Siyabonga ngokuvakashela i-Nature.com.Inguqulo yesiphequluli oyisebenzisayo inokusekelwa okulinganiselwe kwe-CSS.Ukuze uthole ulwazi olungcono kakhulu, sincoma ukuthi usebenzise isiphequluli esibuyekeziwe (noma uvale imodi yokuhambisana ku-Internet Explorer).Okwamanje, ukuze siqinisekise ukwesekwa okuqhubekayo, sizobonisa isayithi ngaphandle kwezitayela ne-JavaScript.
Ama-Gene vectors okwelashwa kwe-cystic fibrosis lung disease kufanele aqondise emigwaqweni yomoya eqhubayo ngoba ukudluliswa kwamaphaphu e-peripheral akuhlinzeki ngenzuzo yokwelapha.Ukusebenza kahle kwe-viral transduction kuhlobene ngokuqondile nesikhathi sokuhlala kwe-vector.Nokho, uketshezi lokulethwa olunjengama-gene carriers lusakazeka ngokwemvelo ku-alveoli ngesikhathi sokuphefumulelwa, futhi izinhlayiya zokwelashwa zisulwa ngokushesha ngayiphi indlela yokuthutha. isikhathi sabathwali bofuzo emigwaqweni yomoya sibalulekile kodwa kunzima ukusifeza.Izinhlayiya kazibuthe ezixhunywe kwi-Gene carrier-conjugated ezingaqondiswa ebusweni bemigudu yomoya zingathuthukisa ukuqondiswa kwesifunda.Ngenxa yezinselelo zokuboniswa kwe-vivo, ukuziphatha kwezinhlayiya zikazibuthe ezincane kangako endaweni yomoya lapho kukhona inkambu kazibuthe esetshenziswayo akuqondakali kahle.Inhloso yalolu cwaningo kwakuwukusebenzisa uchungechunge lukazibuthe ku-vivo izinhlayiya kuqhoqhoqho wamagundane anezinzwa zokuhlola amandla namaphethini okuziphatha kwezinhlayiyana zomuntu ngamunye kanye nenqwaba ku-vivo.Sabe sesihlola ukuthi ingabe ukulethwa kwezinhlayiya kazibuthe ze-lentiviral phambi kwendawo kazibuthe kungakhuphula ukusebenza kahle kokudluliselwa ku-trachea yamagundane.I-Synchrotron X-ray imaging yembula ukuziphatha kwezinhlayiya zikazibuthe endaweni enyakazayo futhi ezinyakazayo ngeke zibe lula. idonswe phezu komzila womoya ophilayo ngozibuthe, kodwa ngesikhathi sokuthutha, amadiphozithi agxile endaweni yokubuka lapho amandla kazibuthe aqine kakhulu.Ukusebenza kahle kokudluliswa kwe-transduction nakho kwanda ngokuphindwe kasithupha lapho izinhlayiya kazibuthe ze-lentiviral zilethwa phambi kwendawo kazibuthe.Ndawonye, ​​le miphumela iphakamisa ukuthi izinhlayiya kazibuthe ze-lentiviral kanye nezizibuthe kungase kube izindlela ezibalulekile zokuthuthukisa amazinga e-victor yokuthuthukisa i-air transduction.
I-Cystic fibrosis (CF) ibangelwa ukuhlukahluka kwesakhi sofuzo esisodwa esibizwa ngokuthi i-CF transmembrane conductance regulator (CFTR).Iphrotheni ye-CFTR iyisiteshi se-ion esikhona kumaseli amaningi e-epithelial emzimbeni wonke, kuhlanganise nemigudu yomoya eqhubayo, indawo enkulu ye-CF pathogenesis.Ukukhubazeka kweCFTR kuholela ekuthuthweni kwamanzi okungavamile, ukunciphisa ukujula kwamanzi omgwaqo we-airway (SL) ungqimba.Lokhu kuphinde kukhinyabeze ikhono lohlelo lwezokuthutha lwe-mucociliary (MCT) lokususa izinhlayiya ezihogeliwe kanye namagciwane emigwaqweni yomoya.Umgomo wethu uwukwenza ukwelapha kofuzo lwe-lentiviral (LV) ukuletha ikhophi efanele yofuzo lwe-CFTR futhi kuthuthukiswe i-ASL, MCT, nempilo yamaphaphu, nokuqhubeka nokuthuthukisa ubuchwepheshe obusha obukwazi ukulinganisa le mitha.
Ama-LV vectors angomunye wabaholi abahamba phambili be-CF airway gene therapy, ikakhulukazi ngenxa yokuthi bangakwazi ukuhlanganisa unomphela isakhi sofuzo sokwelapha emangqamuzaneni ayisisekelo omoya (airway stem cells) .Lokhu kubalulekile ngoba angakwazi ukubuyisela ukugeleza okujwayelekile kanye nokukhishwa kwamafinyila ngokuhlukanisa izakhi zofuzo ezihlotshaniswa ne-CF-ezihlotshaniswa ne-CF ehlobene nomzila womoya womoya, okuholela ekubhekaneni nalokhu amangqamuzana omoya wokuphila konke kufanele kube khona. Isifo samaphaphu e-CF siyaqala.Ukulethwa kwe-vector ekujuleni kwephaphu kungase kubangele ukuguqulwa kwe-alveolar, kodwa lokhu akunayo inzuzo yokwelapha ku-CF.Nokho, uketshezi olufana nabathwali bezakhi zofuzo ngokwemvelo luthuthela ku-alveoli lapho ugqozi ngemva kokubeletha3,4 futhi izinhlayiya zokwelapha zisulwa ngokushesha emgodini we-oral transduction ukuya kumaseli ahlobene ngokuqondile ne-MCT ehlobene ngqo ne-MCT. vumela ukutholwa kwamaselula - "isikhathi sokuhlala" 5 - esincishiswa kalula ngokugeleza komoya kwesifunda okujwayelekile kanye nokubanjwa kwezinhlayiyana ze-mucus ezididiyelwe kanye ne-MCT.Ku-CF, ikhono lokwelula isikhathi sokuhlala kwe-LV ngaphakathi komgwaqo womoya kubalulekile ukuze kuzuzwe amazinga aphezulu okudluliselwa kulesi sifunda, kodwa kuze kube manje kube yinselele.
