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Ingirabuzimafatizo za Gene zo kuvura indwara y’ibihaha ya cystic fibrosis zigomba kwibasira inzira z’ubuhumekero kuko gutanga ibihaha mu buryo bwo ku ruhande nta nyungu zo kuvura. Ingufu zo gutanga virusi zifitanye isano itaziguye n’igihe cyo gutura kwa vector. Ariko, amazi yo gutanga nk’afite gene akwirakwira mu buryo busanzwe mu gihe cyo guhumekwa, kandi uduce two kuvura twose duhita dukurwa vuba binyuze mu gutwara mu mucociliary. Kongera igihe cyo gutura kwa gene mu nzira z’ubuhumekero ni ingenzi ariko biragoye kugeraho. Uduce twa magnetic twa Gene-conjugated Gene-conjugated Gene-conjugated Resistance Particles dushobora kwerekezwa ku buso bw’ubuhumekero bishobora kunoza uburyo bwo kugenzurwa mu karere. Bitewe n’imbogamizi zo kubona mu mubiri, imyitwarire y’uduce duto twa magnetic ku buso bw’ubuhumekero mu gihe hari imbaraga za magnetic zikoreshwa ntabwo byumvikana neza. Intego y’ubu bushakashatsi kwari ugukoresha synchrotron imaging kugira ngo hagaragazwe uburyo urukurikirane rw’uduce twa magnetic mu mubiri w’imbeba zatewe anesthetised rugenda kugira ngo hasuzumwe imiterere n’imiterere y’imyitwarire y’uduce twa magnetic mu mubiri w’imbeba. Hanyuma twasuzumye niba gutanga uduce twa magnetic twa lentiviral mu gihe hari imbaraga za magnetic byakongera ubushobozi bwo gutanga mu mubiri w’imbeba. trachea. Ishusho ya X-ray ya Synchrotron igaragaza imyitwarire y'uduce twa rukuruzi mu miyoboro ya rukuruzi idahagarara kandi igenda muri vitro na vivo. Uduce ntidushobora gukururwa byoroshye hejuru y'inzira y'umwuka ikora hakoreshejwe rukuruzi, ariko mu gihe cyo gutwara, ubwinshi bw'uduce tuba twinshi mu gice cy'ishusho aho imbaraga rukuruzi ziba zikomeye cyane. Ubushobozi bwo kwanduzanya uduce nabwo bwiyongereye inshuro esheshatu igihe uduce twa rukuruzi twa lentiviral twatangwaga imbere y'imbaraga rukuruzi. Ibi bisubizo byose hamwe bigaragaza ko uduce twa rukuruzi twa lentiviral n'imbaraga rukuruzi bishobora kuba uburyo bw'ingirakamaro bwo kunoza uburyo bwo kwita ku ngirabuzimafatizo no kongera urugero rw'uburyo umwuka ugenda mu mubiri.
Indwara ya cystic fibrosis (CF) iterwa n'ihindagurika rya gene imwe yitwa CFTR transmembrane conductance regulator (CFTR). Poroteyine ya CFTR ni umuyoboro wa iyoni uboneka mu turemangingo twinshi tw’impyiko mu mubiri wose, harimo n’inzira z’umwuka ziyobora, ahantu hanini hatera indwara ya CF. Ubusembwa bwa CFTR butuma amazi anyura mu buryo budasanzwe, bugatuma amazi asohoka mu kirere kandi bukagabanya ubujyakuzimu bw’urusobe rw’amazi yo mu kirere (ASL). Ibi kandi bibangamira ubushobozi bwa sisitemu yo gutwara mucociliary (MCT) bwo gukuraho uduce duhumekwa n’udukoko mu nzira z’umwuka. Intego yacu ni ugukora ubuvuzi bwa lentiviral (LV) kugira ngo butange kopi ikwiye ya gene ya CFTR no kunoza ubuzima bwa ASL, MCT, n’ibihaha, no gukomeza guteza imbere ikoranabuhanga rishya rishobora gupima ibi bipimo mu mubiri.
Vectors za LV ni zimwe mu zigomba kugaragara ku isonga mu kuvura gene y’umwuka wa CF, ahanini kuko zishobora gushyira burundu gene ivura mu turemangingo tw’umwuka (uturemangingo tw’umwuka). Ibi ni ingenzi kuko zishobora gusubiza amazi meza no gukuraho ururenda binyuze mu gutandukanya uturemangingo tw’umwuka dufitanye isano na CF, bigatanga inyungu z’ubuzima bwose. Vectors za LV zigomba kwerekezwa ku nzira y’umwuka iyobora, kuko ariho indwara y’ibihaha ya CF itangirira. Gutanga vector mu bihaha bishobora gutuma habaho ururenda rw’amaraso mu maraso, ariko ibi nta nyungu bigira muri CF. Ariko, amazi nk’ay’uturemangingo asanzwe yimukira mu maraso nyuma yo guhumekwa3,4 kandi uduce duvura twinjira vuba mu kanwa na MCT. Ingufu zo gutanga LV zifitanye isano itaziguye n’igihe vector iguma iruhande rw’uturemangingo kugira ngo itume uturemangingo twinjira - "igihe cyo gutura"5 - kigabanuka byoroshye bitewe n’umwuka usanzwe wo mu karere ndetse no gufata uturemangingo tw’amaraso mu maraso na MCT. Kuri CF, ubushobozi bwo kongera igihe cyo gutura kwa LV mu nzira y’umwuka ni ingenzi kugira ngo hagerwe ku rwego rwo hejuru rwo gutanga amashanyarazi muri aka gace, ariko bifite byari bigoye cyane.
Kugira ngo dutsinde iyi mbogamizi, turasaba ko uduce twa LV magnetic (MPs) dushobora gufasha mu buryo bubiri bwuzuzanya. Ubwa mbere, dushobora kuyoborwa hakoreshejwe magnetism kugera ku buso bw'umwuka kugira ngo tunoze uburyo bwo kwibandaho no gufasha uduce twa gene dutwara gene kuba mu gace k'umwuka wifuza; na ASL) kwimukira ku rwego rwa 6 rw'uturemangingo. MPs zakoreshejwe cyane nk'ibinyabiziga bitanga imiti iyo zifatanye n'udukoko, imiti ivura indwara za chimiotherapeutic, cyangwa utundi duce duto dufata ku ruhu rw'uturemangingo cyangwa tugafata ku ruhu rw'uturemangingo rukenewe hanyuma zikirundanya ahantu hafite kanseri mu gihe hari amashanyarazi adahindagurika. Ubuhanga bwa Magnetic mu kuvura kanseri 7. Ubundi buryo bwa "hyperthermal" bugamije gushyushya MPs iyo zihuye n'imbaraga za rukuruzi zizunguruka, bityo zikangiza uturemangingo twa kanseri. Ihame ryo guhindura imiterere ya rukuruzi, aho imbaraga za rukuruzi zikoreshwa nk'ikintu gihindura imiterere kugira ngo ADN yongere uburyo bwo kohereza ADN mu turemangingo, rikunze gukoreshwa muri vitro hakoreshejwe ubwoko butandukanye bwa vectors za gene zitari virusi na virusi ku miterere y'uturemangingo bigoye guhindura imiterere. Ingufu za magnetotransfection ya LV zaragaragajwe, aho gutanga LV-MPs muri vitro ku murongo w'uturemangingo tw'impyiko y'umuntu imbere y'umurima wa rukuruzi udahinduka, byongera ubushobozi bwo guhindura imiterere ku nshuro 186 ugereranije na vector ya LV yonyine. LV-MP yanakoreshejwe kuri moderi ya CF muri vitro, aho guhindura imiterere ya rukuruzi byongera LV mu miterere y'umwuka n'amazi inshuro 20 imbere y'amase ya CF10. Ariko, guhindura imiterere ya magnetotransfection mu mubiri w'uturemangingo byagaragaye ko bitaweho cyane kandi byasuzumwe gusa mu nyamaswa nke. inyigo11,12,13,14,15, cyane cyane mu bihaha16,17. Nyamara, amahirwe yo guterwa amashanyarazi mu buvuzi bw'ibihaha bya CF arasobanutse. Tan et al. (2020) bavuze ko "inyigo y'igihamya cy'uko amashanyarazi atangwa neza mu bihaha bizategura ingamba zo guhumeka za CFTR mu gihe kizaza kugira ngo habeho kunoza umusaruro w'ubuvuzi ku barwayi ba CF"6.
