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Ama-vectors ezifuyo ekwelapheni isifo samaphaphu se-cystic fibrosis kufanele aqondise izindlela zomoya ezihambayo ngoba ukudluliselwa kwamaphaphu angaphandle akunikezi izinzuzo zokwelapha. Ukusebenza kahle kokudluliselwa kwegciwane kuhlobene ngqo nesikhathi sokuhlala kwe-vector. Kodwa-ke, uketshezi lokulethwa olufana nabathwali bezakhi zofuzo lusakazeka ngokwemvelo ku-alveoli ngesikhathi sokuphefumulelwa, futhi izinhlayiya zokwelapha zanoma yiluphi uhlobo zisuswa ngokushesha ngokuthuthwa kwe-mucociliary. Ukwandisa isikhathi sokuhlala kwabathwali bezakhi zofuzo emigwaqweni yomoya kubalulekile kodwa kunzima ukukufeza. Izinhlayiya zamagnetic ezihlanganiswe nabathwali bezakhi zofuzo ezingaqondiswa ebusweni bezindlela zomoya zingathuthukisa ukuqondiswa kwesifunda. Ngenxa yezinselele zokubona ngeso lengqondo, ukuziphatha kwezinhlayiya ezincane zamagnetic ebusweni bendlela yomoya lapho kukhona insimu yamagnetic esetshenzisiwe akuqondakali kahle. Inhloso yalolu cwaningo kwakuwukusebenzisa i-synchrotron imaging ukubona ngeso lengqondo ukunyakaza kwe-in vivo kochungechunge lwezinhlayiya zamagnetic ku-trachea yamagundane aphethwe yi-anesthesia ukuhlola amandla kanye namaphethini okuziphatha kwezinhlayiya ngazinye kanye nobuningi emzimbeni. Sabe sesihlola nokuthi ukulethwa kwezinhlayiya zamagnetic ze-lentiviral lapho kukhona insimu yamagnetic kuzokhulisa ukusebenza kahle kokudluliselwa egundwini. I-trachea. Ukuthwebula izithombe ze-X-ray ze-Synchrotron kwembula ukuziphatha kwezinhlayiya ze-magnetic emasimini ashukumisayo nahambayo ku-vitro kanye naku-vivo. Izinhlayiya azikwazi ukudonswa kalula ebusweni bomoya ophilayo ngamagnethi, kodwa ngesikhathi sokuthuthwa, amadiphozithi agxila ensimini yokubuka lapho insimu yamagnetic inamandla kakhulu khona. Ukusebenza kahle kokudluliselwa kwe-transduction nakho kwanda ngokuphindwe kasithupha lapho izinhlayiya ze-magnetic ze-lentiviral zilethwa phambi kwensimu yamagnetic. Ndawonye, ​​le miphumela iphakamisa ukuthi izinhlayiya ze-magnetic ze-lentiviral kanye nezinhlayiya ze-magnetic kungaba izindlela eziwusizo zokuthuthukisa ukuqondiswa kwe-genetic vector kanye nokwandisa amazinga okudluliselwa kwe-transduction ekuqhubeni imigudu yomoya ku-vivo.
I-Cystic fibrosis (CF) ibangelwa ukushintshashintsha kwezakhi zofuzo ezibizwa ngokuthi i-CF transmembrane conductance regulator (CFTR). Iphrotheni ye-CFTR iyisiteshi se-ion esikhona kumaseli amaningi e-epithelial kuwo wonke umzimba, kufaka phakathi imigudu yomoya eqhubayo, indawo enkulu ye-CF pathogenesis. Amaphutha e-CFTR aholela ekuthuthweni kwamanzi okungajwayelekile, okuqeda amanzi ebusweni bomoya futhi kuncishiswe ukujula kwengqimba yoketshezi lomoya (ASL). Lokhu futhi kuphazamisa ikhono lesistimu yokuthutha i-mucociliary (MCT) lokususa izinhlayiya eziphefumulelwe kanye namagciwane emigudwini yomoya. Umgomo wethu ukuthuthukisa ukwelashwa kwezakhi zofuzo kwe-lentiviral (LV) ukuze kunikezwe ikhophi efanele yezakhi zofuzo ze-CFTR futhi kuthuthukiswe impilo ye-ASL, MCT, kanye namaphaphu, kanye nokuqhubeka nokuthuthukisa ubuchwepheshe obusha obukwazi ukulinganisa lezi zilinganiso ku-vivo1.