Ukuze sinqobe lesi sithiyo, siphakamisa ukuthi izinhlayiya kazibuthe ze-LV (MPs) zingasiza ngezindlela ezimbili ezihambisanayo.Okokuqala, zingaqondiswa kazibuthe endaweni yomoya ukuze kuthuthukiswe ukukhomba nokusiza izinhlayiya ze-gene carrier zihlale endaweni oyifunayo yomzila womoya; kanye ne-ASL) ukuthuthela kungqimba lwamaseli 6.MPs asetshenziswe kabanzi njengezimoto eziqondiswe ekuletheni izidakamizwa lapho ebopha amasosha omzimba, izidakamizwa ze-chemotherapeutic, noma amanye ama-molecule amancane anamathela kulwelwesi lwamaseli noma abophe kuma-cell surface receptors afanelekile futhi anqwabelene ezindaweni zesimila lapho kukhona ugesi ongashintshi. I-Magnetic Fields for Cancer Treatment 7. Amanye amasu “e-hyperthermal” ahlose ukushisa ama-MPs lapho evezwe kumagnetic fields oscillating, ngaleyo ndlela acekele phansi ama-tumor cells.Isimiso sokudluliswa kazibuthe, lapho i-magnetic field isetshenziswa njenge-agent yokudlulisela ukuze kuthuthukiswe ukudluliswa kwe-DNA kumaseli, isetshenziswa ngokujwayelekile ku-vitro kusetshenziswa uhla lwe-non-viral kanye ne-viral cell-cell ye-LV I-magnetotransfection isisunguliwe, ngokulethwa kwe-in vitro kwe-LV-MPs kulayini weseli ye-bronchial epithelial phambi kwensimu kazibuthe emile, okwandisa ukusebenza kahle kokudluliselwa ngokuphindwe izikhathi ezingu-186 uma kuqhathaniswa ne-LV vector kuphela. isikhwehlela10.Noma kunjalo, ku-vivo magnetotransfection yezitho zithole ukunakwa okuncane futhi kuye kwahlolwa kuphela ezifundweni ezimbalwa zezilwane11,12,13,14,15, ikakhulukazi emaphashini16,17.Noma kunjalo, amathuba okudluliswa kazibuthe ekwelapheni kwamaphaphu e-CF acacile.Tan et20ubufakazi bokuthi i-econfficient20-of-20 yocwaningo olufanele lwe-20. ukulethwa kwe-nanoparticle pulmonary kuzovula indlela yamasu okuhogela esikhathi esizayo e-CFTR ukuze kuthuthukiswe imiphumela yomtholampilo ezigulini ezine-CF”6.
Ukuziphatha kwezinhlayiya zikazibuthe ezincane endaweni yomoya lapho kukhona inkambu kazibuthe esetshenziswayo kunzima ukukubona ngeso lengqondo nokufunda, futhi ngaleyo ndlela kungaqondakali kahle.Kwezinye izifundo, sakha indlela ye-synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) ukuze ubone ngeso lengqondo ngokungabonakali futhi ulinganise umzuzu wezinguquko ze-vivo ku-ASL kuya ku-ASL yokujula kwe-198 kanye nokujula kwe-MAC20 ye-CanCTal ngqo kanye nokujula kwe-M192 kwe-ASL. esetshenziswa njengenkomba yakuqala yokuphumelela kokwelashwa.Ngaphezu kwalokho, indlela yethu yokuhlola ye-MCT isebenzisa izinhlayiya eziyi-10–35 µm ububanzi ezakhiwe nge-alumina noma ingilazi yenkomba ecwebezelayo njengezimpawu ze-MCT ezibonakalayo kusetshenziswa i-PB-PCXI21.Zombili lezi zindlela zobuchwepheshe zifanele ukubonwa kwezinhlobonhlobo zezinhlayiyana, kuhlanganise ne-MP.
Ngenxa yokulungiswa kwayo okuphezulu kwendawo kanye nesikhashana, izindlela zethu zokuhlaziya ezisekelwe ku-PB-PCXI ze-ASL kanye ne-MCT zifaneleka kahle ekuhloleni amandla namaphethini okuziphatha kwezinhlayiyana zoketshezi olulodwa nenqwaba ku-vivo ukuze usisize siqonde futhi sithuthukise amasu okulethwa kofuzo lwe-MP. imigudu yomoya yamagundane emakhaleni kanye namaphaphu ukuze isize ukuchaza amaphethini wethu wokusho izakhi zofuzo ezingalingani ezibonwe esifundweni sethu sesilwane esithwala izakhi zofuzo 3,4.
Inhloso yalolu cwaningo bekuwukusebenzisa i-synchrotron PB-PCXI ukuze ubone ngeso lengqondo ukunyakaza kwe-vivo kochungechunge lwamaLungu ePhalamende kuqhoqhoqho lamagundane aphilayo. Lezi zifundo zokucabanga ze-PB-PCXI zaziklanyelwe ukuhlola uchungechunge lwamaLungu ePhalamende, amandla kazibuthe, nezindawo ukuze kutholwe umphumela wawo ekunyakazeni kwe-MP. ukuhlonza ukucushwa kukazibuthe okwandisa inani lezinhlayiya ezigcinwe kuqhoqhoqho ngemva kokufakwa.Ochungechungeni lwesibili lwezifundo, sifuna ukusebenzisa lokhu kulungiselelwa okuphelele ukuze sibonise iphethini yokudlulisela ewumphumela wokulethwa kwe-vivo kwama-LV-MPs kumzila womoya wamagundane, ngokusekelwe ekucabangeni ukuthi ukulethwa kwama-LV-MPs kumongo wokuhanjiswa kwe-LV kuzokwenza ngcono ukusebenza kahle komzila womoya.
Zonke izifundo zezilwane zenziwe ngokuvumelana nezinqubo ezigunyazwe yiNyuvesi yase-Adelaide (M-2019-060 kanye ne-M-2020-022) kanye neKomidi Lokuziphatha Lezilwane le-SPring-8 Synchrotron. Ukuhlolwa kwenziwa ngokuvumelana neziqondiso ze-ARRIVE.
Konke ukuthwebula kwe-X-ray kwenziwa kumugqa wokukhanya we-BL20XU ku-synchrotron ye-SPring-8 e-Japan, kusetshenziswa ukusetha okufanayo nalokho okuchazwe ngaphambilini21,22.Kafushane, ibhokisi lokuhlola lalitholakala ku-245 m ukusuka kwendandatho yokugcina ye-synchrotron.Ibanga lesampula ukuya kumtshina lika-0.6 m lisetshenziselwa izifundo ze-0. Kwasetshenziswa amandla e-monochromatic beam engama-25 keV.Izithombe zithathwe kusetshenziswa isiguquli se-X-ray sokulungiswa okuphezulu (SPring-8 BM3) sihlanganiswe nomtshina we-sCMOS.Isiguquli siguqula ama-X-ray abe ukukhanya okubonakalayo kusetshenziswa i-scintillator engu-10 µm ewugqinsi (Gd3Al2Ga3C3C eqondiswe ku-microscope × 12), okuyi-microscope eqondiswe ku-mic × (NA 0.3).I-sCMOS detector bekungu-Orca-Flash4.0 (Hamamatsu Photonics, Japan) enosayizi we-array wamaphikiseli angu-2048 × 2048 kanye nosayizi wephikseli eluhlaza ongu-6.5 × 6.5 µm. Lokhu kusetha kunikeza usayizi wephikiseli we-isotropic osebenzayo ongu-0.51 × inkambu engu-1 × yokubuka. mm.Ubude bokuchayeka obungu-100 ms bukhethiwe ukuze kukhuliswe isilinganiso sesignali-kumsindo wezinhlayiya kazibuthe ngaphakathi nangaphandle kwendlela yomoya kuyilapho kunciphisa ama-artifacts abangelwa ukuphefumula.Ocwaningweni lwe-vivo, isivalo se-X-ray esisheshayo safakwa endleleni ye-X-ray ukuze kukhawulwe umthamo wemisebe ngokuvimbela i-X-ray yokukhanya phakathi kokuchayeka.