Imyitwarire y'uduce duto twa rukuruzi ku buso bw'imiyoboro y'umwuka mu gihe hari imbaraga rukuruzi zikoreshwa iragoye kuyireba no kuyiga, bityo ikaba idasobanutse neza. Mu zindi nyigo, twakoze uburyo bwa synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) kugira ngo tubone mu buryo butaziguye kandi dupime impinduka z'iminota mu mubiri mu bujyakuzimu bwa ASL18 n'imyitwarire ya MCT19,20 kugira ngo dupime neza amazi y'ubuso bw'imiyoboro ya gaze kandi dukoreshwe nk'ikimenyetso cya mbere cy'ubushobozi bwo kuvura. Byongeye kandi, uburyo bwacu bwo gusuzuma MCT bukoresha uduce twa 10–35 µm tw'umurambararo tugizwe na alumina cyangwa ikirahure kinini cya refractive nk'ibimenyetso bya MCT bigaragara hakoreshejwe PB-PCXI21. Ubwo buryo bwombi bukwiriye mu kwerekana ubwoko butandukanye bw'uduce, harimo na MP.
Bitewe n’uko ifite ubushobozi bwo kureba ahantu hatandukanye no mu gihe runaka, uburyo bwacu bwo gusesengura bwa PB-PCXI na MCT bukoreshwa mu gusesengura bwarakwiriye cyane mu gusuzuma imiterere n’imiterere y’imyitwarire y’uduce duto n’utw’ibice byinshi mu mubiri kugira ngo bidufashe gusobanukirwa no kunoza uburyo bwo gutanga gene ya MP. Uburyo dukoresha hano bukomoka ku bushakashatsi bwacu dukoresheje SPring-8 BL20B2 beamline, aho twabonye uburyo amazi agenda nyuma yo gutanga dose ya vector mu mazuru no mu bihaha by’imbeba kugira ngo bidufashe gusobanura imiterere yacu yo kugaragaza gene idahuye yagaragaye mu bushakashatsi bwacu ku nyamaswa bujyanye n’ingirabuzimafatizo 3,4.
Intego y'ubu bushakashatsi kwari ugukoresha synchrotron PB-PCXI kugira ngo hagaragazwe ingendo za MP mu mubiri mu gice cy'imbeba nzima. Ubu bushakashatsi bwa PB-PCXI bwagenewe gupima ubwoko butandukanye bwa MP, imbaraga za rukuruzi, n'aho ziherereye kugira ngo hamenyekane ingaruka zabyo ku migendekere ya MP. Twatekereje ko imbaraga rukuruzi zikoreshwa hanze zafasha MP yatanzwe kuguma cyangwa kwimukira mu gace kagenewe. Ubu bushakashatsi kandi bwatwemereye kumenya imiterere ya rukuruzi yongera umubare w'uduce twagumanye mu gice cy'imbeba nyuma yo gushyirwamo. Mu cyiciro cya kabiri cy'ubushakashatsi, twashatse gukoresha ubu buryo bwiza bwo kwerekana imiterere yo gukwirakwiza amashanyarazi ituruka ku gutanga LV-MP mu mubiri mu nzira y'umwuka y'imbeba, hashingiwe ku gitekerezo cy'uko gutanga LV-MP mu rwego rwo gukwirakwiza amashanyarazi mu nzira y'umwuka byatuma habaho kunoza imikorere ya LV.
Inyigo zose ku nyamaswa zakozwe hakurikijwe amabwiriza yemejwe na Kaminuza ya Adelaide (M-2019-060 na M-2020-022) na Komite Ishinzwe Imyitwarire y'inyamaswa ya SPring-8 Synchrotron. Igerageza ryakozwe hakurikijwe amabwiriza ya ARRIVE.
Ishusho yose ya X-ray yakorewe kuri BL20XU beamline kuri SPring-8 synchrotron mu Buyapani, hakoreshejwe uburyo busa n'ubwavuzwe mbere21,22. Muri make, agasanduku k'igerageza kari kari muri metero 245 uvuye ku mpeta yo kubika synchrotron. Intera yo kuva kuri detector kugeza kuri meta 0.6 ikoreshwa mu bushakashatsi bwo gufata amashusho y'uduce duto na metero 0.3 mu bushakashatsi bwo gufata amashusho y'ibintu mu mubiri kugira ngo haboneke ingaruka zo kunyuranya kw'ibyiciro. Hakoreshejwe ingufu z'imirasire ya monochromatic ya 25 keV. Amashusho yafashwe hakoreshejwe converter ya X-ray ifite ubushobozi bwo hejuru (SPring-8 BM3) ihujwe na detector ya sCMOS. Converter ihindura X-rays urumuri rugaragara ikoresheje scintillator ifite ubugari bwa 10 µm (Gd3Al2Ga3O12), hanyuma ijyanwa kuri sensor ya sCMOS ikoresheje objective ya mikorosikopi ya × 10 (NA 0.3). Detector ya sCMOS yari Orca-Flash4.0 (Hamamatsu Photonics, Ubuyapani) ifite ingano ya array ya Pikseli 2048 × 2048 n'ingano ya pikseli 6.5 × 6.5 µm. Iyi gahunda itanga ingano ya pikseli ya isotropic ingana na 0.51 µm n'aho umuntu ashobora kureba hafi 1.1 mm × 1.1 mm. Uburebure bwo kureba bwa 100 ms bwatoranyijwe kugira ngo hakorwe ikigereranyo cy'urusaku hagati y'ibimenyetso n'urusaku by'uduce twa rukuruzi imbere no hanze y'inzira y'umwuka mu gihe bigabanyije uburyo bwo guhumeka buterwa n'umwuka. Ku bushakashatsi bwakozwe mu mubiri, icyuma gifunga X-ray cyihuta cyashyizwe mu nzira ya X-ray kugira ngo kigabanye urugero rw'imirasire binyuze mu guhagarika urumuri rwa X-ray hagati y'aho umuntu ashobora guhura n'urusaku.
