP5 saa 0:09, hepfo iburyo).Imiterere yimikorere yarahindutse mugihe magnet yimuwe kuruhande hejuru ya trachea (nukuvuga ibumoso cyangwa iburyo bwinyamaswa, aho kuba muburebure bwa trachea).Ibice byari bikigaragara neza mugihe cyagendaga, ariko mugihe magneti yakuwe muri trachea, inama zumugozi wibice zaragaragaye (reba Video yinyongera S8: MP5, guhera 0:08).Ibi biremeranya nimyitwarire igaragara yumurima wa magneti munsi yumurimo wa magneti ukoreshwa mumirahuri ya capillary.
Icyitegererezo cyerekana MP5 muri trachea yimbeba nzima.(a) Imashini ikoreshwa kugirango ibone amashusho hejuru no ibumoso bwa trachea, hanyuma (b) nyuma yo kwimura rukuruzi iburyo.Agasanduku gatukura kerekana ubunini bwongera itandukaniro.Aya mashusho akomoka kuri videwo igaragara muri S7′s Yinyongera: MP5.
Iyo nkingi zombi zahujwe mu cyerekezo cyo mu majyaruguru-y'amajyepfo hejuru no munsi ya trachea (ni ukuvuga gukurura; Ishusho 3b), inanga z'abadepite zagaragaye igihe kirekire kandi zari ku rukuta rw'uruhande rwa trachea aho kuba hejuru ya dorsal ya trachea (reba Umugereka).Video S9: MP5).Nyamara, kwibumbira hamwe kwinshi mubice bimwe (ni ukuvuga hejuru yubutaka bwa trachea) ntibyamenyekanye nyuma yubuyobozi bwamazi hakoreshejwe ibikoresho bibiri bya magneti, bikunze kugaragara hamwe nigikoresho kimwe cya rukuruzi.Hanyuma, mugihe rukuruzi imwe yashizweho kugirango isubize inkingi zinyuranye (Igicapo 3c), umubare wibice bigaragara murwego rwo kureba ntiwiyongereye nyuma yo kubyara.Gushiraho ibice bibiri bya magneti biragoye kubera imbaraga za magneti ndende zikurura cyangwa zisunika magnesi.Imiterere noneho yahinduwe kuri magneti imwe ihwanye nu mwuka ariko inyura mu kirere ku mpande ya dogere 90 ku buryo imirongo y’ingufu yambukije urukuta rwa tracheal orthogonally (Ishusho 3d), icyerekezo kigamije kumenya niba bishoboka guteranya ibice kuri urukuta rw'uruhande.kubahirizwa.Nyamara, muri iyi miterere, ntihariho kumenyekana kwegeranya MF cyangwa kugenda kwa magneti.Ukurikije ibisubizo byose, iboneza rifite rukuruzi imwe hamwe nicyerekezo cya dogere 30 byatoranijwe kubushakashatsi bwa vivo bwabatwara gene (Ishusho 3a).
Iyo inyamanswa yashushanijwe inshuro nyinshi ako kanya nyuma yo gutambwa kubantu, kutagira urujya n'uruza rwimitsi byasobanuraga ko imirongo yoroheje, ngufi ishobora gutahurwa mumurima usobanutse hagati yimiterere itandukanye, 'kunyeganyega' ukurikije icyerekezo cya magneti.reba neza kuboneka no kugenda kwa MP6 ibice.
Titer ya LV-LacZ yari 1.8 x 108 IU / mL, hanyuma imaze kuvanga 1: 1 na MP CombiMag MP (MP6), inyamaswa zatewe inshinge 50 µl za dose ya tracheal ya 9 x 107 IU / ml yimodoka ya LV (ni ukuvuga 4.5 x 106 TU / imbeba).).).Muri ubu bushakashatsi, aho kwimura magneti mugihe cyakazi, twashyizeho magneti mumwanya umwe kugirango tumenye niba transduction ya LV ishobora (a) kunonosorwa ugereranije no gutanga vector mugihe hatabayeho umurima wa rukuruzi, na (b) niba inzira yumuyaga ishobora kwibanda.Ingirabuzimafatizo zihererekanyirizwa mu gice cya magnetiki zigenewe imyanya y'ubuhumekero yo 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.Amashusho yimbere yakarere ka tracheal yatewe no gukanika imashini (Ishusho yinyongera. 1) yerekanaga ko itsinda ryavuwe na LV-MP ryagize urwego rwinshi rwo kwanduza imbere ya rukuruzi (Ishusho 9a).Gusa umubare muto wubururu LacZ irangi yari ihari mumatsinda yo kugenzura (Igicapo 9b).Umubare w’uturere dusanzwe twerekanwe na X-Gal werekanye ko imiyoborere ya LV-MP imbere yumurima wa rukuruzi yatumye habaho iterambere ryikubye inshuro 6 (Ishusho 9c).