Isithwali se-LV asizange sisetshenziswe kunoma yiziphi izifundo zokucabanga ze-SPring-8 PB-PCXI ngoba igumbi lokucabanga le-BL20XU aliqinisekisiwe i-Biosafety Level 2. Esikhundleni salokho, sikhethe uchungechunge lwamaLungu ePhalamende anezimpawu ezinhle kubahlinzeki bezentengiselwano ababili—okuhlanganisa uhla losayizi, izinto zokwakha, ukugxila kwensimbi, kanye nezinhlelo zokusebenza ezingaphakathi kwensimu enyakazayo ye-MP, kuqala ukuze siqonde ukuthi i-MP izindlela zomoya. ebusweni.Ama-MP ahluka ngosayizi asuka ku-0.25 kuya ku-18 μm futhi enziwa ngezinto ezihlukahlukene (bheka Ithebula 1), kodwa ukwakheka kwesampula ngayinye, kuhlanganise nobukhulu bezinhlayiya zikazibuthe ngaphakathi kwe-MP, akwaziwa.Ngokusekelwe ocwaningweni lwethu olubanzi lwe-MCT 19, 20, 21, 23, 24, silindele ukuthi ama-MP emgwaqeni abonakale njenge-trache encane njengesibonelo somoya. ukukhipha amafreyimu alandelanayo ukuze kubonakale ukubonakala okuthuthukisiwe kokunyakaza kwe-MP.I-MP eyodwa engu-0.25 μm-usayizi incane kunokulungiswa kwedivayisi yokuthwebula, kodwa i-PB-PCXI ilindeleke ukuthi ithole ukuhluka kwevolumu yazo kanye nokunyakaza koketshezi olungaphezulu lapho zifakwa khona ngemva kokumiswa.
Amasampula e-MP ngayinye kuThebula 1 alungiselelwa ku-20 μl glass capillaries (Drummond Microcaps, PA, USA) enobubanzi obungaphakathi obungu-0.63 mm.Izinhlayiya zeCorpuscular ziyatholakala emanzini, kuyilapho izinhlayiya ze-CombiMag zitholakala kuketshezi oluphathelene nomkhiqizi.Ishubhu ngalinye ligcwele uhhafu ngoketshezi (cishe 1 bheka isampula) 1).Ama-capillary engilazi abekwe avundlile esiteji sesampula ebhokisini lokuthwebula, ngokulandelana, futhi abeka imiphetho yoketshezi.A 19 mm ububanzi (28 mm ubude) igobolondo le-nickel elingavamile umhlaba i-neodymium iron boron (NdFeB) i-N35, ikati. no. ixhunywe esigabeni esihlukile sokuhumusha ukuze kuzuzwe Shintsha indawo yayo ukude ngesikhathi sokuthwebula.Ukuthola isithombe se-X-ray kuqala lapho uzibuthe umi cishe ngo-30 mm ngaphezu kwesampula, futhi izithombe zitholwa ngenani lamafreyimu angu-4 ngomzuzwana. Ngesikhathi sokuthwebula, uzibuthe wasondezwa eduze kweshubhu le-capillary yengilazi (cishe ngo-1 mm) bese ihunyushwa ngokuhambisana neshubhu kuya emandleni.
Ukusethwa kwe-in vitro imaging equkethe amasampula e-MP kuma-capillary engilazi esigabeni sokuhumusha i-xy. Umzila we-X-ray imakwe ngomugqa onedeshi ebomvu.
Lapho ukubonakala kwe-in vitro kwamaLungu ePhalamende sekusunguliwe, i-subset yawo yahlolwa ku-vivo kumagundane wesifazane we-albino Wistar wohlobo lwasendle (~amaviki angu-12 ubudala, ~ 200 g).0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, mg4/Japan orphaorpha) buts (I-Vetorphale®, Meiji Seika) Amagundane alaliswa ngengxube ye-Pharma), e-Japan) ngomjovo we-intraperitoneal.Ngemva kwe-anesthesia, alungiselelwa ukuthwebula izithombe ngokususa uboya obuzungeze uqhoqhoqho, afake ishubhu le-endotracheal (ET; 16 Ga iv cannula, Terumo BCT) kanye nepuleti eliwaqinisa ngokwezifiso ukuze agcine izinga lokushisa lomzimba ngokwezifiso. 22 .I-imaging plate yabe isinamathiselwa esigabeni sokuhumusha sesampula ebhokisini lesithombe nge-engeli encane ukuze kuqondiswe uqhoqhoqho ngokuvundlile esithombeni se-X-ray, njengoba kuboniswe kuMfanekiso 2a.
(a) Ukusethwa kwesithombe se-vivo ebhokisini lesithombe le-SPring-8, indlela ye-X-ray imakwe ngomugqa odeshi obomvu.(b,c) Ukwenziwa kwendawo kazibuthe kuqhoqhoqho kwenziwa ukude kusetshenziswa amakhamera e-IP afakwe nge-orthogonally.Ohlangothini lwesobunxele lwesithombe sesikrini, iluphu yocingo ebambe ikhanda ingabonwa, futhi ishubhu le-cannula lokulethwa liyabonakala.
Isistimu yepompo yesirinji elawulwa kude (UMP2, World Precision Instruments, Sarasota, FL) esebenzisa isirinji yengilazi engu-100 μl yayixhunywe ku-PE10 tubing (OD 0.61 mm, ID 0.28 mm) ngenaliti engu-30 Ga. Maka ithubhu ukuze uqinisekise ukuthi ithiphu isendaweni efanele. I-syringe plunger yahoxiswa ngenkathi ichopho leshubhu licwiliswa kusampula ye-MP eyayizolethwa.Ishubhu yokulethwa elayishiwe yabe ifakwa epayipini le-endotracheal, labeka ithiphu ngaphakathi kwengxenye eqine kakhulu yamandla kazibuthe asetshenziswayo. ukutholwa) zarekhodwa kusetshenziswa i-Powerlab ne-LabChart (AD Instruments, Sydney, Australia) 22. Lapho imaging Lapho i-enclosure ingafinyeleleki, amakhamera amabili e-IP (Panasonic BB-SC382) abekwe cishe ku-90 ° komunye nomunye futhi asetshenziselwa ukuqapha isikhundla sikazibuthe ngokuhlobene nomzila we-trachea, i-Migmize . ama-artifacts, isithombe esisodwa satholwa umoya ngamunye ngesikhathi sethafa lokugeleza kolwandle.