Iyi mashini itwara amashanyarazi ya LV ntiyigeze ikoreshwa mu bushakashatsi ubwo aribwo bwose bwo gufata amashusho bwa SPring-8 PB-PCXI kuko icyumba cyo gufata amashusho cya BL20XU kidafite icyemezo cy’ubuziranenge ku rwego rwa 2. Ahubwo, twahisemo ubwoko butandukanye bwa MP zizwi neza mu bacuruza ubucuruzi babiri—bafite ingano zitandukanye, ibikoresho, ingano y’icyuma, n’ikoreshwa—kubanza gusobanukirwa uburyo imbaraga za rukuruzi zigira ingaruka ku mikorere ya MP mu miyoboro y’ikirahure, hanyuma no mu nzira z’umwuka zirimo. ku buso. MP zifite ingano iri hagati ya 0.25 na 18 μm kandi zikozwe mu bikoresho bitandukanye (reba Imbonerahamwe ya 1), ariko imiterere ya buri cyitegererezo, harimo n'ingano y'uduce twa rukuruzi turi muri MP, ntabwo izwi. Dushingiye ku bushakashatsi bwacu bwa MCT bwagutse 19, 20, 21, 23, 24, twiteze ko MP ntoya nka 5 μm zishobora kugaragara ku buso bw'inzira y'umwuka, urugero nko gukuramo uduce dukurikiranye kugira ngo tubone uko MP igenda neza. MP imwe ifite 0.25 μm ni nto ugereranyije n'ubushobozi bw'igikoresho gifata amashusho, ariko PB-PCXI byitezwe ko izamenya itandukaniro ry'ingano yazo n'uburyo amazi yo hejuru ashyirwaho nyuma yo gushyirwaho.
Ingero za buri MP muri Table 1 zateguwe mu miyoboro y'ibirahuri ya 20 μl (Drummond Microcaps, PA, USA) ifite umurambararo w'imbere wa mm 0.63. Uduce twa corpuscular turaboneka mu mazi, mu gihe uduce twa CombiMag tuboneka mu mazi yihariye y'uruganda. Buri muyoboro wuzuyemo amazi (hafi 11 μl) hanyuma ushyirwa ku gikoresho cy'icyitegererezo (reba Ishusho ya 1). Uduce twa glass capillaries twashyizwe mu buryo butambitse ku cyiciro cy'icyitegererezo mu gasanduku k'ifoto, uko bikurikirana, maze dushyirwa ku nkengero z'amazi. Ingufu ya nickel shell rare earth neodymium iron boron (NdFeB) ya 19 mm (uburebure bwa mm 28) (N35, cat. no. LM1652, Jaycar Electronics, Ositaraliya) ifite magnetization ya 1.17 Tesla yashyizwe ku cyiciro gitandukanye cyo guhindura kugira ngo ihindure aho iherereye kure mu gihe cyo gufata amashusho. Gufata amashusho ya X-ray bitangira iyo magnet ishyizwe hafi ya mm 30 hejuru y'icyitegererezo, kandi amashusho aboneka ku gipimo cya frame 4 ku isegonda. Mu gihe cyo gufata amashusho, magnet yaregeranyijwe hafi y'ikirahuri. umuyoboro wa capillary (uri hafi mm 1) hanyuma uhindurirwa ku muyoboro kugira ngo harebwe ingaruka z'imbaraga z'umurima n'aho uherereye.
Ishusho yakozwe muri vitro irimo ingero za MP mu miyoboro y'ikirahure ku cyiciro cyo guhindura xy. Inzira y'imirasire ya X-ray irangwa n'umurongo utukura ufite utudomo.
Ubwo hamaze kugaragara uburyo MPs zigaragara mu buryo bwa vitro, itsinda ryazo ryapimwe mu buryo bwa vivo mu mbeba z’ingore zo mu bwoko bwa Wistar (zifite ~ ibyumweru 12, ~ 200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Ubuyapani), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Ubuyapani) na 4 mg/kg butorphanol (Vetorphale®, Meiji Seika) Imbeba zatewe ikinya hifashishijwe uruvange rwa Pharma), Ubuyapani) binyuze mu gutera ikinya imbere mu nda. Nyuma yo guterwa ikinya, zateguriwe gufata amashusho zikuraho ubwoya bukikije trachea, zishyiramo umuyoboro wa endotracheal (ET; 16 Ga iv cannula, Terumo BCT) hanyuma zigahagarara ku isahani y’ishusho yakozwe mu buryo bwihariye irimo agafuka gashyuha kugira ngo ubushyuhe bw’umubiri bukomeze 22. Hanyuma isahani y’ishusho yashyizwe ku cyiciro cyo guhindura icyitegererezo mu gasanduku k’ishusho ku nguni nto kugira ngo trachea ihuze neza mu ishusho ya X-ray, nk'uko bigaragara ku Ishusho ya 2a.
(a) Gushyiraho amashusho mu buryo bw'umubiri mu gasanduku k'amashusho ka SPring-8, inzira y'imirasire ya X-ray irangwa n'umurongo utukura ufite akadomo. (b, c) Gushyira magneti ku gice cy'imiyoboro y'amazi mu buryo bwa "trachea" byakozwe hifashishijwe kamera ebyiri za IP zishyizwe mu buryo bwa orthogonally. Ku ruhande rw'ibumoso rw'ishusho ya ecran, umurongo w'insinga ufata umutwe urashobora kugaragara, kandi "cannula" iri mu mwanya wayo mu muyoboro wa ET.
Sisitemu yo gupompa syringe igenzurwa kure (UMP2, World Precision Instruments, Sarasota, FL) ikoresheje syringe y'ikirahure ya 100 μl yahujwe n'umuyoboro wa PE10 (OD 0.61 mm, ID 0.28 mm) binyuze mu rushinge rwa Ga 30. Shyira akamenyetso kuri uyu muyoboro kugira ngo urebe neza ko umutwe uri mu mwanya ukwiye muri trachea mugihe ushyiramo umuyoboro wa ET. Hakoreshejwe micropompe, syringe plunger yakuweho mu gihe umutwe w'umuyoboro winjijwe mu gipimo cya MP kugira ngo gishyirwemo. Umuyoboro w'amazi washyizwe mu muyoboro wa endotracheal, ushyira umutwe mu gice gikomeye cy'imbaraga rukuruzi twari twiteze gukoresha. Gufata amashusho byagenzuwe hakoreshejwe icyuma gipima umwuka gihujwe n'agasanduku kacu gashingiye kuri Arduino, kandi ibimenyetso byose (urugero: ubushyuhe, guhumeka, gufungura/gufunga shutter no kubona amashusho) byafashwe hakoreshejwe Powerlab na LabChart (AD Instruments, Sydney, Ositaraliya) 22. Mu gihe cyo gufata amashusho Igihe agace kari katagerwaho, kamera ebyiri za IP (Panasonic BB-SC382) zashyizwe kuri dogere 90 kugeza byakoreshejwe mu kugenzura aho rukuruzi iherereye ugereranije n'aho trachea iherereye mu gihe cyo gufata amashusho (Ishusho ya 2b, c). Kugira ngo bigabanye uburyo bwo kugenda, habonetse ishusho imwe kuri buri mwuka mu gihe cy'amazi atemba mu mpera z'umugezi.
