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Ama-gene vectors okwelapha i-pulmonary cystic fibrosis kufanele aqondiswe emigwaqweni yomoya ehambisa umoya, njengoba ukudluliswa kwamaphaphu e-peripheral akunawo umphumela wokwelapha.Ukusebenza kahle kokudluliselwa kwegciwane kuhlobene ngokuqondile nesikhathi sokuhlala komthwali.Kodwa-ke, uketshezi lokulethwa olufana nabathwali bezakhi zofuzo ngokwemvelo basakazeka ku-alveoli ngesikhathi sokuhogela, futhi izinhlayiya zokwelapha zanoma yikuphi ukuma zisuswa ngokushesha ngokuthuthwa kwe-mucociliary.Ukwandisa isikhathi sokuhlala kwabathwali bezakhi zofuzo emgudwini wokuphefumula kubalulekile kodwa kunzima ukufeza.Izinhlayiya kazibuthe ezihlanganiswe nenkampani yenethiwekhi ezingaqondiswa endaweni yokuphefumula zingathuthukisa ukukhomba kwesifunda.Ngenxa yezinkinga nge-invivo imaging, ukuziphatha kwezinhlayiya zikazibuthe ezincane kangaka endaweni yomoya lapho kukhona indawo kazibuthe esetshenziswayo akuqondakali kahle.Inhloso yalolu cwaningo kwakuwukusebenzisa imaging ye-synchrotron ukuze ubone ngeso lengqondo ukunyakaza kochungechunge lwezinhlayiya zikazibuthe kuqhoqhoqho wamagundane anesthetized ukuze kufundwe amandla kanye namaphethini okuziphatha kwezinhlayiya ezingabodwa neziyinqwaba ku-vivo.Sibe sesihlola ukuthi ingabe ukulethwa kwezinhlayiya kazibuthe ze-lentviral lapho kukhona indawo kazibuthe kuzokwandisa ukusebenza kahle kokudluliselwa kuqhoqhoqho lamagundane.I-synchrotron imaging X-ray ibonisa ukuziphatha kwezinhlayiya kazibuthe ezindaweni ezimile nezinyakazayo zikazibuthe ku-vitro naku-vivo.Izinhlayiya azikwazi ukuhudulwa kalula ebusweni bemigudu yomoya ephilayo kusetshenziswa odonsa, kodwa ngesikhathi sokuthutha, amadiphozithi agxiliswa endaweni yokubuka, lapho inkambu kazibuthe iqine kakhulu.Ukusebenza kahle kokudlulisa nakho kwandiswa ngokuphindwe kasithupha lapho izinhlayiya kazibuthe ze-lentiviral zilethwa phambi kwendawo kazibuthe.Ihlanganiswe ndawonye, ​​le miphumela iphakamisa ukuthi izinhlayiya kazibuthe ze-lentiviral kanye nezindawo kazibuthe zingase zibe izindlela ezibalulekile zokuthuthukisa ukuqondiswa kwe-gene vector kanye namazinga okudlulisa emigwaqweni yomoya e-vivo.
I-Cystic fibrosis (CF) ibangelwa ukuhlukahluka kofuzo olulodwa olubizwa ngokuthi i-CF transmembrane conductance regulator (CFTR).Iphrotheni ye-CFTR iyishaneli ye-ion ekhona kumaseli amaningi e-epithelial kuwo wonke umzimba, kuhlanganise ne-airways, indawo enkulu ku-pathogenesis ye-cystic fibrosis.Ukukhubazeka ku-CFTR kuholela ekuthuthweni kwamanzi okungavamile, ukuphelelwa amandla kwamanzi endaweni yomoya, kanye nokuncipha kongqimba lwe-airway surface fluid (ASL) ukujula.Iphinde iphazamise ikhono le-mucociliary transport (MCT) uhlelo lokusula imigudu yomoya yezinhlayiya ezihogeliwe kanye namagciwane.Umgomo wethu uwukwenza ukwelashwa kofuzo lwe-lentviral (LV) ukuletha ikhophi elungile yofuzo lwe-CFTR nokuthuthukisa i-ASL, i-MCT, nempilo yamaphaphu, nokuqhubeka nokuthuthukisa ubuchwepheshe obusha obungakwazi ukukala le mingcele ku-vivo1.
Ama-LV vectors angabanye abahola phambili ekwelashweni kofuzo kwe-cystic fibrosis, ikakhulukazi ngenxa yokuthi bangakwazi ukuhlanganisa unomphela isakhi sofuzo sokwelapha kumaseli asisekelo omoya (amaseli e-airway stem).Lokhu kubalulekile ngoba angakwazi ukubuyisela ukugeleza kwamanzi okuvamile kanye nokukhishwa kwamafinyila ngokuhlukanisa amaseli angaphezulu omzila womoya asebenza ngofuzo ahambisana ne-cystic fibrosis, okuholela ezinzuzweni zokuphila konke.Ama-LV vectors kufanele aqondiswe emigwaqweni yomoya ehambisa umoya, njengoba kulapho ukubandakanyeka kwamaphaphu ku-CF kuqala khona.Ukulethwa kwe-vector ekujuleni kwephaphu kungase kubangele ukuguqulwa kwe-alveolar, kodwa lokhu akunawo umphumela wokwelapha ku-cystic fibrosis.Kodwa-ke, uketshezi olufana nabathwali bezakhi zofuzo ngokwemvelo lufudukela ku-alveoli lapho luhogelwa ngemva kokubeletha3,4 futhi izinhlayiya zokwelapha zikhishelwa ngokushesha emgodini womlomo ngama-MCT.Ukusebenza kahle kokudluliselwa kwe-LV kuhlobene ngokuqondile nobude besikhathi i-vector ehlala iseduze namaseli okuhlosiwe ukuze avumele ukutholwa kwamaselula - "isikhathi sokuhlala" 5 esifinyezwa kalula ngokugeleza komoya kwesifunda okujwayelekile kanye nokuthathwa okuhlanganisiwe kwamafinyila kanye nezinhlayiya ze-MCT.Nge-cystic fibrosis, ikhono lokwelula isikhathi sokuhlala kwe-LV emigwaqweni yomoya kubalulekile ukuze kuzuzwe amazinga aphezulu okudluliselwa kule ndawo, kodwa kuze kube manje kube yinselele.
Ukuze sinqobe lesi sithiyo, siphakamisa ukuthi izinhlayiya kazibuthe ze-LV (MPs) zingasiza ngezindlela ezimbili ezihambisanayo.Okokuqala, bangaqondiswa uzibuthe endaweni yomoya ukuze bathuthukise ukukhomba futhi basize izinhlayiya ezithwala ufuzo zibe sendaweni efanele yomzila womoya;kanye ne-ASL) zidlulela kungqimba lwamaseli 6. Ama-MP asetshenziswa kakhulu njengezimoto eziqondiswe ekuletheni izidakamizwa lapho ebophezela kumasosha omzimba, emithini ye-chemotherapy, noma kwamanye ama-molecule amancane anamathela kulwelwesi lwamangqamuzana noma abophe kuma-cell surface receptors awo futhi anqwabelane ezindaweni zesimila ukuba khona kukagesi omile.Izinkambu zikazibuthe zokwelashwa komdlavuza 7. Ezinye izindlela “ze-hyperthermic” zihloselwe ukubulala amangqamuzana e-tumor ngokufudumeza ama-MP lapho evezwe ezindaweni ezinozibuthe ezinyakazayo.Umgomo wokudluliswa kukazibuthe, lapho indawo kazibuthe isetshenziswa khona njenge-ejenti yokudlulisela ukuze kuthuthukiswe ukudluliswa kwe-DNA emangqamuzaneni, ivamise ukusetshenziswa ku-vitro kusetshenziswa uhla lwamavektha wezakhi zofuzo ezingenalo igciwane kanye negciwane kwemigqa yamaseli okunzima ukuyidlulisela. ..Ukusebenza kahle kwe-LV magnetotransfection ngokulethwa kwe-LV MP in vitro emgqeni weseli we-bronchial epithelium phambi kwendawo kazibuthe emile kwasungulwa, okwandisa ukusebenza kahle kokudluliselwa izikhathi ezingu-186 uma kuqhathaniswa ne-LV vector iyodwa.I-LV MT iphinde yasetshenziswa kumodeli ye-in vitro ye-cystic fibrosis, lapho ukudluliselwa kazibuthe kukhulise ukudluliselwa kwe-LV emasikweni okuxhumana oketshezi lomoya ngesici esingu-20 lapho kukhona i-cystic fibrosis sputum10.Nokho, ku-vivo organ magnetotransfection ithole ukunakwa okuncane futhi iye yahlolwa kuphela ezifundweni ezimbalwa zezilwane11,12,13,14,15, ikakhulukazi emaphashini16,17.Kodwa-ke, amathuba okudluliselwa kazibuthe ekwelapheni kwamaphaphu ku-cystic fibrosis acacile.Tan et al.(2020) yathi "ucwaningo lokuqinisekisa ekulethweni kwamaphaphu okusebenzayo kwama-nanoparticles kazibuthe luzovula indlela yamasu okuhogela esikhathi esizayo e-CFTR ukuze kuthuthukiswe imiphumela yomtholampilo ezigulini ezine-cystic fibrosis"6.