Urugero rwamashusho yibintu byerekana tracheal transduction hamwe na LV-MP (a) imbere yumurima wa magneti na (b) mugihe magneti adahari..
Ibice bitagira aho bibogamiye bitukura (urugero rwerekanwe ku gishushanyo cya 2) byerekanaga ko selile zifite LacZ zari zihari murugero rumwe kandi ahantu hamwe nkuko byavuzwe mbere.
Ikibazo cy'ingutu mu kuvura indwara ya gene yo mu kirere gikomeje kuba ahantu nyaburanga h’ibice bitwara abantu mu nyungu no kugera ku rwego rwo hejuru rwogukwirakwiza neza mu bihaha bigendanwa imbere y’umwuka uhumeka neza.Ku batwara LV bagenewe kuvura indwara zubuhumekero muri fiboside ya cystic, kongera igihe cyo gutura ibice byabatwara mumihanda ihumeka kugeza ubu byari intego itagerwaho.Nkuko byagaragajwe na Castellani n'abandi, gukoresha imirima ya magneti mu kuzamura transduction bifite ibyiza kurenza ubundi buryo bwo gutanga gene nka amashanyarazi kuko bishobora guhuza ubworoherane, ubukungu, gutanga aho biherereye, kongera imikorere, hamwe nigihe gito cyo gukora.kandi birashoboka igipimo cyo hasi cyimodoka10.Nyamara, muri vivo gushira hamwe nimyitwarire yibice bya magnetique mumuyaga uhumeka imbaraga za rukuruzi zo hanze ntabwo byigeze bisobanurwa, kandi mubyukuri ubushobozi bwubu buryo bwo kongera imiterere ya gene mumyuka mibi itagaragara ntabwo bwerekanwe muri vivo.
Ubushakashatsi bwakozwe na vitro kuri synchrotron ya PCXI bwerekanye ko ibice byose twagerageje, usibye MP polystirene, byagaragaye mumashusho twakoresheje.Imbere yumurima wa magneti, imirima ya magneti ikora imirongo, uburebure bwayo bujyanye nubwoko bwibice n'imbaraga z'umurima wa magneti (ni ukuvuga hafi no kugenda kwa rukuruzi).Nkuko bigaragara ku gishushanyo cya 10, imirongo tureba irashirwaho uko buri gice kigizwe na magnetique kandi kigatera umurego wacyo wa magneti.Iyi mirima itandukanye itera ibindi bice bisa gukusanya no guhuza hamwe nitsinda ryimigozi yitsinda bitewe nimbaraga zaho ziva mumbaraga zaho zo gukurura no kwanga ibindi bice.
Igishushanyo cyerekana (a, b) iminyururu yibice bigize imbere ya capillaries yuzuye amazi na (c, d) trachea yuzuye umwuka.Menya ko capillaries na trachea bidashushanyije kurwego.Ikibaho (a) kirimo kandi ibisobanuro bya MF irimo ibice bya Fe3O4 bitondekanye muminyururu.
Iyo rukuruzi yimukiye hejuru ya capillary, inguni yumugozi wibice yageze kumurongo wambere kuri MP3-5 irimo Fe3O4, nyuma yumugozi wuduce ntukiguma mumwanya wambere, ahubwo wimukiye hejuru yubuso ujya mumwanya mushya.rukuruzi.Izi ngaruka zishobora kubaho kubera ko hejuru yikirahuri capillary yoroshye bihagije kugirango iyi mikorere ibeho.Igishimishije, MP6 (CombiMag) ntabwo yitwaye gutya, wenda kubera ko ibice byari bito, byari bifite igifuniko gitandukanye cyangwa cyashizwe hejuru, cyangwa amazi yikwirakwiza nyirizina yagize ingaruka kubushobozi bwabo bwo kugenda.Itandukaniro riri mu ishusho y’ibice bya CombiMag naryo rifite intege nke, byerekana ko amazi n’ibice bishobora kugira ubucucike bumwe bityo ntibishobora kugenda byoroshye.Ibice birashobora kandi kwizirika mugihe rukuruzi rwihuta cyane, byerekana ko imbaraga zumurima wa magneti zidashobora gutsinda buri gihe guterana hagati yibice biri mumazi, byerekana ko imbaraga zumurima wa magneti hamwe nintera iri hagati ya magneti n'ahantu hagenewe bitagomba kuza nka a gutungurwa.ngombwa.Ibisubizo birerekana kandi ko nubwo magnesi zishobora gufata microparticles nyinshi zinyura ahantu hagenewe, ntibishoboka ko magnesi zishobora gushingirwaho kugirango zimure ibice bya CombiMag hejuru ya trachea.Rero, twanzuye ko muri vivo LV MF ubushakashatsi bugomba gukoresha imirima ihagaze kugirango igere ku gice cyihariye cyigiti cyumuyaga.