Uzibuthe unamathiselwe esigabeni sesibili esingatholakala ukude ngaphandle kwendawo yokuthwebula izithombe.Izindawo zikazibuthe ezihlukahlukene kanye nokuhlelwa kwahlolwa, okuhlanganisa: Kufakwe nge-engeli ecishe ibe ngu-30° ngaphezu koqhoqhoqho (ukulungiswa kuboniswe kuMfanekiso 2a no-3a); omunye uzibuthe ngaphezu kwesilwane kanti omunye ngezansi, enezigxobo ezibekwe ukuheha (Umfanekiso 3b); omunye uzibuthe ngaphezu kwesilwane kanti omunye ngezansi, izigxobo zimiselwe ukuxosha (Umfanekiso 3c); kanye nozibuthe owodwa ngenhla kanye ne-perpendicular kuqhoqhoqho (Umdwebo 3d).Uma isilwane nozibuthe sekulungisiwe futhi i-MP ezohlolwa ilayishwa empompini yesirinji, letha umthamo ongu-50 μl ngesilinganiso esingu-4 μl/sec ngenkathi uthola izithombe.Uzibuthe ube esehlehliswa emuva naphambili ngenkathi eqhubeka nomzila womzila.
Ukucushwa kukazibuthe kwesithombe se-vivo (a) uzibuthe owodwa ngaphezu koqhoqhoqho nge-engeli ecishe ibe ngu-30°, (b) omagnethi ababili abasethelwe ukuheha, (c) omagnethi ababili abasethelwe ukuxosha, (d) uzibuthe oyedwa ngaphezulu kanye ne-perpendicular kuqhoqho. side.Uzibuthe uhanjiswa ngobude bomzila womoya noma kwesokunxele bese kwesokudla ngaphezu koqhoqhoqho ngendlela ye-X-ray.
Siphinde safuna ukunquma ukubonakala nokuziphatha kwezinhlayiya emigwaqweni yomoya lapho kungabikho ukuphefumula okudidayo nokunyakaza kwenhliziyo.Ngakho-ke, ekupheleni kwesikhathi sokucabanga, izilwane zabulawa ngobuntu ngenxa ye-pentobarbital overdose (i-Somnopentil, i-Pitman-Moore, i-Washington Crossing, e-USA; ~ 65 mg / kg / kg, i-ip yenhliziyo ishiywe kanye kanye ne-imaging platform kanye nezilwane ezicatshangelwayo). inqubo yokucabanga yaphindwa, kwengeza umthamo owengeziwe we-MP uma lingekho i-MP elibonakalayo endaweni yomoya.
Izithombe ezitholiwe beziyinkundla eyisicaba kanye nenkundla emnyama zalungiswa zase zihlanganiswa zaba imuvi (amafreyimu angama-20 ngomzuzwana; 15-25 × isivinini esivamile kuye ngezinga lokuphefumula) kusetshenziswa umbhalo wangokwezifiso obhalwe ku-MATLAB (R2020a, The Mathworks).
Zonke izifundo zokulethwa kwezakhi zofuzo ze-LV zenziwa e-Laboratory Animal Research Facility eNyuvesi yase-Adelaide futhi kuhloswe ngazo ukusebenzisa imiphumela yokuhlolwa kwe-SPring-8 ukuhlola ukuthi ukulethwa kwe-LV-MP lapho kukhona amandla kazibuthe kungathuthukisa ukudluliswa kwezakhi zofuzo ku-vivo. uzibuthe.
Izakhi zofuzo ze-LV zakhiwe kusetshenziswa izindlela ezichazwe ngaphambilini 25, 26 .Ivektha ye-LacZ iveza isakhi sofuzo se-beta-galactosidase esenziwe endaweni yenuzi esiqhutshwa umgqugquzeli we-MPSV (LV-LacZ), okhiqiza umkhiqizo wokusabela okuluhlaza okwesibhakabhaka kumaseli ashintshiwe, obonakala emaphethelweni ezicubu zamaphaphu nasezigabeni zezicubu. i-hemocytometer ukubala i-titer ku-TU/ml.Izithwali zigcinwa ku-cryopreserved ku--80 °C, zincibilikisiwe ngaphambi kokusetshenziswa, futhi ziboshwe ku-CombiMag ngokuxuba ngesilinganiso esingu-1:1 futhi zifukamele eqhweni okungenani imizuzu engu-30 ngaphambi kokulethwa.
Amagundane e-Sprague Dawley ajwayelekile (n = 3/iqembu, ~2-3 alaliswa intraperitoneally ngengxube ye-0.4 mg/kg medetomidine (Domitor, Ilium, Australia) kanye ne-60 mg/kg ketamine (Ilium, Australia) enenyanga ubudala) ip) umjovo kanye nokungahlinzeki kokuphefumula komlomo16Ukuqinisekisa ukuthi i-cannulation yomlomo ingakwazi ukuvuza. ithola ukuguqulwa kwe-LV, yafakwa esimweni kusetshenziswa iphrothokholi yethu yokuphazanyiswa komshini echazwe ngaphambilini, lapho umzila womoya woqhoqhoqho wawuhlikihlwa nge-axially ngobhasikidi wocingo (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Ikhabhinethi ye-tracheal yenziwa cishe ngemva kwemizuzu yokuphepha ye-LV-0MP ye-LV-0MP.
Inkambu kazibuthe esetshenziswe kulokhu kuhlola yahlelwa ngendlela efanayo nesifundo se-in vivo X-ray imaging, nozibuthe abafanayo ababanjwe ngenhla koqhoqhoqho besebenzisa iziqeshana ze-distillation stent (Umfanekiso 4) .Ivolumu engu-50 μl (2 × 25 μl aliquots) ye-LV-MP yalethwa kuqhoqhoqho (n = A = I-tiptte yezilwane ezichazwe ngaphambilini) = Izilwane ezi-3) zithole ama-LV-MP afanayo ngaphandle kokusebenzisa uzibuthe.Ngemva kokuba ukumnika kuqediwe, i-cannula iyasuswa eshubhuni ye-ET futhi isilwane sikhishwe.Uzibuthe uhlala endaweni imizuzu engu-10, bese uyasuswa.Amagundane athola umthamo ongaphansi kwe-meloxicam (1 ml/kg) (Ilium, Australia, umjovo we-atipsle we-atipazo) ulandelwa umjovo we-atipazo. i-hydrochloride (i-Antisedan, i-Zoetis, i-Australia) .Amagundane ayegcinwa efudumele futhi eqashwe kuze kube yilapho eselulame ngokugcwele ekubulaweni kwezinzwa.