Ukuziphatha kwezinhlayiya ezincane kazibuthe ebusweni bomgudu wokuphefumula lapho kukhona inkambu kazibuthe esetshenziswayo kunzima ukukubona ngeso lengqondo nokufunda, ngakho-ke akuqondakali kahle.Kwezinye izifundo, sithuthukise indlela ye-Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) yokuthwebula okungahlanyisi kanye nokulinganisa umzuzu wezinguquko ze-vivo ekujuleni kwe-ASL18 kanye nokuziphatha kwe-MCT19,20 ukukala ngokuqondile ukugeleza kwamanzi kwesiteshi segesi. futhi isetshenziswa njengenkomba yokuqala ukusebenza ngempumelelo kokwelashwa.Ngaphezu kwalokho, indlela yethu yokufaka amaphuzu ye-MCT isebenzisa izinhlayiya ezinobubanzi obungu-10–35 µm ezakhiwe nge-alumina noma ingilazi yenkomba ecwebezelayo njengezimpawu ze-MCT ezibonakala nge-PB-PCXI21.Zombili izindlela zifanele ukuthwebula uhla lwezinhlayiyana, okuhlanganisa nama-MP.
Ngenxa yokulungiswa okuphezulu kwendawo kanye nesikhashana, ukuhlolwa kwethu okusekelwe ku-PB-PCXI ye-ASL kanye ne-MCT kulungele kahle ukutadisha amandla kanye namaphethini okuziphatha ezinhlayiya ezilodwa neziyinqwaba ku-vivo ukuze usisize siqonde futhi sithuthukise izindlela zokulethwa kofuzo lwe-MP.Indlela esiyisebenzisayo lapha isekelwe ezifundweni zethu sisebenzisa umugqa wokukhanya we-SPring-8 BL20B2, lapho sabona khona ukunyakaza koketshezi kulandela ukulethwa komthamo we-dummy vector emoyeni wamagundane wamakhala kanye namaphaphu ukuze sisize ukuchaza amaphethini wethu wokuvezwa kofuzo abonwayo. kufuzo lwethu.izifundo zezilwane ezinomthamo we-carrier of 3.4.
Inhloso yalolu cwaningo bekuwukusebenzisa i-synchrotron ye-PB-PCXI ukubona ngeso lengqondo ukunyakaza kwe-vivo kochungechunge lwama-MP ku-trachea yamagundane aphilayo.Lezi zifundo ze-PB-PCXI zokucabanga zaklanyelwe ukuhlola uchungechunge lwe-MP, amandla kazibuthe, kanye nendawo ukuze kutholwe umthelela wazo ekuhambeni kwe-MP.Sicabange ukuthi inkambu kazibuthe yangaphandle izosiza i-MF ethunyelwe ukuthi ihlale noma iye endaweni eqondiwe.Lezi zifundo ziphinde zasivumela ukuthi sinqume ukucushwa kukazibuthe okwandisa inani lezinhlayiya ezisele kuqhoqhoqho ngemva kokufakwa.Ochungechungeni lwesibili lwezifundo, sasihlose ukusebenzisa lokhu kumiswa okuphelele ukuze sibonise iphethini yokudlulisela ewumphumela wokulethwa kwe-vivo kwe-LV-MPs emigwaqweni yomoya yamagundane, ngokucabangela ukuthi ukulethwa kwama-LV-MPs esimweni sokuqondiswa komgwaqo womoya kuzophumela. ekwandiseni ukusebenza kahle kokudluliselwa kwe-LV..
Zonke izifundo zezilwane zenziwe ngokuhambisana nemithethonqubo egunyazwe iNyuvesi yase-Adelaide (M-2019-060 kanye ne-M-2020-022) kanye neKomidi Lokuziphatha Lezilwane le-SPring-8 Synchrotron.Ukuhlola kwenziwe ngokuhambisana nezincomo ze-ARRIVE.
Zonke izithombe ze-x-ray zithathwe kumugqa wokukhanya we-BL20XU ku-SPring-8 synchrotron e-Japan kusetshenziswa ukusetha okufana nalokhu okuchazwe ngaphambilini21,22.Kafushane, ibhokisi lokuhlola lalitholakala ku-245 m ukusuka endandeni yokugcina i-synchrotron.Ibanga lesampula ukuya kumtshina elingu-0.6 m lisetshenziselwa izifundo zokuthatha izithombe zezinhlayiyana kanye no-0.3 m ocwaningweni lwe-in vivo imaging ukuze kudalwe imiphumela yesigaba sokungafani.Kusetshenziswe insimbi ye-monochromatic enamandla angu-25 keV.Izithombe zitholwe kusetshenziswa i-X-ray transducer enesinqumo esiphezulu (SPring-8 BM3) ehlanganiswe nomtshina we-sCMOS.I-transducer iguqula ama-X-ray abe ukukhanya okubonakalayo kusetshenziswa i-scintillator engu-10 µm obukhulu (Gd3Al2Ga3O12), ebese iqondiswa kunzwa ye-sCMOS kusetshenziswa inhloso yesibonakhulu esingu-×10 (NA 0.3).Umtshina we-sCMOS wawuyi-Orca-Flash4.0 (Hamamatsu Photonics, Japan) enosayizi ofanayo wamaphikiseli angu-2048 × 2048 kanye nosayizi wephikseli eluhlaza ongu-6.5 × 6.5 µm.Lesi silungiselelo sinikeza usayizi wephikseli ye-isotropic ephumelelayo engu-0.51 µm kanye nenkambu yokubuka ecishe ibe ngu-1.1 mm × 1.1 mm.Ubude besikhathi sokuchayeka obungu-100 ms bukhethiwe ukuze kukhuliswe isilinganiso sesignali-kumsindo wezinhlayiya kazibuthe ngaphakathi nangaphandle kwemigudu yomoya kuyilapho kuncishiswa ama-artifact anyakazayo okubangwa ukuphefumula.Ezifundweni ze-vivo, isivalo se-X-ray esisheshayo safakwa endleleni ye-X-ray ukuze kukhawulwe umthamo wemisebe ngokuvimbela ugongolo lwe-X-ray phakathi kokuchayeka.