Isumaku ifatanye n'icyiciro cya kabiri gishobora kuba kiri kure y'aho amashusho aherereye. Ahantu hatandukanye ho gushyira isumaku n'imiterere byapimwe, harimo: Yashyizwe ku nguni ya dogere 30 hejuru ya trachea (imiterere igaragara ku Ishusho ya 2a na 3a); isumaku imwe hejuru y'inyamaswa n'indi hepfo, hamwe n'inkingi zigomba gukurura (Ishusho ya 3b); isumaku imwe hejuru y'inyamaswa n'indi hepfo, hamwe n'inkingi zigomba kwirukana (Ishusho ya 3c); na isumaku imwe hejuru kandi igororotse kuri trachea (Ishusho ya 3d). Iyo inyamaswa na sumaku bimaze gushyirwaho kandi MP igomba gupimwa imaze gushyirwa mu ipompo ya syringe, tanga dose ya 50 μl ku gipimo cya 4 μl/segonda mugihe ushakisha amashusho. Isumaku irajyanwa inyuma no inyuma cyangwa ku ruhande rwa trachea mugihe ukomeza kubona amashusho.
Imiterere ya rukuruzi yo gushushanya mu mubiri (a) rukuruzi imwe hejuru ya trachea ku nguni ya dogere 30, (b) rukuruzi ebyiri zishyirwaho kugira ngo zikure, (c) rukuruzi ebyiri zishyirwaho kugira ngo zigarure, (d) rukuruzi imwe hejuru kandi igororotse muri trachea. Uwarebaga yarebye hasi kuva mu kanwa kugera mu bihaha anyuze muri trachea, maze imirasire ya X-ray inyura ibumoso bw'imbeba igasohoka iburyo. Rukuruzi yimurirwa ku burebure bw'inzira y'umwuka cyangwa ibumoso n'iburyo hejuru ya trachea mu cyerekezo cy'imirasire ya X-ray.
Twashatse kandi kumenya uko uduce tw’imitsi tugaragara n’uko twitwara mu nzira z’ubuhumekero mu gihe nta guhumeka no gukora k’umutima bitera urujijo. Kubwibyo, mu mpera z’igihe cyo gufata amashusho, inyamaswa zishwe mu buryo bwa kimuntu kubera gukoresha pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~ 65 mg/kg ip). Hari inyamaswa zasigaye ku rubuga rwo gufata amashusho, kandi iyo guhumeka no gutera k’umutima byahagararaga, igikorwa cyo gufata amashusho cyasubiwemo, hongerwaho indi dose ya MP niba nta MP yagaragaraga ku buso bw’ubuhumekero.
Amashusho yabonetse yakosowe mu gice cy’ubutaka (flat-field) n’igice cy’umwijima (dark-field) hanyuma ateranywa muri filime (amafoto 20 ku isegonda; umuvuduko usanzwe wa 15-25 × bitewe n’umuvuduko w’ubuhumekero) hakoreshejwe inyandiko yihariye yanditswe muri MATLAB (R2020a, The Mathworks).
Inyigo zose zo gutanga jeni za LV zakorewe mu kigo cy’ubushakashatsi ku nyamaswa muri Kaminuza ya Adelaide, zigamije gukoresha ibyavuye mu igerageza rya SPring-8 kugira ngo harebwe niba gutanga jeni za LV-MP imbere y’umurima wa rukuruzi bishobora kongera ihererekanya rya jeni mu mubiri. Kugira ngo hasuzumwe ingaruka za MP n’umurima wa rukuruzi, havuwe amatsinda abiri y’inyamaswa: itsinda rimwe ryahawe LV-MP rifite rukuruzi, irindi itsinda rihabwa itsinda rigenzura rifite LV-MP ridafite rukuruzi.
Vekteri za LV zakozwe hakoreshejwe uburyo bwavuzwe mbere 25, 26. Vekteri ya LacZ igaragaza gene ya beta-galactosidase ikoreshwa na LPSV promoter (LV-LacZ), ikora umusaruro w'ubururu mu turemangingo twahinduwe, tugaragara imbere mu bice by'ibihaha no mu bice by'uturemangingo. Titration yakozwe mu turemangingo habarwa umubare w'uturemangingo twa LacZ positive hakoreshejwe hemocytometer kugira ngo harebwe titer muri TU / ml. Uturemangingo turabikwa kuri -80 °C, tugashonga mbere yo gukoreshwa, hanyuma tugahambirwa kuri CombiMag mu kuvanga ku gipimo cya 1:1 hanyuma tugashyirwa ku rubura nibura iminota 30 mbere yo gutanga.
Imbeba zisanzwe za Sprague Dawley (n = 3/itsinda, ~ 2-3 zatewe ikinya mu nda y'umura hamwe n'uruvange rwa 0.4 mg/kg medetomidine (Domitor, Ilium, Ositaraliya) na 60 mg/kg ketamine (Ilium, Ositaraliya) y'ukwezi kumwe) ip) hamwe no guterwa mu kanwa nta kubagwa hakoreshejwe cannula ya 16 Ga iv. Kugira ngo harebwe ko inyama z'umwuka wo mu nda zibona LV transduction, byakozwe hakoreshejwe uburyo bwacu bwo guterwa mu maraso, aho ubuso bw'umwuka wo mu nda bwasutswe ku buryo burambuye hakoreshejwe agasanduku k'insinga (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. Gutanga LV-MP mu maraso byakorewe mu kabati k'umutekano w'ibinyabuzima nyuma y'iminota 10 nyuma yo guterwa.
Imbaraga rukuruzi zakoreshejwe muri ubu bushakashatsi zakozwe mu buryo busa n’ubwakozwe mu isuzuma rya X-ray, hamwe na rukuruzi zimwe zari hejuru y’uruti rw’umuyoboro hakoreshejwe uduce twa distillation stent clips (Ishusho ya 4). Ingano ya 50 μl (2 × 25 μl aliquots) ya LV-MP yashyizwe muri trachea (n = inyamaswa 3) hakoreshejwe pipette irimo agace ka gel nkuko byasobanuwe mbere. Itsinda ry’abagenzuzi (n = inyamaswa 3) ryahawe LV-MP zimwe nta kurutiza. Nyuma yo kuyitera irangiye, cannula ikurwa mu muyoboro wa ET maze inyamaswa irakurwamo. Rutiza iguma aho iri mu mwanya wayo mu gihe cy’iminota 10, hanyuma irakurwaho. Imbeba zahawe dose ya meloxicam (1 ml/kg) (Ilium, Ositaraliya) hagakurikiraho gusimbuza ikinya hakoreshejwe ip ya 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Ositaraliya). Imbeba zafunzwe kandi zigakurikiranwa kugeza zikize burundu nyuma yo gusimbuza ikinya.