Idivayisi yokulethwa kwe-LV-MP kukhabhinethi yokuphepha yebhayoloji.Ihabhu le-Luer elimpunga ngokukhanyayo leshubhu le-ET lingabonakala liphuma emlonyeni futhi ichopho lejeli lepayipi eliboniswe esithombeni lifakwa ngeshubhu le-ET liye ekujuleni okufunayo kuqhoqhoqho.
Isonto elilodwa ngemva kwenqubo yokudosa i-LV-MP, izilwane zabulawa ngobuntu nge-100% CO2 inhalation kanye nenkulumo ye-LacZ yahlolwa kusetshenziswa ukwelashwa kwethu okujwayelekile kwe-X-gal. Izindandatho ezintathu ze-caudal kakhulu ze-cartilaginous zisusiwe ukuze kuqinisekiswe ukuthi noma yikuphi ukulimala komshini noma ukugcinwa kwamanzi okuvela ekubekweni kwe-endotracheal tube akuzange kufakwe ekuhlaziyweni. isitsha esiqukethe irabha ye-silicone (i-Sylgard, i-Dow Inc) kusetshenziswa inaliti ye-Minutien (Amathuluzi Esayensi Enhle) ukuze ubone ngeso lengqondo indawo ekhanyayo. Ukusatshalaliswa kanye nephethini yamaseli ashintshiwe kuqinisekiswe ukuthwebula izithombe kwangaphambili kusetshenziswa isibonakhulu se-Nikon (SMZ1500) ngekhamera ye-DigiLite kanye nesofthiwe ye-TCapture (Tucsen Photonics, China). ububanzi boqhoqhoqho), nabo bonke ubude boqhoqhoqho obufanekiswe isinyathelo ngesinyathelo, okuqinisekisa ukugqagqana okwanele phakathi kwesithombe ngasinye ukuze kuvumeleke "ukuthungwa" kwesithombe.Izithombe eziphuma kuqhoqhoqho zazibe sezihlanganiswa zibe yinhlanganisela yesithombe esisodwa kusetshenziswa i-Image Composite Editor v2.0.3 (Microsoft Research) kusetshenziswa i-algorithm yokunyakaza okuhleliwe. okuzenzakalelayo kweskripthi se-MATLAB (R2020a, MathWorks) njengoba kuchazwe ngaphambilini, kusetshenziswa izilungiselelo zokuthi 0.35 < Hue < 0.58, Saturation > 0.15, kanye neValue < 0.7. Ngokulandela umkhondo wethishu, imaskhi yenziwa mathupha ku-GIMP v2.10.24 ukuze kuvinjwe ukutholwa kwezicubu ezingaphandle ku-oda lendawo ngayinye ehlanganisiwe ukuze kuvinjelwe ukutholwa kwendawo ngayinye ye-composite ku-oda lendawo ngayinye eyinhlanganisela. izicubu zoqhoqhoqho.Izindawo ezinamabala ezivela kuzo zonke izithombe eziyinhlanganisela ezivela esilwaneni ngasinye zafingqwa ukuze kukhiqizwe ingqikithi yendawo enamabala yaleso silwane.Indawo enamabala yabe isihlukaniswa ngesamba sendawo yemaski ukuze kukhiqizwe indawo evamile.
I-trachea ngayinye yayishumekwe kupharafini futhi izigaba ezingu-5 μm zasikwa. Izigaba zaphikisana nokubomvu okusheshayo okungathathi hlangothi imizuzu engu-5 futhi izithombe zatholwa kusetshenziswa isibonakhulu se-Nikon Eclipse E400, ikhamera ye-DS-Fi3 kanye nesofthiwe yokuthwebula isici se-NIS (inguqulo 5.20.00).
Konke ukuhlaziya kwezibalo kwenziwe ku-GraphPad Prism v9 (I-GraphPad Software, Inc.).Ukubaluleka kwezibalo kwamiswa kokuthi p ≤ 0.05.Okuvamile kwaqinisekiswa kusetshenziswa ukuhlolwa kwe-Shapiro-Wilk, futhi umehluko ekungcoleni kwe-LacZ kwahlolwa kusetshenziswa i-t-test engabhanqiwe.
AmaLungu ePhalamende ayisithupha achazwe kuThebula 1 ahlolwe kusetshenziswa i-PCXI, futhi ukubonakala kuchazwe kuThebula 2.Ama-MP1 e-polystyrene amabili (MP1 kanye ne-MP2; 18 μm kanye no-0.25 μm, ngokulandelana) ayengabonakali ngaphansi kwe-PCXI, kodwa amanye amasampuli ayebonakala (izibonelo zikhonjisiwe ku-5 (i-5% ye-MP; i-MP1% ye-5; i-MP1% ye-4). 0.25 μm kanye no-0.9 μm, ngokulandelana) zibonakala kancane.Nakuba iqukethe ezinye zezinhlayiya ezincane kakhulu ezihloliwe, i-MP5 (98% Fe3O4; 0.25 μm) iyona evelele kakhulu.Umkhiqizo we-CombiMag MP6 kunzima ukuwubona. i-capillary.Lapho omagnethi besuka ku-capillary, izinhlayiya zanda ngezintambo ezinde, kodwa njengoba odonsa besondela futhi amandla kazibuthe ekhula, izintambo zezinhlayiya zaba mfushane njengoba izinhlayiya zifudukela endaweni engaphezulu ye-capillary (bheka i-Supplementary Video S1: MP4), ikhulisa Ukuminyana kwezinhlayiyana ku-Conpillary, lapho amandla e-capilla ekhishwa phezu kwensimu. ancipha futhi amaLungu ePhalamende ahlele kabusha abe yizintambo ezinde ezisuka endaweni engaphezulu ye-capillary (bona i-Supplementary Video S2:MP4).Ngemuva kokuthi uzibuthe uyeke ukunyakaza, izinhlayiya ziyaqhubeka nokuhamba isikhathi esifushane ngemva kokufinyelela endaweni yokulinganisa.Njengoba i-MP ihamba ibheke futhi isuka endaweni engaphezulu ye-capillary, ngokuvamile idonsa uketshezi oluyizibuthe.
Ukubonakala kwe-MP ngaphansi kwe-PCXI kuyahlukahluka kakhulu phakathi kwamasampuli.(a) MP3, (b) MP4, (c) MP5 kanye (d) MP6.Zonke izithombe eziboniswe lapha zithathwe ngozibuthe otholakala cishe ngo-10 mm ngaphezu kwe-capillary.Izindingilizi ezisobala ezinkulu zingamabhamuza omoya avaleleke kuma-capillaries, okubonisa ngokucacile ukugqama kwesigaba esimnyama nesimhlophe esiqukethe unqenqema lwesigaba esibomvu se-imaging box. ukukhulisa.Qaphela ukuthi amadayamitha wezinhlelo zikazibuthe kuzo zonke izibalo akufanele zikalwe futhi cishe zinkulu ngokuphindwe kayi-100 kunalokho okubonisiwe.