Imidiya ye-LV ayizange isetshenziswe kunoma yiziphi izifundo zokucabanga ze-SPring-8 PB-PCXI ngoba igumbi lokuthwebula le-BL20XU aliqinisekisiwe i-Biosafety Level 2.Esikhundleni salokho, sikhethe uhla lwamaLungu ePhalamende anezimpawu ezinhle kubathengisi ababili bezentengiselwano abahlanganisa izinhlobonhlobo zosayizi, izinto zokwakha, ukugxiliswa kwensimbi, kanye nezicelo , - okokuqala ukuze siqonde ukuthi amandla kazibuthe akuthinta kanjani ukunyakaza kwama-MP kuma-capillaries engilazi, bese kuthi umoya ophilayo.phezulu.Usayizi we-MP uyahluka ukusuka ku-0.25 kuye ku-18 µm futhi wenziwa ngezinto ezihlukahlukene (bona Ithebula 1), kodwa ukwakheka kwesampula ngayinye, kuhlanganise nosayizi wezinhlayiya kazibuthe ku-MP, akwaziwa.Ngokusekelwe ocwaningweni lwethu olubanzi lwe-MCT 19, 20, 21, 23, 24, silindele ukuthi amaLungu ePhalamende afinyelele ku-5 µm angabonwa endaweni yomzila womoya we-tracheal, isibonelo, ngokukhipha amafreyimu alandelanayo ukuze sibone ukubonakala okuthuthukisiwe kokunyakaza kwe-MP.I-MP eyodwa engu-0.25 µm incane kunokulungiswa kwedivayisi yokuthwebula, kodwa i-PB-PCXI ilindeleke ukuthi ithole ukuhluka kwevolumu yayo kanye nokunyakaza koketshezi olungaphezulu lapho zifakwa khona ngemva kokufakwa.
Amasampula e-MP ngayinye etafuleni.I-1 yalungiswa ngama-capillaries engilazi angama-20 μl (i-Drummond Microcaps, PA, USA) enobubanzi bangaphakathi obungu-0.63 mm.Izinhlayiya ze-Corpuscular ziyatholakala emanzini, kuyilapho izinhlayiya ze-CombiMag zitholakala oketshezini olusemthethweni lomkhiqizi.Ishubhu ngalinye ligcwaliswa uhhafu ngoketshezi (cishe u-11 µl) futhi libekwe esibambi sesampula (bona Umfanekiso 1).Ama-capillaries engilazi abekwe ngokuvundlile esiteji egumbini lokuthwebula izithombe, ngokulandelana, futhi abekwe emaphethelweni oketshezi.Uzibuthe we-nickel-shell ongu-19 mm ububanzi (28 mm ubude) owenziwe ngomhlaba ongavamile, i-neodymium, insimbi ne-boron (NdFeB) (N35, ikati no. LM1652, Jaycar Electronics, Australia) enensalela engu-1.17 T hlukanisa ithebula lokudlulisa ukuze uzuze ukude ushintshe indawo yakho phakathi nokunikezela.I-X-ray imaging iqala lapho uzibuthe umi cishe ngo-30 mm ngaphezu kwesampula futhi izithombe zitholwa kumafreyimu angu-4 ngomzuzwana.Ngesikhathi sokuthwebula, uzibuthe wasondezwa eduze kweshubhu lengilazi le-capillary (ebangeni elingaba ngu-1 mm) wabe esehambisa eduze kweshubhu ukuhlola umphumela wamandla wensimu nesimo.
Ukusethwa kwe-in vitro imaging equkethe amasampula e-MP kuma-capillary engilazi esigabeni sokuhunyushwa kwesampula ye-xy.Indlela yohlaka lwe-X-ray imakwe ngomugqa wamachashazi obomvu.
Uma ukubonakala kwe-in vitro kwamaLungu ePhalamende sekusunguliwe, isethi engaphansi yawo yahlolwa nge-vivo kumagundane angama-Wistar albino esifazane asendle (~amaviki angu-12 ubudala, ~200 g).I-Medetomidine 0.24 mg/kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg/kg (Dormicum®, Astellas Pharma, Japan) kanye ne-butorphanol 4 mg/kg (Vetorphale®, Meiji Seika).Amagundane alaliswa izinzwa ngengxube ye-Pharma (Japan) ngomjovo we-intraperitoneal.Ngemuva kwe-anesthesia, balungiselelwa ukuthwebula izithombe ngokususa uboya obuzungeze uqhoqhoqho, bafake ishubhu le-endotracheal (ET; 16 Ga intravenous cannula, Terumo BCT), futhi bangakwazi ukuhamba endaweni yokulala epuleti lokucabanga elenziwe ngokwezifiso eliqukethe isikhwama esishisayo. ukugcina izinga lokushisa lomzimba.22. Ipuleti lokuthwebula labe selinamathiselwa esiteji sesampula ebhokisini lesithombe nge-engeli encane ukuze kuqondiswe uqhoqhoqho ngokuvundlile esithombeni se-x-ray njengoba kukhonjisiwe kuMfanekiso 2a.
(a) Ukusethwa kwesithombe se-vivo kuyunithi yokucabanga ye-SPring-8, umzila we-X-ray wamachashazi omakwe ngomugqa wamachashazi obomvu.(b,c) Ukwenziwa kwasendaweni kazibuthe we-tracheal kwenziwa ukude kusetshenziswa amakhamera we-IP afakwe nge-orthogonally.Kuhlangothi lwesobunxele lwesithombe esikrinini, ungabona iluphu yocingo ebambe ikhanda kanye ne-cannula yokulethwa efakwe ngaphakathi kweshubhu ye-ET.
Isistimu yephampu yesirinji elawulwa kude (UMP2, World Precision Instruments, Sarasota, FL) esebenzisa isirinji yengilazi engu-100 µl yayixhunywe kushubhu le-PE10 (0.61 mm OD, 0.28 mm ID) kusetshenziswa inaliti engu-30 Ga.Maka ishubhu ukuze uqinisekise ukuthi ichopho lisendaweni efanele kuqhoqhoqho lapho ufaka ishubhu le-endotracheal.Kusetshenziswa i-micropump, ipayipi lesirinji lakhishwa futhi ichopho leshubhu lacwiliswa kusampula ye-MP ezolethwa.Ishubhu yokulethwa elayishiwe yabe isifakwa kushubhu endotracheal, kwabeka ithiphu engxenyeni eqinile yenkambu yethu kazibuthe elindelwe.Ukutholwa kwesithombe kwakulawulwa kusetshenziswa umtshina wokuphefumula oxhunywe kubhokisi lethu lesikhathi elisekelwe ku-Arduino, futhi wonke amasignali (isb, izinga lokushisa, ukuphefumula, ukuvuleka/ukuvala isivalo, nokutholwa kwesithombe) zarekhodwa kusetshenziswa i-Powerlab ne-LabChart (AD Instruments, Sydney, Australia) 22 Uma Kufanekiswa Lapho indlu ingatholakali, amakhamera we-IP amabili (i-Panasonic BB-SC382) ayebekwe cishe ku-90 ° komunye nomunye futhi asetshenziselwa ukulawula indawo kazibuthe ehlobene noqhoqhoqho ngesikhathi sokuthwebula (Umfanekiso 2b, c).Ukuze kuncishiswe ama-artifacts anyakazayo, kutholwe isithombe esisodwa sokuphefumula ngakunye ngesikhathi sethafa lethafa lokugeleza kokuphefumula.
Umazibuthe unamathiselwe esigabeni sesibili, esingase sibekwe ukude ngaphandle komzimba wesithombe.Izikhundla ezihlukahlukene kanye nokuhlelwa kukazibuthe kwahlolwa, okuhlanganisa: ibekwe nge-engeli engaba ngu-30 ° ngaphezu kwe-trachea (ukulungiswa kuboniswa ku-Figures 2a no-3a);omunye uzibuthe ngaphezu kwesilwane kanti omunye ngezansi, izigxobo zimiselwe ukukhangwa (Umfanekiso 3b)., uzibuthe owodwa ngaphezu kwesilwane nenye engezansi, enezigxobo ezilungiselelwe ukuxoshwa (Umfanekiso 3c), kanye nozibuthe owodwa ngenhla kanye ne-perpendicular to trachea (Figure 3d).Ngemva kokusetha isilwane nomazibuthe kanye nokulayisha i-MP ngaphansi kokuhlolwa empompini yesirinji, letha umthamo ongu-50 µl ngenani elingu-4 µl/isekhondi lapho kutholwa izithombe.Uzibuthe ube usuhanjiswa emuva naphambili kanye noma ngaphesheya koqhoqhoqho ngenkathi uqhubeka nokuthola izithombe.