Ama-vector e-LV angenye yezindlela ezihamba phambili zokwelapha izakhi zofuzo zomoya ze-CF, ikakhulukazi ngoba angahlanganisa unomphela izakhi zofuzo zokwelapha kumaseli ayisisekelo omoya (amaseli okuqala omoya). Lokhu kubalulekile ngoba angabuyisela umswakama ojwayelekile kanye nokuhlanzwa kwe-mucus ngokuhlukanisa kumaseli omoya ahlobene ne-CF asebenza kahle, okuholela ezinzuzweni zokuphila konke. Ama-vector e-LV kufanele aqondiswe endleleni yomoya eqhubayo, njengoba kulapho isifo samaphaphu se-CF siqala khona. Ukulethwa kwe-vector ngokujulile emaphashini kungabangela ukudluliselwa kwe-alveolar, kodwa lokhu akunanzuzo yokwelapha ku-CF. Kodwa-ke, uketshezi olufana nabathwali bezakhi zofuzo luthuthela ngokwemvelo ku-alveoli ngemva kokuphefumulelwa ngemva kokubeletha3,4 futhi izinhlayiya zokwelapha zisuswa ngokushesha emlonyeni yi-MCT. Ukusebenza kahle kokudluliselwa kwe-LV kuhlobene ngqo nobude besikhathi i-vector ehlala eduze kwamaseli aqondiwe ukuvumela ukumuncwa kwamaseli - "isikhathi sokuhlala"5 - esincishiswa kalula ukuhamba komoya okuvamile kwesifunda kanye nokubanjwa kwe-mucus yezinhlayiya ezihlanganisiwe kanye ne-MCT. Ku-CF, ikhono lokwandisa isikhathi sokuhlala se-LV ngaphakathi kwendlela yomoya kubalulekile ukufeza amazinga aphezulu okudluliselwa kulesi sifunda, kodwa kube njalo. kube nzima kakhulu.
Ukuze sinqobe lesi sithiyo, siphakamisa ukuthi izinhlayiya ze-LV magnetic (MPs) zingasiza ngezindlela ezimbili ezihambisanayo. Okokuqala, zingaqondiswa ngogesi endaweni yomoya ukuze kuthuthukiswe ukuqondiswa nokusiza izinhlayiya zezakhi zofuzo ukuthi zihlale endaweni yomoya efiselekayo; kanye ne-ASL) ukuze zithuthele kungqimba lweseli 6. Ama-MP asetshenziswe kabanzi njengezimoto zokulethwa kwemithi eziqondiwe lapho ebopha kuma-antibodies, izidakamizwa ze-chemotherapy, noma amanye ama-molecule amancane anamathela kuma-membrane eseli noma abopha kuma-receptor afanele ebusweni beseli futhi aqoqeke ezindaweni zesimila lapho kukhona ugesi omile. Amasimu Amagnetic Okwelapha Umdlavuza 7. Amanye amasu “aphezulu” ahlose ukushisa ama-MP lapho evezwa emasimu kazibuthe ajikelezayo, ngaleyo ndlela abhubhise amangqamuzana esimila. Isimiso sokudluliselwa kwamandla kazibuthe, lapho insimu kazibuthe isetshenziswa khona njenge-ejenti yokudluliselwa kwamandla ukuze kuthuthukiswe ukudluliselwa kwe-DNA kumaseli, sivame ukusetshenziswa kwi-vitro kusetshenziswa izinhlobo ezahlukene zama-vectors ezakhi zofuzo ezingezona ezamagciwane kanye nezamagciwane emigqeni yamaseli okunzima ukuwadlulisela. Ukusebenza kahle kwe-LV magnetotransfection sekuqinisekisiwe, ngokulethwa kwe-LV-MPs kwi-vitro kumugqa weseli we-epithelial womuntu we-bronchial phambi kwensimu kazibuthe engaguquki, okwandisa ukusebenza kahle kokudluliselwa kwamandla ngama-186 uma kuqhathaniswa ne-LV vector yodwa. I-LV-MP iphinde yasetshenziswa kumodeli ye-CF ye-vitro, lapho ukudluliselwa kwamandla kazibuthe kwandisa ukudluliselwa kwe-LV kuma-interface cultures okuxhumana nomoya ngama-20 uma kukhona i-CF sputum10. Kodwa-ke, ukudluliselwa kwamandla kazibuthe kwe-in vivo kwezitho kuthole ukunakwa okuncane kakhulu futhi kuhlolwe kuphela ezilwaneni ezimbalwa. izifundo11,12,13,14,15, ikakhulukazi emaphashini16,17. Noma kunjalo, amathuba okufakelwa kwamagnetic ekwelashweni kwamaphaphu e-CF acacile. UTan et al.(2020) bathi “ucwaningo lobufakazi bomqondo wokulethwa kwamaphaphu e-nanoparticle magnetic okuphumelelayo luzovula indlela yamasu okuphefumula e-CFTR esikhathini esizayo ukuthuthukisa imiphumela yezokwelapha ezigulini ze-CF”6.