Igikoresho cyo gutanga LV-MP mu kabati k’umutekano w’ibinyabuzima. Urusobe rw’umukara woroheje rwa Luer rw’umuyoboro wa ET rugaragara ruturutse mu kanwa kandi umutwe wa gel w’umuyoboro ugaragara ku ifoto ushyirwa mu muyoboro wa ET kugeza ku burebure wifuza mu muyoboro wa trachea.
Nyuma y'icyumweru kimwe, inyamaswa zapfuye mu buryo busanzwe zihumeka CO2 100%, maze LacZ igaragara hakoreshejwe uburyo bwacu busanzwe bwo kuvura X-gal. Impeta eshatu zikomeye cyane zo mu bwoko bwa cartilaginous zakuweho kugira ngo hamenyekane ko ibyangiritse cyangwa amazi yakuwe mu miyoboro y'amaraso bitashyizwe mu isesengura. Buri trachea yaciwe mu buryo burambuye kugira ngo hakorwe ibice bibiri byo gusesengura, maze bishyirwa mu isahani irimo silicone rubber (Sylgard, Dow Inc) hakoreshejwe urushinge rwa Minutien (Fine Science Tools) kugira ngo harebwe ubuso bw'urumuri. Ikwirakwizwa n'imiterere y'uturemangingo twahinduwe byemejwe n'ifoto yo imbere hakoreshejwe mikorosikopi ya Nikon (SMZ1500) hakoreshejwe kamera ya DigiLite na porogaramu ya TCapture (Tucsen Photonics, Ubushinwa). Amashusho yabonetse ku gipimo cya 20x magnification (harimo n'ahantu henshi cyane ku bugari bwose bwa trachea), hamwe n'uburebure bwose bwa trachea bufatwa intambwe ku yindi, bishimangira ko buri shusho ihurirana bihagije kugira ngo habeho "kudoda". Amashusho yo kuri buri Nyuma yaho trachea yakusanyijwe mu ishusho imwe ivanze hakoreshejwe Image Composite Editor v2.0.3 (Microsoft Research) hakoreshejwe algorithme y'ingendo isanzwe. Uduce twa LacZ twerekana mu mashusho avanze ya trachea ya buri nyamaswa twapimwe hakoreshejwe script ya MATLAB yikora (R2020a, MathWorks) nk'uko byasobanuwe mbere, hakoreshejwe imiterere ya 0.35 < Hue < 0.58, Saturation > 0.15, na Value < 0.7. Mu gukurikirana imiterere y'uruti, mask yakorewe intoki muri GIMP v2.10.24 kuri buri shusho ivanze kugira ngo hamenyekane agace k'uruti no gukumira ko haboneka ibitari byo hanze y'uruti rw'uruti. Uduce twanduye twavuye mu mashusho yose avanze ya buri nyamaswa twakusanyijwe kugira ngo haboneke agace kose k'uruti rw'iyo nyamaswa. Agace kanduye kagabanyijwemo agace kose k'uruti kugira ngo habeho agace gasanzwe.
Buri trachea yashyizwe muri parafini hanyuma ibice bya μm 5 biracibwa. Ibice byashyizwemo ibara ry'umutuku wihuta mu gihe cy'iminota 5, kandi amashusho yabonetse hakoreshejwe mikorosikopi ya Nikon Eclipse E400, kamera ya DS-Fi3 na porogaramu yo gufata ibintu bya NIS (version 5.20.00).
Isesengura ryose ry’ibarurishamibare ryakozwe muri GraphPad Prism v9 (GraphPad Software, Inc.). Akamaro k’ibarurishamibare kashyizwe kuri p ≤ 0.05. Ubusanzwe bwemejwe hakoreshejwe ikizamini cya Shapiro-Wilk, kandi itandukaniro mu gusiga irangi rya LacZ ryasuzumwe hakoreshejwe ikizamini cya t kidashyizwe hamwe.
MP esheshatu zasobanuwe muri Table 1 zasuzumwe hakoreshejwe PCXI, kandi uko bigaragara bivugwa muri Table 2. MP ebyiri za polystyrene (MP1 na MP2; 18 μm na 0.25 μm, uko bikurikirana) ntizagaragaraga muri PCXI, ariko izindi ngero zari zizwi (ingero zigaragara ku Ishusho ya 5). MP3 na MP4 (10-15% Fe3O4; 0.25 μm na 0.9 μm, uko bikurikirana) ziragaragara gato. Nubwo harimo uduce duto twapimwe, MP5 (98% Fe3O4; 0.25 μm) ni yo yagaragaye cyane. Igicuruzwa cya CombiMag MP6 kiragoye kugitahura. Mu bihe byose, ubushobozi bwacu bwo kumenya MP bwarushijeho kwiyongera bitewe no guhindura magneti inyuma no inyuma ijyanye na capillary. Iyo magneti zavaga kuri capillary, uduce twagutse mu migozi miremire, ariko uko magneti zegerezaga n'imbaraga za magnetic field ziyongera, imigozi y'uduce yagabanutse uko uduce twimukiraga hejuru ya capillary. (reba Videwo y'inyongera S1: MP4), byongera ubucucike bw'uduce tw'ubuso. Ku rundi ruhande, iyo rukuruzi ikuwe mu gacupa k'imiyoboro, imbaraga z'umurima ziragabanuka maze MP zigasubiramo imigozi miremire iva hejuru y'agacupa k'imiyoboro (reba Videwo y'inyongera S2: MP4). Iyo rukuruzi ihagaritse kugenda, uduce dukomeza kugenda igihe gito nyuma yo kugera ku mwanya w'uburinganire. Uko MP igenda yerekeza hejuru y'agacupa k'imiyoboro no kure y'aho, uduce tw'ingufu dukunze gukurura imyanda mu mazi.
Uburyo MP igaragara muri PCXI buratandukanye cyane hagati y’ingero. (a) MP3, (b) MP4, (c) MP5 na (d) MP6. Amashusho yose agaragara hano yafashwe hakoreshejwe rukuruzi iri hafi mm 10 hejuru y’umuyoboro w’amaraso. Utuziga tunini tugaragara ni utubumbe tw’umwuka dufungiye mu miyoboro y’amaraso, bigaragaza neza imiterere y’umukara n’umweru y’ishusho y’itandukaniro ry’impinduka. Agasanduku gatukura karimo ubwiyongere bw’itandukaniro. Menya ko uburebure bw’ibishushanyo bya rukuruzi mu mashusho yose atari ubunini kandi bunini inshuro zigera ku 100 kuruta uko byagaragajwe.