Ukucushwa kukabuthe ku-vivo imaging (a) uzibuthe owodwa ngaphezu koqhoqho nge-engeli ecishe ibe ngu-30°, (b) omagnethi ababili abahlelelwe ukuheha, (c) omagnethi ababili abalungiselwe ukucisha, (d) uzibuthe oyedwa ngaphezulu kanye ne-perpendicular to the uqhoqhoqho.Owayebukele wabheka phansi kusukela emlonyeni kuya emaphashini ngoqhoqhoqho futhi ugongolo lwe-X-ray lwadlula ohlangothini lwesobunxele lwegundane lwaphuma ngakwesokudla.Umazibuthe uhanjiswa ngobude bomzila womoya noma kwesokunxele bese kwesokudla ngaphezu koqhoqhoqho ngendlela ye-X-ray.
Siphinde safuna ukunquma ukubonakala nokuziphatha kwezinhlayiya emigwaqweni yomoya ngaphandle kokuxutshwa kokuphefumula nokushaya kwenhliziyo.Ngakho-ke, ekupheleni kwesikhathi sokucabanga, izilwane zahlukunyezwa ngokobuntu ngenxa ye-pentobarbital overdose (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~ 65 mg/kg ip).Ezinye izilwane zashiywa endaweni yokuthwebula izithombe, futhi ngemva kokunqamuka kokuphefumula nokushaya kwenhliziyo, inqubo yokuthwebula yaphindwa, yengeza umthamo owengeziwe we-MP uma kungekho MP ebonakala endaweni yomoya.
Izithombe ezaba umphumela zalungiselelwa inkambu eyisicaba nemnyama zase zihlanganiswa zaba imuvi (amafreyimu angu-20 ngomzuzwana; 15–25 × isivinini esivamile kuye ngezinga lokuphefumula) kusetshenziswa umbhalo wangokwezifiso obhalwe ku-MATLAB (R2020a, The Mathworks).
Zonke izifundo zokulethwa kwe-LV gene vector zenziwa eNyuvesi yase-Adelaide Laboratory Animal Research Center futhi zazihloswe ukusebenzisa imiphumela yokuhlolwa kwe-SPring-8 ukuhlola ukuthi ukulethwa kwe-LV-MP phambi kwendawo kazibuthe kungathuthukisa ukudluliswa kofuzo ku-vivo. .Ukuhlola imiphumela ye-MF kanye nenkundla kazibuthe, amaqembu amabili ezilwane alashwa: iqembu elilodwa lajovwa nge-LV MF ngokubeka uzibuthe, kanti elinye iqembu lajovwa neqembu elilawulayo eline-LV MF ngaphandle kukazibuthe.
Ama-LV gene vectors akhiqizwa kusetshenziswa izindlela ezichazwe ngaphambilini 25, 26.Ivektha ye-LacZ iveza isakhi sofuzo se-beta-galactosidase senuzi esiqhutshwa yi-MPSV constitutive promoter (LV-LacZ), ekhiqiza umkhiqizo wokusabela oluhlaza okwesibhakabhaka kumaseli ashintshiwe, obonakala emaphethelweni nasezingxenyeni zezicubu zamaphaphu.I-Titration yenziwa kumasiko amaseli ngokubala mathupha inombolo yamaseli e-LacZ-positive kusetshenziswa i-hemocytometer ukuze kubalwe i-titer ku-TU/ml.Izithwali zigcinwa ku-cryopreserved ku -80°C, zincibilikisiwe ngaphambi kokusetshenziswa, futhi ziboshwe ku-CombiMag ngokuxuba u-1:1 futhi zifukamele eqhweni okungenani imizuzu engama-30 ngaphambi kokulethwa.
Amagundane avamile we-Sprague Dawley (n = 3/iqembu, ~2-3 ip enesthetized enenhlanganisela ye-0.4mg/kg medetomidine (Domitor, Ilium, Australia) kanye ne-60mg/kg ketamine (Ilium, Australia) enenyanga engu-1 ubudala) ip ) umjovo kanye ne-cannula yomlomo engahlinzeki nge-cannula engu-16 Ga emithanjeni.Ukuqinisekisa ukuthi izicubu ze-tracheal airway zithola ukushintshwa kwe-LV, kufakwe isimo kusetshenziswa iphrothokholi yethu echazwe ngaphambilini yokuphazamiseka lapho umzila womoya we-tracheal wawuhlikihlwa nge-axially ngobhasikidi wocingo (N-Circle, i-nitinol extractor ngaphandle kwethiphu NTSE-022115 ) -UDH , Cook Medical, USA) 30 p28.Khona-ke, cishe imizuzu eyi-10 ngemva kokuphazamiseka kukhabhinethi ye-biosafety, ukuphathwa koqhoqhoqho kwe-LV-MP kwenziwa.
Inkambu kazibuthe esetshenziswe kulokhu kuhlolwa yalungiswa ngokufana nocwaningo lwe-x-ray lwe-vivo, nozibuthe abafanayo ababanjwe phezu koqhoqhoqho ngezinsimbi ze-distillation stent (Umfanekiso 4).Ivolumu engu-50 µl (ama-aliquots angu-2 x 25 µl) we-LV-MP ilethwe kuqhoqhoqho (n = izilwane ezi-3) kusetshenziswa i-piette enesihloko sejeli njengoba kuchazwe ngaphambilini.Iqembu elilawulayo (n = izilwane ezi-3) lithole i-LV-MP efanayo ngaphandle kokusebenzisa uzibuthe.Ngemva kokuphothulwa kwe-infusion, i-cannula ikhishwa ku-tube endotracheal futhi isilwane sikhishwe.Uzibuthe uhlala endaweni imizuzu eyi-10 ngaphambi kokususwa.Amagundane afakwa ngaphansi kwesikhumba nge-meloxicam (1 ml/kg) (I-Ilium, e-Australia) elandelwa ukuhoxiswa kwezinzwa ngomjovo we-intraperitoneal we-1 mg/kg i-atipamazole hydrochloride (Antisedan, Zoetis, Australia).Amagundane ayegcinwa efudumele futhi agcinwe kuze kube yilapho eselulame ngokuphelele ku-anesthesia.
Idivayisi yokulethwa kwe-LV-MP kukhabhinethi yokuphepha yebhayoloji.Ungabona ukuthi umkhono we-Luer-lock ompunga okhanyayo weshubhu ye-ET uphuma emlonyeni, futhi ithiphu le-pipette yejeli eliboniswe emfanekisweni lifakwa ngeshubhu le-ET liye ekujuleni okufunayo kuqhoqhoqho.