Ukuziphatha kwezinhlayiya ezincane zikagesi ezikhungweni zomoya lapho kukhona insimu kazibuthe esetshenzisiwe kunzima ukukubona nokufunda, ngakho-ke akuqondwa kahle.Kwezinye izifundo, sakha indlela ye-synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) yokubona ngeso lengqondo futhi silinganise izinguquko zemizuzu e-vivo ekujuleni kwe-ASL18 kanye nokuziphatha kwe-MCT19,20 ukukala ngqo igesi Ukumuncwa kwamanzi kobuso bemisele futhi kwasetshenziswa njengesibonakaliso sokuqala sokusebenza kahle kokwelashwa.Ngaphezu kwalokho, indlela yethu yokuhlola i-MCT isebenzisa izinhlayiya ezinobubanzi obungu-10–35 µm ezakhiwe nge-alumina noma ingilazi ye-refractive index ephezulu njengezimpawu ze-MCT ezibonakalayo kusetshenziswa i-PB-PCXI21.Zombili lezi zindlela zifanelekile ekubonisweni kwezinhlobo ezahlukene zezinhlayiya, kufaka phakathi i-MP.
Ngenxa yokuxazululeka kwayo okuphezulu kwendawo kanye nesikhathi, amasu ethu okuhlaziya e-ASL kanye ne-MCT asekelwe ku-PB-PCXI afaneleka kahle ekuhloleni ukuguquguquka kanye namaphethini okuziphatha kwezinhlayiya ezilodwa kanye nezinkulu emzimbeni ukuze asisize siqonde futhi sithuthukise amasu okulethwa kwezakhi zofuzo ze-MP. Indlela esiyisebenzisayo lapha ivela ezifundweni zethu sisebenzisa i-beamline ye-SPring-8 BL20B2, lapho sibone khona ukunyakaza koketshezi ngemuva kokulethwa kwedosi ye-vector ye-sham emigundwini yomoya yamagundane ekhaleni nasephaphu ukuze kusizwe ekuchazeni amaphethini ethu okubonakaliswa kwezakhi zofuzo angalingani abonwe ezifundweni zethu zezilwane zedosi ye-gene carrier dose 3,4.
Inhloso yalolu cwaningo kwakuwukusebenzisa i-synchrotron PB-PCXI ukubona ngeso lengqondo ukunyakaza kwe-in vivo kochungechunge lwama-MP ku-trachea yamagundane aphilayo. Lezi zifundo zokuthwebula izithombe ze-PB-PCXI zenzelwe ukuhlola uhla lwama-MP, amandla ensimu yamagnetic, kanye nezindawo ukuze kutholakale umphumela wazo ekuhambeni kwe-MP. Sacabanga ukuthi insimu yamagnetic esetshenziswa ngaphandle ingasiza i-MP ethunyelwe ukuthi ihlale noma ithuthele endaweni eqondiwe. Lezi zifundo zisivumele futhi ukuthi sithole ukwakheka kwamagnet okwandisa inani lezinhlayiya ezigcinwe ku-trachea ngemuva kokufakwa. Ochungechungeni lwesibili lwezifundo, sifuna ukusebenzisa lokhu kulungiselelwa okuhle kakhulu ukuze sibonise iphethini yokudluliselwa ebangelwa ukulethwa kwama-LV-MP ku-in vivo ku-airway yamagundane, ngokusekelwe ekucabangeni ukuthi ukulethwa kwama-LV-MP kumongo wokuqondiswa komoya kuzoholela ekusebenzeni kahle kokudluliselwa kwe-LV okuthuthukisiwe.
Zonke izifundo zezilwane zenziwe ngokwemigomo evunyelwe yi-University of Adelaide (M-2019-060 kanye ne-M-2020-022) kanye ne-SPring-8 Synchrotron Animal Ethics Committee. Ukuhlolwa kwenziwe ngokwemihlahlandlela ye-ARRIVE.