Iyo ibice bimaze gushyikirizwa umubiri, biragoye kubimenya murwego rwimikorere igoye yumubiri, ariko ubushobozi bwabo bwo gutahura bwarushijeho kuba bwiza bwo kwimura magneti mu buryo butambitse hejuru ya trachea kugirango "uzunguze" imirya yabadepite.Mugihe amashusho nyayo ashoboka, biroroshye gutahura uduce duto nyuma yinyamaswa zimaze kwicwa.Ubusanzwe abadepite bari hejuru cyane aha hantu mugihe rukuruzi rwashyizwe hejuru yerekana amashusho, nubwo uduce tumwe na tumwe wasangaga munsi ya trachea.Bitandukanye nubushakashatsi bwa vitro, ibice ntibishobora gukururwa na trachea nukugenda kwa rukuruzi.Ubu bushakashatsi burahuye nuburyo ururenda rutwikiriye ubuso bwa trachea rusanzwe rutunganya uduce duto twahumetse, tukawufata mu mucyo hanyuma tukawukuraho binyuze mu buryo bwo gukuraho muco-ciliary.
Twakekereje ko gukoresha magnesi hejuru no munsi ya trachea kugirango bikururwe (Igishusho 3b) bishobora kuvamo umurima umwe rukuruzi wa magneti, aho kuba umurima wa rukuruzi wibanze cyane kumwanya umwe, bikaba byavamo gukwirakwiza ibice bimwe..Nyamara, ubushakashatsi bwibanze ntabwo bwabonye ibimenyetso bifatika bishyigikira iyi hypothesis.Mu buryo nk'ubwo, gushiraho ama magneti kugirango yange (Igishusho 3c) ntabwo byaviriyemo ibice byinshi gutura mumashusho.Ibyavuye mu bushakashatsi byombi byerekana ko uburyo bubiri bwa magneti budatera imbere cyane kugenzura aho abadepite berekana, kandi ko imbaraga za rukuruzi zavuyemo zigoye kuyihuza, bigatuma ubu buryo budakorwa neza.Mu buryo nk'ubwo, kwerekeza kuri magneti hejuru no hakurya ya trachea (Igishusho 3d) nabyo ntabwo byongereye umubare wibice bisigaye mumashusho.Bimwe muribi bikoresho bindi ntibishobora kugerwaho kuko bivamo kugabanuka kwingufu za magnetique mumwanya wo kubitsa.Rero, iboneza rya rukuruzi imwe kuri dogere 30 (Igishusho 3a) bifatwa nkibyoroshye kandi byiza muburyo bwo gupima vivo.
Ubushakashatsi bwa LV-MP bwerekanye ko iyo LV vectors zahujwe na CombiMag hanyuma zigatangwa nyuma yo guhungabana kumubiri imbere yumurima wa rukuruzi, urwego rwa transduction rwiyongereye cyane muri trachea ugereranije nubugenzuzi.Ukurikije ubushakashatsi bwerekana amashusho ya synchrotron hamwe nibisubizo bya LacZ, umurima wa magneti wasaga nkuwashoboye kugumana LV muri trachea no kugabanya umubare wibice bya vector byahise byinjira mubihaha.Iterambere nk'iryo rishobora kuganisha ku gukora neza mugihe ugabanije titeri yatanzwe, transduction itagenewe, ingaruka ziterwa no gukingira indwara, hamwe nigiciro cyo kohereza gene.Icy'ingenzi, ukurikije uwabikoze, CombiMag irashobora gukoreshwa hamwe nubundi buryo bwo kohereza gene, harimo nizindi virusi (nka AAV) na acide nucleic.