Uko magneti ihindurwa ibumoso n'iburyo hejuru y'agace k'umuyoboro, inguni y'umugozi wa MP irahinduka kugira ngo ihuze na magneti (reba Ishusho ya 6), bityo bigasobanura imirongo y'umuyoboro wa rukuruzi. Kuri MP3-5, nyuma y'uko agace k'umuyoboro kageze ku nguni y'umupaka, uduce dukururwa hejuru y'agace k'umuyoboro. Ibi akenshi bituma MP zihurira mu matsinda manini hafi y'aho umuyoboro wa rukuruzi ukomeye cyane (reba Videwo y'inyongera S3: MP5). Ibi kandi bigaragara cyane iyo hafashwe amashusho hafi y'umusozo wa kapilari, bigatuma MP zihurira hamwe kandi zigashyira imbaraga ku murongo w'amazi n'umwuka. Uduce muri MP6, twari bigoye kubona kurusha MP3-5, ntitwakururwaga uko magneti yagiye ku gace k'umuyoboro, ahubwo imigozi ya MP yaratandukanye, bigatuma uduce tuba mu murima wo kureba (reba Videwo y'inyongera S4: MP6). Hari igihe, iyo umuyoboro wa rukuruzi wakoreshejwe wagabanukaga bitewe no kwimura magneti intera nini uvuye aho yafatiwe amashusho, MP zisigaye zamanukaga buhoro buhoro ku buso bwo hasi bw'umuyoboro bitewe n'imbaraga rukuruzi mu gihe ziguma mu mugozi (reba Videwo y'inyongera S5: MP3).
Inguni y'umugozi wa MP irahinduka uko rukuruzi ihindurirwa iburyo hejuru y'umuyoboro wa capillary. (a) MP3, (b) MP4, (c) MP5 na (d) MP6. Agasanduku gatukura karimo uburyo bwo kongera itandukaniro. Menya ko videwo z'inyongera zitanga amakuru kuko zigaragaza imiterere y'uduce tw'ingenzi n'amakuru ahindagurika adashobora kugaragara muri aya mashusho adahinduka.
Ibizamini byacu byagaragaje ko kwimura magneti buhoro buhoro ugana inyuma no hejuru ku gice cy'imiyoboro y'amaraso byoroshya ishusho ya MP mu rwego rwo kugenda mu buryo bugoye mu mubiri. Ikizamini cya "in vivo" nticyakozwe kuko amasaro ya polystyrene (MP1 na MP2) atagaragaraga mu gasanduku k'amaraso. Buri MPi imwe muri enye zasigaye yageragejwe mu mubiri hamwe n'umurongo muremure wa magneti washyizweho hejuru ya trachea ku nguni ya 30° kugera ku ruhande ruhagaze (reba Ishusho ya 2b na 3a), kuko ibi byatanze iminyururu miremire ya MP kandi byari ingirakamaro kurusha uko imiterere ya magneti yarangiye. MP3, MP4 na MP6 ntibyabonetse mu gice cy'imiyoboro y'amaraso cy'inyamaswa nzima. Iyo inzira z'umwuka z'imbeba zafatwaga amashusho nyuma y'uko inyamaswa zishwe nk'abantu, utwo duce twakomeje kugaragara nubwo hongewemo ingano y'inyongera hakoreshejwe pompe ya syringe. MP5 yari ifite icyuma kinini cyane kandi ni yo gace konyine kagaragaraga, bityo ikaba yarakoreshejwe mu gusuzuma no kugaragaza imyitwarire ya MP mu mubiri.
Gushyira magneti hejuru y'umuyoboro w'amaraso mu gihe cyo gutanga MP byatumye MP nyinshi, ariko atari zose, zishyirwa mu rwego rwo kureba. Uduce twinjira muri trachea tugaragara neza mu nyamaswa zatanzweho abantu. Ishusho ya 7 na Videwo y'inyongera S6: MP5 yerekana gufata vuba no guhuza uduce duto ku buso bwa trachea yo mu nda, bigaragaza ko MP zishobora kwerekezwa mu turere twifuzwa twa trachea. Mu gihe cyo gushakisha kure kuri trachea nyuma yo gutanga MP, bamwe mu MP babonetse hafi ya carina, bigaragaza ko imbaraga za magnetique zitari zihagije kugira ngo zikusanye kandi zigumane MP zose, kuko zatangwaga mu gace k'imbaraga za magnetique nyinshi mu gihe cyo gutanga amazi. Nyamara, ingano ya MP nyuma yo kubyara yari hejuru hirya no hino mu gace kafashweho amashusho, bigaragaza ko MP nyinshi zagumye mu turere tw'imiyoboro y'umwuka aho imbaraga za magnetique zakoreshejwe zari nyinshi.
Amashusho ya (a) mbere na (b) nyuma yo kohereza MP5 mu muhogo w'imbeba iherutse kwicwa, rukuruzi ishyizwe hejuru y'agace gafatiweho amashusho. Agace gafatiweho amashusho kari hagati y'impeta ebyiri za cartilage. Mbere yo kohereza MP, hari amazi mu nzira y'ubuhumekero. Agasanduku gatukura karimo uburyo bwo kongera ibara ry'uruhu. Aya mashusho ni ayo muri videwo yerekanywe muri Videwo y'inyongera S6: MP5.
Guhindura magneti ku gice cy'imiyoboro y'amazi mu mubiri byatumye umuyoboro wa MP uhindura inguni mu gice cy'imiyoboro y'amazi mu buryo busa n'ubugaragara mu miyoboro y'amazi (reba Ishusho ya 8 na Videwo y'inyongera S7: MP5). Ariko, mu bushakashatsi bwacu, imiyoboro ya MP ntiyashoboraga gukururwa ku gice cy'imiyoboro y'amazi nk'uko byashobokaga ku miyoboro y'amazi. Hari igihe umuyoboro wa MP urushaho kuba muremure uko magneti igenda ibumoso n'iburyo. Igishimishije ni uko twasanze kandi umugozi w'imiyoboro usa n'aho uhindura uburebure bw'urusobe rw'amazi yo hejuru iyo magneti yimuwe mu buryo burambuye ku gice cy'imiyoboro y'amazi, kandi ukaguka iyo magneti yimuwe hejuru y'umuyoboro w'amazi kandi umugozi w'imiyoboro y'amazi ukazunguruka ukagera ahantu hahagaze (reba Videwo y'inyongera S7). : MP5 kuri 0:09, hepfo iburyo). Imiterere y'ingendo yahindutse ubwo rukuruzi yashyirwaga hejuru ya trachea ku ruhande (ni ukuvuga ibumoso cyangwa iburyo bw'inyamaswa aho kuba ku burebure bwa trachea). Uduce twakomezaga kugaragara neza uko twagendaga, ariko igihe rukuruzi yakurwaga muri trachea, imitwe y'imigozi y'uduce yagaragaye (reba Videwo y'inyongera S8: MP5, guhera kuri 0:08). Ibi bihuye n'imyitwarire ya MP twabonye munsi y'imbaraga rukuruzi zikoreshwa mu gasanduku k'ikirahure.
Urugero rw'amashusho agaragaza MP5 mu muhogo w'imbeba irwaye anesthetize. (a) Ingufu zikoreshwa mu kubona amashusho ari hejuru n'ibumoso bw'umuhogo, hanyuma (b) nyuma yuko ingufu zimuriwe iburyo. Agasanduku gatukura karimo uburyo bwo kongera itandukaniro. Aya mashusho ni ayo muri videwo yerekanywe muri Videwo y'inyongera S7: MP5.