Zonke izithombe ze-X-ray zenziwe kumugqa we-BL20XU ku-synchrotron ye-SPring-8 eJapane, kusetshenziswa isethaphu esifana naleyo echazwe ngaphambilini. Ngamafuphi, ibhokisi lokuhlola lalitholakala ngamamitha angu-245 ukusuka endandatho yokugcina ye-synchrotron. Ibanga lesampula-kuya-kumtholi lisetshenziswa ezifundweni zokuthwebula izinhlayiya kanye nama-0.3 m ezifundweni zokuthwebula izithombe ze-in vivo ukukhiqiza imiphumela yokungafani kwesigaba. Kwasetshenziswa amandla okukhanya kwe-monochromatic angu-25 keV. Izithombe zathathwa kusetshenziswa i-converter ye-X-ray enesinqumo esiphezulu (i-SPring-8 BM3) ehlanganiswe ne-sCMOS detector. I-converter iguqula ama-X-ray abe ukukhanya okubonakalayo kusetshenziswa i-scintillator engu-10 µm obukhulu (i-Gd3Al2Ga3O12), eqondiswa ku-sensor ye-sCMOS kusetshenziswa i-× 10 microscope objective (NA 0.3). I-sCMOS detector yayiyi-Orca-Flash4.0 (i-Hamamatsu Photonics, eJapan) enosayizi we-array ongu- Amaphikseli angu-2048 × 2048 kanye nosayizi wephikseli ongahluziwe ongu-6.5 × 6.5 µm. Lokhu kusetha kuveza usayizi wephikseli we-isotropic osebenzayo ongu-0.51 µm kanye nensimu yokubuka engaba ngu-1.1 mm × 1.1 mm. Ubude bokuvezwa obungu-100 ms bukhethiwe ukuze kwandiswe isilinganiso sesiginali-kuya-kumsindo sezinhlayiya zamagnetic ngaphakathi nangaphandle kwendlela yomoya ngenkathi kunciphisa ukunyakaza okubangelwa ukuphefumula. Ezifundweni ze-in vivo, i-shutter ye-X-ray esheshayo yabekwa endleleni ye-X-ray ukuze kuncishiswe umthamo wokukhanya ngokuvimbela umugqa we-X-ray phakathi kokuvezwa.
Isithwali se-LV asizange sisetshenziswe kunoma yiziphi izifundo ze-imaging ze-SPring-8 PB-PCXI ngoba igumbi le-imaging le-BL20XU alinaso isitifiketi se-Biosafety Level 2. Esikhundleni salokho, sikhethe uhla lwama-MP aziwayo kahle kubahlinzeki ababili bezentengiselwano—ahlanganisa uhla lobukhulu, izinto zokwakha, ukugcwala kwensimbi, kanye nezicelo—okokuqala ukuqonda ukuthi amasimu kazibuthe athinta kanjani ukunyakaza kwe-MP ngaphakathi kwama-capillaries engilazi, bese kuba semigwaqweni yomoya ephilayo. ebusweni. Ama-MP asukela ku-0.25 kuya ku-18 μm ngobukhulu futhi enziwe ngezinto ezahlukahlukene (bheka Ithebula 1), kodwa ukwakheka kwesampula ngayinye, okuhlanganisa nosayizi wezinhlayiya ezisebenza ngogesi ngaphakathi kwe-MP, akwaziwa. Ngokusekelwe ezifundweni zethu ezibanzi ze-MCT 19, 20, 21, 23, 24, silindele ukuthi ama-MP amancane njengo-5 μm angabonakala ebusweni bomoya we-tracheal, isibonelo ngokususa ozimele abalandelanayo ukuze kubonakale ukubonakala okuthuthukisiwe kokunyakaza kwe-MP. I-MP eyodwa enobukhulu obungu-0.25 μm incane kunesinqumo sedivayisi yokuthwebula izithombe, kodwa i-PB-PCXI kulindeleke ukuthi ibone umehluko wevolumu yayo kanye nokunyakaza koketshezi olungaphezulu lapho ibekwe khona ngemva kokufakwa.