Njengoba uzibuthe uhunyushwa kwesokunxele nakwesokudla ngaphezu kwe-capillary, i-engeli yentambo ye-MP iyashintsha ukuze iqondanise uzibuthe (bheka Umfanekiso 6), ngaleyo ndlela ichaza imigqa yamagnetic field.Ku-MP3-5, ngemva kokuba i-chord ifinyelele i-angle yombundu, izinhlayiya zihudulwa endaweni engaphezulu ye-capillary. I-Supplementary Video S3:MP5).Lokhu kubonakala futhi ikakhulukazi lapho uthwebula isithombe eduze kokuphela kwe-capillary, okubangela ukuthi amalungu ePhalamende ahlanganise futhi agxilise ku-fluid-air interface.Izinhlayiya ku-MP6, obekunzima kakhulu ukuzibona kune-MP3-5, azihudulwanga njengoba uzibuthe uhamba eduze kwe-capillary, kodwa i-MP ishiya ingxenyana yokuhlukanisa i-Video. S4:MP6).Kwezinye izimo, lapho inkambu kazibuthe esetshenzisiwe incishiswa ngokuhambisa uzibuthe ibanga elikhulu ukusuka endaweni yokuthwebula izithombe, noma imaphi amaLungu ePhalamende asele ehlela kancane kancane aye endaweni engaphansi yeshubhu ngamandla adonsela phansi kuyilapho ehlala entanjeni (bona Ividiyo Eyengeziwe S5: MP3).
I-engeli yeyunithi yezinhlamvu ze-MP iyashintsha njengoba uzibuthe uhunyushwa kwesokudla ngaphezu kwe-capillary.(a) MP3, (b) MP4, (c) MP5 kanye (d) ne-MP6. Ibhokisi elibomvu liqukethe ukukhulisa okugqamisayo.Qaphela ukuthi amavidiyo angeziwe anolwazi njengoba eveza ukwakheka kwezinhlayiyana ezibalulekile nolwazi oluguqukayo olungenakubonwa kulezi zithombe ezimile.
Ukuhlola kwethu kubonise ukuthi ukuhambisa uzibuthe kancane uye phambili emuva naphambili kuqhoqhoqho kusiza ukubonwa kwe-MP esimweni sokunyakaza okuyinkimbinkimbi kwe-vivo.Ukuhlolwa kwe-vivo akwenziwanga njengoba ubuhlalu be-polystyrene (MP1 kanye ne-MP2) babungabonakali ku-capillary.Ilungu ngalinye kwamane e-MP asele ahlolwe ku-vivo nge-axis ende uzibuthe elungiselelwe mayelana ne-engeli e-30 emiswe phezu kwe-engeli Izibalo 2b kanye no-3a), njengoba lokhu kubangele amaketanga e-MP amade futhi kwasebenza kangcono kunokumiswa kukazibuthe okunqanyuliwe.MP3, MP4 kanye ne-MP6 azitholakalanga kuqhoqhoqho wanoma yiziphi izilwane eziphilayo.Lapho imigudu yomoya yamagundane ithwetshulwa ngemva kokubulawa kwezilwane ngokobuntu, izinhlayiya zahlala zingabonakali ngisho nalapho ivolumu eyengeziwe yengezwa kusetshenziswa i-iron oxide ne-MP ebonakalayo kuphela i-syringe ye-syringe. ngakho-ke yasetshenziswa ukuze kuhlolwe futhi kuvezwe indlela yokuziphatha kwe-MP.
Ukubeka uzibuthe phezu koqhoqho ngesikhathi sokulethwa kwe-MP kubangele abaningi, kodwa hhayi wonke, ukuthi amaLungu ePhalamende agxile emkhakheni wokubuka.Izinhlayiya ezingena kuqhoqhoqho zibonwa kangcono ezilwaneni ezihlatshelwe abantu.Umfanekiso 7 kanye ne-Supplementary Video S6: I-MP5 ikhombisa ukuthwebula kazibuthe okusheshayo nokuqondanisa kwezinhlayiya ebusweni bomgudu wokuphefumula owufunayo we-MPA uqondise uqhoqhoqho. ethungatha kude kuqhoqhoqho ngemva kokulethwa kwe-MP, amanye amaLungu ePhalamende atholakale eduzane ne-carina, okuphakamisa ukuthi amandla kazibuthe ayenganele ukuqoqa nokugcina wonke amaLungu ePhalamende, njengoba ayelethwa endaweni yamandla kazibuthe amaningi ngesikhathi soketshezi.
Izithombe ezisuka ku-(a) ngaphambi kanye (b) ngemva kokulethwa kwe-MP5 kuqhoqhoqho legundane elisanda kubulawa nozibuthe obekwe ngenhla kwendawo yokuthwebula izithombe. Indawo esesithombeni itholakala phakathi kwamasongo oqwanga omabili. Ngaphambi kokulethwa kwe-MP, kunoketshezi oluthile endleleni yomoya. Ibhokisi elibomvu liqukethe ukukhuliswa okugqamisa ukugqama.Lezi zithombe zivela kuvidiyo engu-5 ekhonjisiwe ku-Supply 6MP.
Ukuhumusha uzibuthe kuqhoqhoqho ku-vivo kubangele ukuthi iketango le-MP lishintshe i-engeli ngaphakathi kwendawo yomoya ngendlela efana naleyo ebonakala kuma-capillaries (bona Umfanekiso 8 kanye ne-Supplementary Video S7:MP5). Nokho, ocwaningweni lwethu, ama-MP awakwazanga ukuhudulwa phezu komzila womoya ophilayo ngendlela abengenza ngayo ngama-capillaries. iphinde yathola ukuthi iyunithi yezinhlamvu zezinhlayiyana ibonakala ishintsha ukujula kongqimba lwamanzi angaphezulu lapho uzibuthe uhanjiswa ngobude kuqhoqhoqho, futhi uyanwebeka lapho uzibuthe uhanjiswa ngokuqondile phezulu futhi iyunithi yezinhlamvu izungeziswe ibekwe endaweni eqondile (bona Ividiyo Eyengeziwe S7). : MP5 ngo-0:09, phansi kwesokudla).Iphethini yesici yokunyakaza yashintsha lapho uzibuthe uhunyushwa phezulu kuqhoqhoqho ngokusemaceleni (okungukuthi, kwesokunxele noma kwesokudla sesilwane kunokuhambisana nobude boqhoqhoqho).Izinhlayiya zazisabonakala ngokucacile njengoba zinyakaza, kodwa lapho uzibuthe ekhishwa kuqhoqhoqho, amathiphu ezinhlayiyana ze-Video ku-Spplement5: 0:08).Lokhu kuhambisana nokuziphatha kwe-MP esikubonile ngaphansi kwensimu kazibuthe esetshenziswa ku-capillary yengilazi.