Ubwo inkingi ebyiri zashyirwaga mu cyerekezo cy’amajyaruguru-amajyepfo hejuru no munsi ya trachea (ni ukuvuga gukurura; Ishusho ya 3b), imiyoboro ya MP yagaragaraga miremire kandi yari iherereye ku ruhande rwa trachea aho kuba ku buso bw’umuyoboro w’inyuma (reba Videwo y’inyongera S9: MP5). Ariko, ubwinshi bw’uduce turi ahantu hamwe (ni ukuvuga ku buso bw’inyuma bwa trachea) ntibwagaragaye nyuma yo gutanga amazi igihe hakoreshwaga igikoresho cya magnete ebyiri, ibi bikaba bibaho iyo hakoreshejwe igikoresho cya magnete imwe. Hanyuma iyo magnete imwe yashyizweho kugira ngo isubize inyuma inkingi (Ishusho ya 3c), umubare w’uduce tugaragara mu ishami ry’ishusho ntabwo wasaga n’aho wiyongera nyuma yo gutanga. Imiterere y’imiterere yombi ya magnete ebyiri iragoye bitewe n’imbaraga nyinshi za magnete zikurura cyangwa zisunika magnete, uko zikurikirana. Imiterere yahinduwe ihinduka magnete imwe ijyanye n’inzira y’umwuka ariko inyura mu nzira y’umwuka kuri dogere 90 ku buryo imirongo y’ifasi yambukiranyaga urukuta rwa trachea mu buryo bukurikije umurongo (Ishusho ya 3d), icyerekezo cyagenewe kumenya niba uduce twinshi turi ku ruhande. urukuta rwashoboraga kugaragara. Ariko, muri ubu buryo, nta ngendo igaragara y’ubwiyongere bwa MP cyangwa ihindagurika rya magnet. Hashingiwe kuri ibi byose, imiterere y’icyerekezo cya magnet imwe, dogere 30 (Ishusho ya 3a) yatoranijwe mu bushakashatsi ku ngirabuzimafatizo za gene mu mubiri.
Iyo inyamaswa yafatwaga ifoto inshuro nyinshi nyuma yo kwica umuntu, kuba nta migendere y’ingingo iteye urujijo byasobanuraga ko imirongo mito kandi migufi y’uturemangingo yashoboraga kugaragara mu murima usobanutse neza w’interchondral, “ihindagurika” hakurikijwe imigendere y’ingufu. Nyamara, ntabwo irabona neza aho uturemangingo twa MP6 tugeze n’aho tugeze.
Igipimo cya LV-LacZ cyari 1.8 × 108 TU/ml, kandi nyuma yo kuvanga na CombiMag MP (MP6) kuri 1:1, inyamaswa zahawe dose ya 50 μl tracheal ya 9 × 107 TU/ml LV vehicle (ni ukuvuga 4.5 × 106 TU/rat). Muri ubu bushakashatsi, aho guhindura magnet mu gihe cyo kubyara, twashyize magnet mu mwanya umwe kugira ngo tumenye niba LV transduction (a) ishobora kunozwa ugereranije no gutanga vector mu gihe nta ngufu za magnet, na (b) ishobora kuba ishingiye ku ntego Uturemangingo tw’umwuka twoherezwa mu turere tw’inzira ya rukuruzi yo hejuru y’umwuka.
Kuba hari magnets ndetse n'ikoreshwa rya CombiMag hamwe na vectors za LV ntibyagaragaye ko bigira ingaruka mbi ku buzima bw'inyamaswa, kimwe n'uburyo bwacu bwo gutanga vectors za LV. Amashusho y'imbere y'agace k'imitsi yahuye n'ikibazo cy'ingufu (Ishusho y'inyongera ya 1) yagaragaje ko hari urwego rwo hejuru cyane rwo kwanduzanya mu itsinda ry'inyamaswa zavuwe na LV-MP igihe magnets yari ihari (Ishusho ya 9a). Hari ingano ntoya y'ubururu bwa LacZ mu itsinda ryagenzurwaga (Ishusho ya 9b). Ibarura ry'ahantu hasanzwe handuye X-Gal ryagaragaje ko gutanga LV-MP mu gihe hari imbaraga za rukuruzi byatanze umusaruro ugera kuri 6 (Ishusho ya 9c).
Urugero rw'amashusho y'ibice by'uruhererekane agaragaza uburyo bwo gukurura imirasire y'amaraso hakoreshejwe LV-MP (a) mu gihe hari imbaraga za rukuruzi na (b) mu gihe nta rukuruzi ifite. (c) Iterambere rikomeye mu mibare mu gace gasanzwe ka LacZ gakurura imirasire y'amaraso mu gihe hakoreshwa rukuruzi (*p = 0.029, t-test, n = 3 kuri buri tsinda, ikigereranyo ± SEM).
Ibice by’umutuku byihuta kandi bidafite aho biboze (urugero rugaragara ku Ishusho y’inyongera ya 2) byagaragaje ko uturemangingo twa LacZ turi mu buryo busa n’aho duherereye nkuko byatangajwe mbere.
Ikibazo cy'ingenzi ku buvuzi bw'uturemangingo tw'imiyoboro y'umwuka ni ugushyira neza aho uturemangingo tw'imiyoboro y'umwuka duherereye mu turere dukenewe no kugera ku rwego rwo hejuru rw'imikorere myiza mu bihaha bigenda mu gihe hari umwuka utembera neza kandi hari ururenda rukora. Ku barwayi ba LV bagenewe kuvura indwara ya CF yo mu mwuka, kongera igihe cy'imiyoboro y'imiyoboro y'umwuka mu nzira z'umwuka ziyobora byari intego itaraboneka kugeza ubu. Nkuko byagaragajwe na Castellani et al., gukoresha imbaraga za rukuruzi mu kunoza uburyo bwo gutanga ururenda bifite ibyiza ugereranije n'ubundi buryo bwo gutanga ururenda nka electroporation, kuko bishobora guhuza uburyo bworoshye, uburyo buhendutse, uburyo bwo gutanga ururenda, ubushobozi bwo kongera, no kugabanya igihe cyo gutanga ururenda, ndetse wenda n'urugero ruto rw'imiyoboro y'umwuka. Ariko, gushyira uduce twa rukuruzi mu mwuka mu buryo bw'umwuka mu buryo bw'umwuka mu buryo bw'umwuka hifashishijwe imbaraga za rukuruzi zo hanze ntibyigeze bisobanurwa, ndetse n'ubushobozi bw'ubu buryo ntibwagaragajwe mu buryo bw'umwuka mu buryo bw'umwuka mu buryo bw'umwuka muzima budafite ingaruka mbi.