Isonto elilodwa ngemva kwenqubo yokuphatha ye-LV-MP, izilwane zanikelwa ngokobuntu ngokuhogela i-CO2 engu-100% futhi isisho se-LacZ sahlolwa kusetshenziswa ukwelashwa kwethu okujwayelekile kwe-X-gal.Izindandatho ezintathu ze-caudal cartilage zisusiwe ukuze kuqinisekiswe ukuthi noma yikuphi ukulimala kwemishini noma ukugcinwa kwamanzi ngenxa yokubekwa kwe-endotracheal tube ngeke kufakwe ekuhlaziyeni.Uqhoqhoqho lwalusikwa ngobude ukuze kutholwe izingxenye ezimbili zokuhlaziya futhi lufakwe enkomishini equkethe irabha ye-silicone (Sylgard, Dow Inc) kusetshenziswa inaliti ye-Minutien (Amathuluzi Esayensi Enhle) ukuze kubonakale indawo ekhanyayo.Ukusatshalaliswa nohlamvu lwamaseli ashintshiwe kuqinisekiswe ukuthwebula izithombe okungaphambili kusetshenziswa isibonakhulu se-Nikon (SMZ1500) ngekhamera ye-DigiLite kanye nesofthiwe ye-TCapture (Tucsen Photonics, China).Izithombe zitholwe ekukhulisweni okungu-20x (okuhlanganisa nesilungiselelo esiphezulu sobubanzi obugcwele be-trachea), nobude bonke boqhoqhoqho bubonisiwe isinyathelo ngesinyathelo, okunikeza ukugqagqana okwanele phakathi kwesithombe ngasinye ukuze kuvunyelwe izithombe ukuba "zithungwe".Izithombe eziphuma kuqhoqhoqho ngalinye zabe sezihlanganiswa zaba isithombe esiyinhlanganisela eyodwa kusetshenziswa inguqulo engu-2.0.3 ye-Composite Image Editor (I-Microsoft Research) kusetshenziswa i-algorithm ye-planar motion. Indawo yenkulumo ye-LacZ ngaphakathi kwezithombe eziyinhlanganisela yoqhoqho esilwaneni ngasinye yalinganiswa kusetshenziswa iskripthi esizenzakalelayo se-MATLAB (R2020a, MathWorks) njengoba kuchazwe ngaphambilini28, kusetshenziswa izilungiselelo zokuthi 0.35 < Hue < 0.58, Saturation > 0.15, kanye neValue <0.7. Indawo yenkulumo ye-LacZ phakathi kwezithombe eziyinhlanganisela yoqhoqho esilwaneni ngasinye yalinganiswa kusetshenziswa iskripthi esizenzakalelayo se-MATLAB (R2020a, MathWorks) njengoba kuchazwe ngaphambilini28, kusetshenziswa izilungiselelo zokuthi 0.35 < Hue < 0.58, Saturation > 0.15, kanye neValue <0.7. I-LacZ itholakala ngokuzenzakalelayo futhi ihlanganisa i-akhawunti ye-LacZ ne-Massage 2010 thWorks), как описано ранее28, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 and значение <0 ,7. Indawo yenkulumo ye-LacZ ezithombeni zoqhoqhoqho eziyinhlanganisela evela esilwaneni ngasinye yalinganiswa kusetshenziswa umbhalo ozenzakalelayo we-MATLAB (R2020a, MathWorks) njengoba kuchazwe ngaphambilini28 kusetshenziswa izilungiselelo ze-0.350.15 kanye nenani<0 .7.如前所述，使用自动MATLAB 脚本（R2020a，MathWorks) 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 ， Mathworks） 來自 每 只 的 气管 合 图像 的 的 门 . 5 <色调 <0.58,> 0.15 kanye ne- <0.7 的。。. ........ ........ Области экспрессии I-LacZ на составных изображениях трахеи каждого количественно определяли с использованием автоматизезеванs, Matlamatisks, 2 к описано ранее, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0,7 . Izindawo zenkulumo ye-LacZ ezithombeni eziyinhlanganisela zoqhoqhoqho lwesilwane ngasinye zalinganiswa kusetshenziswa umbhalo ozenzakalelayo we-MATLAB (R2020a, MathWorks) njengoba kuchazwe ngaphambilini kusetshenziswa izilungiselelo ze-0.35 < hue < 0.58, saturation > 0.15 kanye nevelu engu-< 0.7 .Ngokulandela umkhondo wamathishu ku-GIMP v2.10.24, imaskhi yadalwa mathupha esithombeni ngasinye esiyinhlanganisela ukuze kuhlonzwe indawo yethishu futhi kuvinjwe noma yikuphi ukutholwa okungamanga ngaphandle kwethishu yoqhoqhoqho.Izindawo ezinamabala kuzo zonke izithombe eziyinhlanganisela ezivela esilwaneni ngasinye zafingqwa ukuze kunikezwe indawo enamabala yaleso silwane.Indawo ependiwe yabe ihlukaniswa ngendawo ephelele yemaski ukuze kutholwe indawo evamile.
I-trachea ngayinye yayishunyekwe kupharafini futhi ihlukaniswe ubukhulu obungu-5 µm.Izigaba zivinjelwe ngokubomvu okusheshayo okungathathi hlangothi imizuzu emi-5 futhi izithombe zatholwa kusetshenziswa isibonakhulu se-Nikon Eclipse E400, ikhamera ye-DS-Fi3 kanye nesofthiwe yokuthwebula yezinto ze-NIS (inguqulo 5.20.00).
Konke ukuhlaziya kwezibalo kwenziwa ku-GraphPad Prism v9 (GraphPad Software, Inc.).Ukubaluleka kwezibalo kusethwe kokuthi p ≤ 0.05.Okujwayelekile kwahlolwa kusetshenziswa ukuhlolwa kwe-Shapiro-Wilk futhi umehluko ekungcoleni kwe-LacZ kwahlolwa kusetshenziswa i-t-test engabhangqiwe.
AmaLungu ePhalamende ayisithupha achazwe kuThebula loku-1 ahlolwe yi-PCXI, futhi ukubonakala kuchazwe kuThebula 2. AmaPhalamende e-polystyrene amabili (MP1 ne-MP2; 18 µm kanye no-0.25 µm, ngokulandelana) ayengabonakali yi-PCXI, kodwa amasampula asele ayengabonwa. (izibonelo zikhonjiswe kuMfanekiso 5).I-MP3 ne-MP4 zibonakala kancane (10-15% Fe3O4; 0.25 µm kanye no-0.9 µm, ngokulandelana).Nakuba i-MP5 (98% Fe3O4; 0.25 µm) iqukethe ezinye izinhlayiya ezincane kakhulu ezihloliwe, ibigqame kakhulu.Umkhiqizo we-CombiMag MP6 kunzima ukuwuhlukanisa.Kuzo zonke izimo, ikhono lethu lokubona ama-MF lathuthukiswa kakhulu ngokuhambisa uzibuthe emuva naphambili ngokuhambisana ne-capillary.Njengoba omazibuthe besuka ku-capillary, izinhlayiya zakhishwa ngamaketanga amade, kodwa njengoba omazibuthe besondela futhi amandla kazibuthe ekhula, amaketanga ezinhlayiyana aba mfushane njengoba izinhlayiya zithuthela endaweni engaphezulu ye-capillary (bheka i-Supplemental Video S1 : MP4), ikhulisa ukuminyana kwezinhlayiyana phezulu.Ngokuphambene, lapho uzibuthe esuswa ku-capillary, amandla ensimu ayancipha futhi amalungu ePhalamende ahlele kabusha amaketanga amade asuka endaweni engaphezulu ye-capillary (bona Ividiyo Eyengeziwe S2: MP4).Ngemva kokuba uzibuthe uyeka ukunyakaza, izinhlayiya ziyaqhubeka nokunyakaza isikhathi esithile ngemva kokufinyelela endaweni yokulinganisa.Njengoba i-MP iya futhi isuka endaweni engaphezulu ye-capillary, izinhlayiya kazibuthe zivame ukudonsa udoti oketshezini.
Ukubonakala kwe-MP ngaphansi kwe-PCXI kuyahluka kakhulu phakathi kwamasampuli.(a) MP3, (b) MP4, (c) MP5 kanye (d) MP6.Zonke izithombe eziboniswe lapha zithathwe ngozibuthe obekwe cishe ngo-10 mm ngaphezu kwe-capillary.Imibuthano emikhulu ebonakala sengathi amabhamuza omoya avaleleke kuma-capillaries, abonisa ngokucacile izici zonqenqema ezimnyama nezimhlophe zesithombe sokungafani kwesigaba.Ibhokisi elibomvu libonisa ukukhuliswa okuthuthukisa ukugqama.Qaphela ukuthi amadiamitha amasekhethi kazibuthe kuzo zonke izibalo akumele akale futhi cishe makhulu ngokuphindwe kayi-100 kunalokho okubonisiwe.