Isibonelo sezithombe ezibonisa i-MP5 kuqhoqhoqho wegundane elibulala izinzwa.(a) Uzibuthe usetshenziselwa ukuthola izithombe ngenhla nakwesokunxele kuqhoqhoqho, bese (b) ngemva kokuba uzibuthe uyiswe kwesokudla. Ibhokisi elibomvu liqukethe ukukhuliswa okugqamisa ukugqama.Lezi zithombe zisuka kuvidiyo eboniswe ku-Supplementary Video S7:MP5.
Lapho izigxobo ezimbili zilungiswa endaweni esenyakatho naseningizimu ngenhla nangaphansi koqhoqhoqho (okungukuthi ukuheha; Umdwebo 3b), ama-MP chords avela isikhathi eside futhi abekwe eceleni komzila woqhoqhoqho esikhundleni sendawo yoqhoqhoqho (bona i-Supplementary Video S9:MP5). Nokho, ukugxila okuphezulu kwezinhlayiya kwakungeyona indawo yomgogodla owodwa (i-trachea). ukulethwa lapho kusetshenziswa idivayisi enozibuthe okabili, okwenzeka ngokuvamile lapho kusetshenziswa idivayisi kazibuthe owodwa.Kwathi uma uzibuthe oyedwa ulungiselelwa ukuxosha izigxobo zihlehliswe (Fig. 3c), inani lezinhlayiya ezibonakalayo endaweni yokubuka alizange likhule ngemva kokulethwa.Ukusethwa kwakho kokubili kozibuthe okabili kuyinselele ngenxa yokuphakama, ukuthi i-push field ishintshe amandla kazibuthe. uzibuthe owodwa uhambisana nomgwaqo womoya kodwa udlula ku-airway ngamadigri angu-90 ukuze imigqa yensimu iwele udonga lwe-tracheal orthogonally (Fig. 3d), i-orientation eklanyelwe ukucacisa ukuthi ukuhlanganisa kwezinhlayiyana odongeni oluseceleni kungabonwa yini. ukucushwa kwe-orientation (Umfanekiso 3a) wakhethelwa izifundo ze-vivo gene carrier.
Lapho isilwane senziwa isithombe ngokuphindaphindiwe ngokushesha ngemva kokubulawa komuntu, ukungabikho kokunyakaza kwezicubu okudidayo kwakusho ukuthi imigqa yezinhlayiyana emincane nemifushane ingabonakala endaweni ecacile ye-interchondral, "ngokuzamazama" ngokuhambisana nokunyakaza kokuhumusha kukazibuthe. Noma kunjalo, namanje akaboni ngokucacile ubukhona nokunyakaza kwezinhlayiya ze-MP6.
I-LV-LacZ titer yayingu-1.8 × 108 TU/ml, futhi ngemva kwe-1: 1 ixutshwe ne-CombiMag MP (MP6), izilwane zithole umthamo we-50 μl we-tracheal we-9 × 107 TU/ml LV imoto (okungukuthi 4.5 × 106 TU/rat). ).Kulezi zifundo, esikhundleni sokuhumusha uzibuthe ngesikhathi sokubeletha, simise uzibuthe endaweni eyodwa ukuze sinqume ukuthi ukuhanjiswa kwe-LV (a) kungase kuthuthukiswe uma kuqhathaniswa nokulethwa kwe-vector lapho ingekho inkambu kazibuthe, futhi (b) amaseli e-Airway angaba agxilile adluliselwa ezifundeni eziqondiwe kazibuthe zomzila womoya ophezulu.
Ukuba khona kwamagnetic kanye nokusetshenziswa kwe-CombiMag kuhlanganiswe nama-LV vectors akuzange kubonakale kunemiphumela emibi empilweni yezilwane, njengoba kwenza iphrothokholi yethu evamile yokulethwa kwevector ye-LV.Izithombe zangaphambili zesifunda soqhoqhoqho ngaphansi kokuphazamiseka kwemishini (I-Supplementary Fig. 1) ibonise ukuthi kwakukhona amazinga aphezulu kakhulu okuguqulwa kweqembu lezilwane lapho i-ogly-MP iphathwa nge-magneti encane ye-LV (LV-MP). inani le-LacZ staining eluhlaza okwesibhakabhaka lalikhona eqenjini lokulawula (Fig. 9b) .Ukulinganisa kwezindawo ezijwayelekile ze-X-Gal ezingcolile kubonise ukuthi ukuphathwa kwe-LV-MP phambi kwendawo kazibuthe kukhiqize ukuthuthukiswa okucishe kube yi-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 yokudlulisa ye-LacZ evamile ngaphakathi kuqhoqhoqho lapho usebenzisa uzibuthe (*p = 0.029, t-test, n = 3 iqembu ngalinye, kusho ± SEM).
Izigaba ezinebala elibomvu ngokushesha ezimaphakathi (isibonelo esiboniswe ku-Supplementary Fig. 2) zibonise amaseli ane-LacZ akhona ngephethini efanayo nasendaweni njengoba kubikwe ngaphambilini.
Inselele eyinhloko yokwelashwa kwezakhi zofuzo zomoya ihlala iwukuba kwasendaweni okunembile kwezinhlayiya zenkampani yenethiwekhi ezifundeni ezithakaselwayo kanye nokufinyelela amazinga aphezulu okusebenza kahle kokudluliselwa ephashini elinyakazayo lapho kukhona ukugeleza komoya kanye nokukhishwa kwamafinyila asebenzayo.Kubathwali be-LV abaklanyelwe ukwelapha isifo somoya we-CF, okwandisa isikhathi sokuhlala kwezinhlayiya zenkampani yenethiwekhi ngaphakathi kwemigudu yomoya eqhubayo kube yinto engakaze ibonwe kuze kube manje. ukuthuthukisa ukuguqulwa kwezakhi zofuzo kunezinzuzo uma kuqhathaniswa nezinye izindlela zokulethwa kwezakhi zofuzo ezifana ne-electroporation, njengoba ingahlanganisa ubulula, ukusebenza kahle kwezindleko, ukulethwa kwasendaweni, ukusebenza kahle okwandisiwe, izikhathi ezimfishane zokufukamela, futhi mhlawumbe ne-dose yenkampani yenethiwekhi encane10. Nokho, ukufakwa kwe-vivo nokuziphatha kwezinhlayiya kazibuthe emigwaqweni yomoya ngaphansi kwethonya lamandla kazibuthe angaphandle akukaze kuchazwe indlela ye-fegene yokubonisa noma ukubonakaliswa kwe-vivo akukaze kuchazwe. amazinga emigudwini yomoya ephilayo engaguquki.