Igerageza ryacu rya "in vitro synchrotron PCXI" ryagaragaje ko uduce twose twapimye, usibye polystyrene MP, twagaragaraga mu ishusho twakoresheje. Iyo hari imbaraga za rukuruzi, MP zikora imigozi ifite uburebure bujyanye n'ubwoko bw'uduce n'imbaraga za rukuruzi (ni ukuvuga kwegerana n'ingendo za rukuruzi). Nkuko bigaragara ku Ishusho ya 10, imigozi tubona ikorwa bitewe nuko buri gace kamwe gafite ingufu za rukuruzi kandi bigatera imbaraga za rukuruzi zako. Iyi migozi itandukanye ituma utundi duce dusa natwo twishyira hamwe kandi tugahuza, hamwe n'ingendo nk'iz'imigozi ziterwa n'imbaraga zo mu gace gaturuka ku mbaraga zikurura kandi ziteye ubwoba z'utundi duce.
Ishusho yerekana (a, b) uduce duto twakorewe mu mitsi yuzuye amazi na (c, d) trachea yuzuye umwuka. Menya ko imitsi n'imitsi bidashushanyijwe ku rugero. Agace (a) gafite kandi ibisobanuro bya MP, karimo uduce twa Fe3O4 dutondetse mu migozi.
Ubwo magnet yimurirwaga hejuru y’umuyoboro w’amashanyarazi, inguni y’umuyoboro w’amashanyarazi yageze ku rugero rw’ingenzi rwa MP3-5 irimo Fe3O4, nyuma yaho umuyoboro w’amashanyarazi ntiwongeye kuguma mu mwanya wayo wa mbere, ahubwo wimukiye ku buso bugana ku mwanya mushya. magnet. Iyi ngaruka ishobora kubaho kuko ubuso bw’umuyoboro w’ikirahure buroroshye bihagije kugira ngo bushobore kwemerera iyi ngendo kubaho. Igishimishije ni uko MP6 (CombiMag) itabyitwayemo muri ubu buryo, birashoboka ko ari uko uduce twari duto, twari dufite irangi ritandukanye cyangwa imbaraga zitandukanye z’ubuso, cyangwa se amazi atwara amashanyarazi yagize ingaruka ku bushobozi bwazo bwo kugenda. Itandukaniro ry’ishusho y’uduce twa CombiMag naryo rirakomeye, bigaragaza ko amazi n’uduce bishobora kugira ubucucike bumwe bityo ntibishobora kwerekeza hamwe byoroshye. Uduce dushobora kandi gufatirwa iyo magnet yihuta cyane, bigaragaza ko imbaraga z’umuyoboro w’amashanyarazi zidashobora guhora zitsinda ubushyamirane hagati y’uduce turi mu mazi, bigaragaza ko wenda bidatangaje ko imbaraga z’umuyoboro w’amashanyarazi n’intera iri hagati ya magnet n’agace kagenewe. Ni ingenzi cyane. Ukurikije hamwe, ibi bisubizo bigaragaza kandi ko, nubwo magneti zishobora gufata MP nyinshi zinyura mu gace kagenewe, ntabwo bishoboka ko magneti zishobora kwiringirwa. kugira ngo tujye twimura uduce twa CombiMag ku buso bwa trachea. Kubera iyo mpamvu, twanzuye ko inyigo za LV-MP muri vivo zigomba gukoresha imbaraga za rukuruzi zitajegajega kugira ngo zigere ku bice byihariye by'igiti cy'umwuka.
Iyo uduce duto twatanzwe mu mubiri, biragoye kubimenya mu rwego rw'imitsi y'umubiri igenda cyane, ariko ubushobozi bwo kubimenya bwarushijeho kwiyongera binyuze mu guhindura rukuruzi mu buryo butambitse hejuru y'umuyoboro w'amaraso kugira ngo "izunguze" imigozi ya MP. Nubwo gufotora mu buryo bw'imbonankubone bishoboka, biroroha kumenya uko uduce duto tugenda iyo inyamaswa imaze kwicwa nk'umuntu. Ubusanzwe igipimo cya MP cyari kinini cyane aho hantu igihe rukuruzi rwashyirwaga hejuru y'agace gafatiweho amashusho, nubwo uduce tumwe na tumwe twakunze kuboneka kure y'umuyoboro w'amaraso. Bitandukanye n'inyigo zakozwe muri vitro, uduce ntidushobora gukururwa ku muyoboro w'amaraso binyuze mu guhindura rukuruzi. Iki cyavumbuwe gihuye n'uburyo umuyoboro utwikira ubuso bwa muyoboro w'amaraso usanzwe utunganya uduce twahumekewe, ukadufata mu muyoboro w'amaraso hanyuma ugakurwamo n'uburyo bwo gusohora mu muyoboro w'amaraso.
Twatekereje ko gukoresha magnets mu gukurura hejuru no munsi ya trachea (Ishusho ya 3b) bishobora gutuma imbaraga rukuruzi zigira ingano imwe, aho kuba imbaraga rukuruzi ziba nyinshi cyane ahantu hamwe, bishobora gutuma ibice bimwe bikwirakwira. Ariko, ubushakashatsi bwacu bw'ibanze ntibwabonye ibimenyetso bifatika bishyigikira iki gitekerezo. Mu buryo nk'ubwo, gushyiraho magnets ebyiri zo kwirukana (Ishusho ya 3c) ntibyatumye ibice byinshi bishyirwa mu gace kafashwe amashusho. Ibi byavumbuwe bibiri bigaragaza ko gushyiraho magnets ebyiri bidateza imbere cyane igenzura rya MP, kandi ko imbaraga zikomeye rukuruzi zivamo zigoye gushyiraho, bigatuma ubu buryo budakora neza. Mu buryo nk'ubwo, kwerekeza magnets hejuru no mu trachea (Ishusho ya 3d) nabyo ntibyongereye umubare w'ibice biguma mu gace kafashwe amashusho. Bimwe muri ibi bipimo bishobora kudatera imbere kuko bituma imbaraga rukuruzi zigira ingano nke mu gace kafashwe amashusho. Kubwibyo, imiterere imwe ya magneti ya dogere 30 (Ishusho ya 3a) ifatwa nk'uburyo bworoshye kandi bwiza bwo gupima mu mubiri.
Inyigo ya LV-MP yagaragaje ko iyo vectors za LV zahujwe na CombiMag zikazanwa nyuma yo guhungabana kw'umubiri mu gihe hari imbaraga za rukuruzi, urugero rwa transduction rwiyongereye cyane mu muyoboro w'amaraso ugereranije n'ibipimo. Hashingiwe ku bushakashatsi bwa synchrotron imaging na LacZ, imbaraga za rukuruzi zashoboraga kubungabunga LV mu muyoboro w'amaraso no kugabanya umubare w'uduce twa vector twahitaga twinjira mu bihaha. Uko kunoza uburyo bwo kwibandaho bishobora gutuma habaho imikorere myiza mu gihe bigabanya titers zatanzwe, transduction idateganyijwe, ingaruka mbi zo kubyimba no kwirinda indwara, hamwe n'ikiguzi cyo gutwara gene. Icy'ingenzi, nk'uko uwakoze iyi gene abivuga, CombiMag ishobora gukoreshwa hamwe n'ubundi buryo bwo kohereza gene, harimo n'izindi vectors za virusi (nka AAV) na aside nucleic.