Njengoba uzibuthe uhamba kwesokunxele nakwesokudla ngaphezu kwe-capillary, i-engeli yentambo ye-MP iyashintsha ukuze iqondanise nomazibuthe (bheka Umfanekiso 6), ngaleyo ndlela ihlukanise imigqa yamagnetic field.Ku-MP3-5, ngemva kokuba i-chord ifinyelele i-engeli yombundu, izinhlayiya zidonsa endaweni engaphezulu ye-capillary.Lokhu kuvame ukuholela ekutheni amaLungu ePhalamende ahlangane abe amaqembu amakhulu eduze kwalapho amandla kazibuthe aqine khona (bona Ividiyo Eyengeziwe S3: MP5).Lokhu kubonakala futhi ikakhulukazi lapho kuthwebula isithombe eduze nasekupheleni kwe-capillary, okubangela ukuthi i-MP ukuthi ihlanganise futhi igxilise kusixhumanisi somoya owuketshezi.Izinhlayiya ku-MP6, obekunzima ukuzihlukanisa kunalezo eziku-MP3-5, azizange zihudule lapho uzibuthe ugudla i-capillary, kodwa izintambo ze-MP zahlukana, zashiya izinhlayiya zibonakala (bona i-Supplementary Video S4: MP6).Kwezinye izimo, lapho inkambu kazibuthe esetshenzisiwe incishiswa ngokuhambisa uzibuthe ibanga elide ukusuka endaweni yokuthwebula izithombe, noma imaphi amaLungu ePhalamende asele ehlela kancane kancane aye endaweni engaphansi yeshubhu ngamandla adonsela phansi, asale entanjeni (bona Ividiyo Eyengeziwe S5: MP3) .
I-engeli yentambo ye-MP iyashintsha njengoba uzibuthe uya kwesokudla ngaphezu kwe-capillary.(a) MP3, (b) MP4, (c) MP5 kanye (d) MP6.Ibhokisi elibomvu libonisa ukukhuliswa okuthuthukisa ukugqama.Sicela uqaphele ukuthi amavidiyo engeziwe enzelwe izinjongo zokwaziswa njengoba eveza ukwakheka kwezinhlayiyana ezibalulekile nolwazi oluguquguqukayo olungenakubonwa kulezi zithombe ezimile.
Ukuhlola kwethu kubonise ukuthi ukuhambisa umazibuthe uye phambili kancane kancane kuqhoqhoqho kusiza ukubonwa kwe-MF kumongo wokunyakaza okuyinkimbinkimbi ku-vivo.Ukuhlolwa kwe-No in vivo kwenziwa ngoba ubuhlalu be-polystyrene (MP1 ne-MP2) babungabonakali ku-capillary.I-MF ngayinye kwemine esele ihlolwe ku-vivo nge-eksisi ende kazibuthe ibekwe phezu koqhoqhoqho nge-engeli engaba ngu-30 ° ukuya mpo (bheka Umfanekiso 2b no-3a), njengoba lokhu kubangele amaketanga amade e-MF futhi kwasebenza kangcono. kunozibuthe..ukumisa kunqanyuliwe.I-MP3, MP4 kanye ne-MP6 azikatholakali kuqhoqhoqho lanoma yiziphi izilwane eziphilayo.Lapho ubuka indlela yokuphefumula yamagundane ngemva kokubulala izilwane ngokobuntu, izinhlayiya zahlala zingabonakali ngisho nalapho ivolumu eyengeziwe yengezwa kusetshenziswa iphampu yesirinji.I-MP5 yayinokuqukethwe okuphezulu kwe-iron oxide futhi bekuwukuphela kwezinhlayiyana ezibonakalayo, ngakho-ke yasetshenziselwa ukuhlola nokuveza isimilo se-MP ku-vivo.
Ukubekwa kukazibuthe phezu koqhoqhoqho ngesikhathi sokufakwa kwe-MF kubangele ukuthi ama-MF amaningi, kodwa hhayi wonke, agxile emkhakheni wokubuka.Ukungena koqhoqhoqho kwezinhlayiya kubonakala kahle ezilwaneni ezihlukunyezwe ngokobuntu.Umfanekiso 7 kanye ne-Supplementary Video S6: I-MP5 ibonisa ukuthwebula kazibuthe okusheshayo nokuqondanisa kwezinhlayiya ebusweni boqhoqhoqho, okubonisa ukuthi ama-MP angaqondiswa ezindaweni ezifunwayo zoqhoqhoqho.Lapho kuseshwa kude kakhulu kuqhoqhoqho ngemva kokulethwa kwe-MF, amanye ama-MF atholakala eduze kwe-carina, okubonisa amandla kazibuthe anganele okuqoqa nokubamba wonke ama-MF, njengoba alethwa endaweni yamandla kazibuthe aphezulu ngesikhathi sokuphathwa koketshezi.inqubo.Kodwa-ke, ukugxila kwe-MP ngemuva kokubeletha kwakuphakeme endaweni yesithombe, okuphakamisa ukuthi amaLungu ePhalamende amaningi ahlala ezindaweni zomoya lapho amandla kazibuthe asetshenzisiwe ayephezulu kakhulu.
Izithombe (a) zangaphambili kanye (b) nangemuva kokulethwa kwe-MP5 kuqhoqhoqho legundane elisanda kubulawa elinozibuthe obekwe ngenhla nje kwendawo yokuthwebula izithombe.Indawo eveziwe itholakala phakathi kwamasongo ama-cartilaginous.Kunoketshezi oluthile emigudwini yomoya ngaphambi kokuba i-MP ilethwe.Ibhokisi elibomvu libonisa ukukhuliswa okuthuthukisa ukugqama.Lezi zithombe zithathwe kuvidiyo efakwe ku-S6: MP5 Supplementary Video.
Ukunyakazisa uzibuthe kuqhoqhoqho ku-vivo kuholele ekushintsheni kwe-engeli yeketango le-MP endaweni yomoya, efana naleyo ebonwa kuma-capillary (bheka uMdwebo 8 kanye nevidiyo Eyengeziwe S7: MP5).Kodwa-ke, ocwaningweni lwethu, amaLungu ePhalamende awakwazanga ukuhudulwa phezu kwamapheshana okuphefumula aphilayo, njengoba ama-capillaries ayengenza.Kwezinye izimo, iketango le-MP liyakhula njengoba uzibuthe uhamba kwesokunxele nakwesokudla.Kuyathakazelisa ukuthi siphinde sathola ukuthi uchungechunge lwezinhlayiyana lushintsha ukujula kongqimba olungaphezulu loketshezi lapho uzibuthe uhanjiswa ngobude kuqhoqhoqho, futhi uyanwebeka lapho uzibuthe ugudluzwa phezulu ngokuqondile futhi iketango lezinhlayiyana lizungeziswa liyiswe endaweni eqondile (bona). Ividiyo Eyengeziwe S7).: MP5 ngo-0:09, ngezansi kwesokudla).Iphethini yokunyakaza okuyisici yashintsha lapho umazibuthe ehanjiswa eceleni enqamula phezulu kuqhoqhoqho (okungukuthi, kwesokunxele noma kwesokudla sesilwane, kunokuhamba ngoqhoqhoqho).Izinhlayiya zazisabonakala ngokucacile phakathi nokunyakaza kwazo, kodwa lapho uzibuthe ekhishwa kuqhoqhoqho, amathiphu ezintambo zezinhlayiyana aqala ukubonakala (bheka i-Supplementary Video S8: MP5, kusukela ngo-0:08).Lokhu kuvumelana nokuziphatha okuqashelwe kwendawo kazibuthe ngaphansi kwesenzo sensimu kazibuthe esetshenziswa ku-capillary yengilazi.