Ukuhlola kwethu kwe-in vitro synchrotron PCXI kubonise ukuthi zonke izinhlayiya esizihlolile, ngaphandle kwe-polystyrene MP, zazibonakala ekusetheni izithombe esizisebenzisile.Ebukhoneni bensimu kazibuthe, ama-MP enza izintambo ubude bazo obuhlobene nohlobo lwezinhlayiya namandla kazibuthe (okungukuthi ukusondela nokunyakaza kukazibuthe).Njengoba kuboniswe kuMfanekiso 10 yizinhlayiyana ezibunjwa ngozibuthe ngamunye. iheha indawo yayo yasendaweni kazibuthe.Lezi zindawo ezihlukene zenza ezinye izinhlayiya ezifanayo zihlangane futhi zixhume, ngokunyakaza okufana neyunithi yeqembu ngenxa yamandla endawo avela kumandla asendaweni akhangayo nanyanyekayo kwezinye izinhlayiya.
Izitimela zezinhlayiya ezibonisa uhlelo (a,b) ezikhiqizwe ngaphakathi kwama-capillary agcwele uketshezi kanye (c,d) noqhoqhoqho ogcwele umoya.Qaphela ukuthi ama-capillaries noqhoqhoqho akudonseleki esikalini.Iphaneli (a) iqukethe nencazelo ye-MP, equkethe izinhlayiya ze-Fe3O4 ezihlelwe ngeyunithi yezinhlamvu.
Lapho uzibuthe ugudluzwa ngaphezu kwe-capillary, i-engeli yochungechunge lwezinhlayiyana yafinyelela embundwini obalulekile we-MP3-5 equkethe i-Fe3O4, okwathi ngemva kwalokho iyunithi yezinhlamvu ayibange isahlala endaweni yokuqala, kodwa yagudluzwa phezulu yaya endaweni entsha.Zibuthe.Lo mphumela ungenzeka ngenxa yokuthi i-glass capillary surface ibushelelezi ngokwanele ukuvumela lokhu kunyakaza ukuba kwenzeke. Kuyathakazelisa ukuthi le MP izinhlayiya bezizincane, zinezimbotshana ezihlukene noma amashaji angaphezulu, noma uketshezi lwenkampani yenethiwekhi kuthinte ikhono lazo lokunyakaza.Ukugqama kwesithombe sezinhlayiya ze-CombiMag nakho kubuthakathaka, okuphakamisa ukuthi uketshezi nezinhlayiyana zingase zibe nokuminyana okufanayo ngakho-ke azihambisani kalula. akumangazi ukuthi amandla kazibuthe kanye nebanga phakathi kukazibuthe nendawo okuhloswe kuyo kubaluleke kakhulu. Uma ihlanganiswa, le miphumela iphinde iphakamise ukuthi, nakuba omagnethi bekwazi ukuthwebula ama-MP amaningi ageleza endaweni okuqondiswe kuyo, mancane amathuba okuthi omazibuthe bangathenjelwe kuzo ukuze bahambise izinhlayiya ze-CombiMag phezu koqhoqhoqho. umuthi.
Lapho izinhlayiya zilethwa emzimbeni, kunzima ukuzibona kumongo wezicubu zomzimba ezihambayo eziyinkimbinkimbi, kodwa ikhono lokuzibona lathuthukiswa ngokuhumusha imagnethi ivundlile ngenhla kuqhoqhoqho ukuze "inyakazise" izintambo ze-MP. Nakuba ukuthwebula izithombe bukhoma kungenzeka, kulula ukubona ukunyakaza kwezinhlayiyana uma isilwane sesibulewe ngobuntu. ezinye izinhlayiya zazivame ukutholakala ngokuqhubekayo eduze kwe-trachea.Ngokuphambene nezifundo ze-in vitro, izinhlayiya azikwazi ukuhudulwa eduze kwe-trachea ngokuhumusha i-magnet.Lokhu kutholakala kuhambisana nendlela i-mucus egqoke ubuso be-trachea ngokuvamile icubungula izinhlayiya eziphefumulayo, ziwabambe ku-mucus futhi kamuva zisuswe yi-mucociliary clearance mechanism.
Sacabanga ukuthi ukusetshenziswa kwamagnet ukuze kuhehe ngenhla nangaphansi koqhoqhoqho (Fig. 3b) kungase kubangele inkambu kazibuthe efanayo, kunokuba inkambu kazibuthe egxile kakhulu endaweni eyodwa, okungenzeka iholele ekusabalaliseni okufanayo kwezinhlayiya. Nokho, ucwaningo lwethu lokuqala alutholanga ubufakazi obucacile bokusekela le nkolelo. ukubekwa kwezinhlayiya ezengeziwe endaweni enemifanekiso.Lokhu okutholakele okubili kubonisa ukuthi ukusethwa kozibuthe okumbaxambili akuthuthukisi kakhulu ukulawulwa kwendawo kokuqondiswa kwe-MP, nokuthi amandla kazibuthe anamandla avelayo anzima ukukulungisa, okwenza le ndlela ingasebenzi kahle.Ngokufanayo, ukuqondisa uzibuthe ngenhla nangoqhoqhoqho (Fig. 3d) nakho akuzange kwenyuse inani lezinhlayiya ezigcinwe endaweni ethile ngenxa yokuthi lezi zimo zingagcinwa endaweni ethile. kumandla kazibuthe aphansi ngaphakathi kwendawo yokubeka.Ngakho-ke, ukucushwa kwezizibuthe ze-angle engu-30-degree eyodwa (Umfanekiso 3a) kubhekwa njengendlela elula nephumelelayo kakhulu yokuhlolwa kwe-vivo.
Ucwaningo lwe-LV-MP lubonise ukuthi lapho amavekhtha e-LV ehlanganiswa ne-CombiMag futhi ehanjiswa ngemva kokuphazamiseka ngokomzimba phambi kwendawo kazibuthe, amazinga okushintshashintsha anyuswa kakhulu kuqhoqhoqho uma kuqhathaniswa nezilawuli.Ngokusekelwe ocwaningweni lwe-synchrotron imaging kanye nemiphumela ye-LacZ, indawo kazibuthe ngokusobala ikwazile ukulondoloza i-LV ngaphakathi kuqhoqhoqho futhi yehlisa inani lezinhlayiya ezijulile ezihloselwe i-vector pectors ngokushesha. kungase kuholele ekusebenzeni okuphakeme ngenkathi kunciphisa ama-titers alethiwe, ukudluliselwa kwe-off-target, imiphumela emibi yokuvuvukala kanye ne-immune, kanye nezindleko ze-gene carrier.Okubalulekile, ngokusho komkhiqizi, i-CombiMag ingasetshenziswa ngokuhambisana nezinye izindlela zokudlulisa izakhi zofuzo, kuhlanganise namanye ama-viral vectors (njenge-AAV) kanye ne-nucleic acids.