Izithombe eziyisampula ezibonisa i-MP5 kuqhoqhoqho wegundane elibulawa izinzwa.(a) Umazibuthe usetshenziselwa ukuthola izithombe ngenhla nakwesokunxele kuqhoqhoqho, bese (b) ngemva kokuhambisa uzibuthe kwesokudla.Ibhokisi elibomvu libonisa ukukhuliswa okuthuthukisa ukugqama.Lezi zithombe zivela kuvidiyo efakwe ku-S7′s Supplementary Video: MP5.
Lapho izigxobo ezimbili zishunwa ku-orientation esenyakatho naseningizimu ngenhla nangaphansi koqhoqhoqho (okungukuthi, ukuheha; Umdwebo 3b), ama-MP chords abonakala amade futhi abekwe odongeni olusemaceleni loqhoqho kunokuba endaweni yomgogodla we-trachea. uqhoqhoqho (bheka Isithasiselo).Ividiyo S9:MP5).Kodwa-ke, ukugxila okuphezulu kwezinhlayiya endaweni eyodwa (okungukuthi, indawo engemuva ye-trachea) ayizange ibonakale ngemva kokuphathwa koketshezi kusetshenziswa idivayisi kazibuthe ekabili, evame ukwenzeka ngedivayisi eyodwa kazibuthe.Khona-ke, lapho uzibuthe owodwa ulungiselelwe ukuxosha izigxobo eziphambene (Umfanekiso 3c), inani lezinhlayiya ezibonakalayo emkhakheni wokubuka alizange likhule ngemva kokulethwa.Ukusetha kokubili ukumiswa kazibuthe kuyinselele ngenxa yamandla kazibuthe aphezulu aheha noma aphushe ozibuthe ngokulandelana kwawo.Ukusetha kwabe sekushintshwa kwaba uzibuthe oyedwa ohambisana nemigudu yomoya kodwa edlula emigwaqweni yomoya nge-engeli engu-90 degree ukuze imigqa yamandla iwele udonga lwe-tracheal orthogonally (Figure 3d), umumo ohloselwe ukunquma ukuthi kungenzeka yini ukuhlanganisa izinhlayiyana udonga olungemuva.kuqashelwe.Kodwa-ke, kulokhu kucushwa, bekungekho umnyakazo wokuqongelela i-MF obonakalayo noma ukunyakaza kazibuthe.Ngokusekelwe kuyo yonke le miphumela, ukucushwa okunozibuthe oyedwa kanye ne-orientation ye-30-degree kukhethwe izifundo ze-vivo zezinkampani zofuzo (Fig. 3a).
Lapho isilwane senziwa isithombe izikhathi eziningi ngokushesha ngemva kokuzinikela ngokobuntu, ukungabikho kokunyakaza kwezicubu eziphazamisayo kwakusho ukuthi imigqa yezinhlayiyana emifushane, emifushane ingabonakala endaweni ecacile ye-intercartilaginous, 'enyakazayo' ngokuvumelana nokunyakaza kokuhumusha kukazibuthe.bona ngokucacile ubukhona nokunyakaza kwezinhlayiya ze-MP6.
I-titer ye-LV-LacZ yayingu-1.8 x 108 IU/mL, futhi ngemva kokuxuba u-1:1 ne-CombiMag MP (MP6), izilwane zajovwa ngo-50 µl wedosi yoqhoqhoqho engu-9 x 107 IU/ml yemoto ye-LV (okungukuthi 4.5 x 106 TU/igundane).)).Kulezi zifundo, esikhundleni sokunyakazisa uzibuthe ngesikhathi sokubeletha, simise umazibuthe endaweni eyodwa ukuze sinqume ukuthi ukuhanjiswa kwe-LV (a) kungathuthukiswa yini uma kuqhathaniswa nokulethwa kwe-vector lapho ingekho inkambu kazibuthe, futhi (b) uma indlela yomoya ingaba ngcono. gxila.Amaseli adluliselwa ezindaweni eziqondiswe kazibuthe zendlela yokuphefumula ephezulu.
Ukuba khona koozibuthe kanye nokusetshenziswa kwe-CombiMag kuhlanganiswe nama-LV vectors akubonakalanga kungakhinyabeza impilo yezilwane, njengoba kwenza isimiso sethu esivamile sokulethwa kwevekhtha ye-LV.Izithombe zangaphambili zesifunda se-tracheal ezingaphansi kokuphazamiseka kwemishini (I-Supplementary Fig. 1) ibonise ukuthi iqembu eliphathwayo le-LV-MP lalinamazinga aphezulu kakhulu okudluliselwa phambi kozibuthe (Fig. 9a).Inani elincane kuphela le-LacZ eluhlaza okwesibhakabhaka lalikhona eqenjini lokulawula (Umfanekiso 9b).Ukulinganisa kwezifunda ezijwayelekile ezinombala we-X-Gal kubonise ukuthi ukuphathwa kwe-LV-MP lapho kukhona indawo kazibuthe kuholele ekuthuthukisweni okuphindwe ka-6 (Fig. 9c).
Isibonelo sezithombe eziyinhlanganisela ezibonisa ukushintshwa koqhoqhoqho nge-LV-MP (a) lapho kukhona inkambu kazibuthe kanye (b) uma ingekho uzibuthe.(c) Ukuthuthukiswa okuphawulekayo ngokwezibalo endaweni evamile yokuguqulwa kwe-LacZ kuqhoqhoqho ngokusetshenziswa kukazibuthe (*p = 0.029, t-test, n = 3 iqembu ngalinye, kusho iphutha ± elijwayelekile lencazelo).
Izigaba ezinamabala abomvu amaphakathi (isibonelo esiboniswe ku-Supplementary Fig. 2) sibonise ukuthi amaseli ane-LacZ-stained abekhona kusampula efanayo futhi endaweni efanayo njengoba ibikwe ngaphambilini.
Inselele eyinhloko ekwelashweni kwezakhi zofuzo zomoya isalokhu iwukuba kwasendaweni okunembayo kwezinhlayiya zenkampani yenethiwekhi ezindaweni ezithakaselwayo kanye nokuzuzwa kwezinga eliphezulu lokusebenza kahle kokudluliselwa ephashini elihambayo lapho kukhona ukugeleza komoya kanye nokuvunyelwa kwamafinyila asebenzayo.Kubathwali be-LV ehloselwe ukwelapha izifo zokuphefumula ku-cystic fibrosis, ukwandisa isikhathi sokuhlala kwezinhlayiya ezithwala emigwaqweni yomoya ehambisa umoya kuze kube manje kube umgomo ongafinyeleleki.Njengoba kuvezwe uCastellani et al., ukusetshenziswa kwezizibuthe ukuze kuthuthukiswe ukudluliswa kwezakhi zofuzo kunezinzuzo ngaphezu kwezinye izindlela zokulethwa kwezakhi zofuzo ezifana ne-electroporation ngoba ingahlanganisa ubulula, umnotho, ukulethwa kwendawo, ukusebenza kahle okwandisiwe, kanye nesikhathi esifushane sokufukamela.futhi mhlawumbe umthamo ophansi wemoto10.Kodwa-ke, ukubekwa kwe-vivo kanye nokuziphatha kwezinhlayiya kazibuthe emigwaqweni yomoya ngaphansi kwethonya lamandla kazibuthe angaphandle akukaze kuchazwe, futhi eqinisweni ikhono lale ndlela lokwandisa amazinga okuveza izakhi zofuzo emigwaqweni yomoya ephilayo ayikaboniswa ku-vivo.
Ukuhlolwa kwethu kwe-in vitro ku-synchrotron ye-PCXI kubonise ukuthi zonke izinhlayiya esizihlolile, ngaphandle kwe-MP polystyrene, zazibonakala ekusethweni kwezithombe esizisebenzisayo.Lapho kukhona insimu kazibuthe, izindawo kazibuthe zakha izintambo, ubude bazo buhlobene nohlobo lwezinhlayiya namandla kazibuthe (okungukuthi, ukusondela nokuhamba kukazibuthe).Njengoba kuboniswe kuMfanekiso 10, izintambo esizibonayo zakheka njengoba inhlayiya ngayinye iba nguzibuthe futhi ingenisa indawo yayo yasendaweni kazibuthe.Lezi zinkambu ezihlukene zenza ukuba ezinye izinhlayiya ezifanayo ziqoqe futhi zixhumane nokunyakaza kwezintambo zeqembu ngenxa yamandla endawo avela kumandla endawo okukhanga kanye nokuxoshwa kwezinye izinhlayiya.
Umdwebo obonisa (a, b) amaketanga ezinhlayiyana akha ngaphakathi kwama-capillary agcwele uketshezi kanye (c, d) noqhoqhoqho olugcwele umoya.Qaphela ukuthi ama-capillaries kanye ne-trachea akudonswa esikalini.Iphaneli (a) iqukethe nencazelo ye-MF equkethe izinhlayiya ze-Fe3O4 ezihlelwe ngamaketanga.
Lapho uzibuthe uhamba phezu kwe-capillary, i-engeli yochungechunge lwezinhlayiyana yafinyelela embundwini obalulekile we-MP3-5 equkethe i-Fe3O4, okwathi ngemva kwalokho iyunithi yezinhlamvu ayibange isahlala endaweni yayo yasekuqaleni, kodwa yagudluza phezulu yaya endaweni entsha.uzibuthe.Lo mphumela cishe uvela ngenxa yokuthi ingaphezulu le-glass capillary libushelelezi ngokwanele ukuvumela lokhu kunyakaza kwenzeke.Kuyathakazelisa ukuthi i-MP6 (i-CombiMag) ayizange iziphathe ngale ndlela, mhlawumbe ngenxa yokuthi izinhlayiya bezizincane, zine-coating ehlukile noma inkokhiso engaphezulu, noma uketshezi lwenkampani yenethiwekhi luthinte ikhono lazo lokunyakaza.Umehluko osesithombeni sezinhlayiyana ze-CombiMag nawo ubuthakathaka, okuphakamisa ukuthi uketshezi nezinhlayiyana zingase zibe nokuminyana okufanayo ngakho azikwazi ukuya ngakunye kalula.Izinhlayiya zingabambeka uma uzibuthe uhamba ngokushesha kakhulu, okubonisa ukuthi amandla kazibuthe awakwazi njalo ukunqoba ukungqubuzana phakathi kwezinhlayiya oketshezini, okuphakamisa ukuthi amandla kazibuthe kanye nebanga phakathi kukazibuthe nendawo okuhlosiwe akufanele kuze ukumangala.kubalulekile.Le miphumela iphinde ikhombise ukuthi nakuba omazibuthe bekwazi ukuthwebula izinhlayiya ezincane eziningi ezigeleza endaweni okuqondiswe kuyo, mancane amathuba okuthi omazibuthe bangathenjelwe kubo ukuze bahambise izinhlayiya ze-CombiMag ebusweni boqhoqhoqho.Ngakho-ke, siphethe ngokuthi izifundo ze-vivo LV MF kufanele zisebenzise izinkambu kazibuthe ezimile ukuze ziqondise ngokomzimba izindawo ezithile zesihlahla somoya.
Uma izinhlayiya zilethwe emzimbeni, kunzima ukuzibona kumongo wezicubu ezinyakazayo eziyinkimbinkimbi zomzimba, kodwa amandla azo okuthola athuthukisiwe ngokuhambisa uzibuthe uvundlile phezu koqhoqhoqho ukuze "unyakaze" izintambo ze-MP.Nakuba kungenzeka ukuthwebula izithombe ngesikhathi sangempela, kulula ukubona ukunyakaza kwezinhlayiyana ngemva kokuba isilwane sibulewe ngokobuntu.Ukugxila kwe-MP ngokuvamile kwakuphakeme kakhulu kule ndawo lapho uzibuthe ubekwe phezu kwendawo yokuthwebula, nakuba ezinye izinhlayiya zazivame ukutholakala ngaphansi kuqhoqhoqho.Ngokungafani nezifundo ze-in vitro, izinhlayiya azikwazi ukuhudulelwa phansi kuqhoqhoqho ngokuhamba kukazibuthe.Lokhu kutholwa kuhambisana nendlela ufinyi olumboze ubuso boqhoqhoqho ngokuvamile olucubungula ngayo izinhlayiya ezihogelwe, lizibambe kumafinyila bese lizisusa ngokusebenzisa indlela yokususa i-muco-ciliary.
Sacabanga ukuthi ukusebenzisa omazibuthe ngenhla nangaphansi koqhoqhoqho ukuze kukhangeke (Fig. 3b) kungabangela inkambu kazibuthe efana kakhulu, kunokuba ibe yizibuthe egxile kakhulu endaweni eyodwa, okungase kubangele ukusatshalaliswa okufanayo kwezinhlayiya..Kodwa-ke, isifundo sethu sokuqala asizange sithole ubufakazi obucacile bokusekela le nkolelo.Ngokufanayo, ukusetha amapheya ozibuthe ukuze aphindiselele (Fig. 3c) akuzange kuphumele ekuzinzeni kwezinhlayiyana eziningi endaweni yesithombe.Lokhu okubili okutholwe kukhombisa ukuthi ukusethwa kozibuthe okukabili akuthuthukisi kakhulu ukulawulwa kwendawo kokukhomba kwe-MP, nokuthi amandla azibuthe anamandla okuba umphumela kunzima ukuwashuna, okwenza le ndlela ingasebenzi.Ngokufanayo, ukuqondisa uzibuthe ngenhla futhi ngaphesheya koqhoqhoqho (Umdwebo 3d) nakho akuzange kukhulise inani lezinhlayiya ezisele endaweni yesithombe.Okunye kwalokhu kulungiselelwa kungase kungaphumeleli njengoba kubangela ukuncipha kwamandla kazibuthe endaweni yokubeka.Ngakho, ukucushwa kwamagnetic okukodwa kuma-degree angu-30 (Fig. 3a) kubhekwa njengendlela elula futhi ephumelelayo kakhulu yokuhlola i-vivo.
Ucwaningo lwe-LV-MP lubonise ukuthi lapho amavekhtha e-LV ehlanganiswa ne-CombiMag futhi alethwa ngemva kokuphazamiseka ngokomzimba lapho kukhona indawo kazibuthe, amazinga okudlulisela akhuphuka kakhulu kuqhoqhoqho uma kuqhathaniswa nezilawuli.Ngokusekelwe ezifundweni ze-synchrotron imaging kanye nemiphumela ye-LacZ, indawo kazibuthe ibonakale ikwazi ukugcina i-LV kuqhoqhoqho futhi yehlise inani lezinhlayiya ze-vector ezingene ngokushesha zijule ephashini.Ukuthuthukiswa okuhlosiwe okunjalo kungaholela ekusebenzeni okuphezulu ngenkathi kunciphisa iziqu ezilethiwe, ukudluliselwa okungahlosiwe, imiphumela emibi yokuvuvukala neyokuzivikela komzimba, kanye nezindleko zokudlulisa izakhi zofuzo.Okubalulekile, ngokomkhiqizi, i-CombiMag ingasetshenziswa ngokuhambisana nezinye izindlela zokudlulisa izakhi zofuzo, okuhlanganisa namanye ama-viral vectors (afana ne-AAV) nama-nucleic acid.