Amasampula e-MP ngayinye kuThebula 1 alungiselelwe kuma-capillary engilazi angu-20 μl (i-Drummond Microcaps, PA, e-USA) anobubanzi bangaphakathi obungu-0.63 mm. Izinhlayiya ze-corpuscular ziyatholakala emanzini, kuyilapho izinhlayiya ze-CombiMag zitholakala oketshezini oluyimfihlo lomkhiqizi. Ithubhu ngalinye ligcwele uketshezi (cishe u-11 μl) futhi libekwe esibambini sesampula (bheka Umfanekiso 1). Ama-capillary engilazi abekwe ngokuvundlile esigabeni sesampula ebhokisini lokuthwebula izithombe, ngokulandelana, futhi abekwa emaphethelweni oketshezi. I-magnet engu-19 mm ububanzi (28 mm ubude) ye-nickel shell rare earth neodymium iron boron (NdFeB) magnet (N35, cat. no. LM1652, Jaycar Electronics, e-Australia) ene-magnetization eseleyo ye-1.17 Tesla inamathiselwe esigabeni sokuhumusha esihlukile ukuze kufezwe Shintsha indawo yayo kude ngesikhathi sokuthwebula izithombe. Ukutholwa kwesithombe se-X-ray kuqala lapho i-magnet ibekwe cishe ku-30 ​​mm ngaphezu kwesampula, futhi izithombe zitholakala ngesivinini samafreyimu angu-4 ngomzuzwana. Ngesikhathi sokuthwebula izithombe, i-magnet yasondezwa eduze kwengilazi. ipayipi le-capillary (cishe i-1 mm ukusuka lapha) bese lihunyushwa eceleni kwepayipi ukuze kuhlolwe imiphumela yamandla ensimu kanye nendawo.
Ukusethwa kwesithombe se-in vitro okuqukethe amasampula e-MP kuma-capillary engilazi esigabeni sokuhumusha sesampula se-xy. Indlela yomsebe we-X-ray imakwe ngomugqa obomvu onezinhlayiya.
Lapho ukubonakala kwama-MPs ngaphakathi kwe-vitro sekuqinisekisiwe, iqembu elincane lawo lahlolwa ngaphakathi kwe-vivo kumagundane ase-Wistar angama-albino asendle (~amasonto ayi-12 ubudala, ~200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Japan) kanye namagundane angu-4 mg/kg butorphanol (Vetorphale®, Meiji Seika) ahlinzwa nge-anesthesia ngengxube ye-Pharma), Japan) ngokujova ngaphakathi kwe-peritoneal. Ngemva kokuhlinzwa, alungiselelwa ukuthwebula izithombe ngokususa uboya obuzungeze i-trachea, afake i-endotracheal tube (ET; 16 Ga iv cannula, Terumo BCT) bese ewavimba ukuba alale phansi epuletini lokuthwebula elenziwe ngokwezifiso eliqukethe isikhwama sokushisa ukuze kugcinwe izinga lokushisa lomzimba 22. Ipuleti lokuthwebula izithombe labe selinamathiselwa esigabeni sokuhumusha isampula ebhokisini lokuthwebula izithombe nge-engeli encane ukuze kuqondiswe i-trachea ngokuvundlile esithombeni se-X-ray, njengoba kuboniswe kuMfanekiso 2a.
(a) Ukusetha izithombe ze-in vivo ebhokisini lokuthwebula izithombe le-SPring-8, indlela yomugqa we-X-ray imakwe ngomugqa obomvu onezinhlayiya.(b,c) Ukubekwa kwe-magnet ku-trachea kwenziwe kude kusetshenziswa amakhamera amabili e-IP afakwe ngendlela eqondile. Ngakwesobunxele sesithombe sesikrini, iluphu yentambo ebambe ikhanda ingabonakala, kanye ne-cannula yokulethwa endaweni yayo ngaphakathi kwe-ET tube.
Uhlelo lokupompa i-syringe olulawulwa kude (UMP2, World Precision Instruments, Sarasota, FL) olusebenzisa i-syringe yengilazi engu-100 μl luxhunywe ku-PE10 tube (OD 0.61 mm, ID 0.28 mm) ngenaliti engu-30 Ga. Maka i-syringe ukuze uqinisekise ukuthi i-tip isendaweni efanele ku-trachea lapho ufaka i-ET tube. Kusetshenziswa i-micropump, i-syringe plunger yakhishwa ngenkathi i-tip ye-tube ifakwa kusampula ye-MP ezolethwa. I-tube yokulethwa elayishiwe yabe isifakwa ku-endotracheal tube, ibeka i-tip engxenyeni eqinile yensimu yethu yamagnetic esetshenziswayo elindelwe. Ukutholwa kwesithombe kwalawulwa kusetshenziswa i-respiration detector exhunywe ebhokisini lethu lesikhathi elisekelwe ku-Arduino, futhi zonke izimpawu (isb. izinga lokushisa, ukuphefumula, ukuvulwa/ukuvalwa kwe-shutter kanye nokutholwa kwesithombe) zaqoshwa kusetshenziswa i-Powerlab kanye ne-LabChart (AD Instruments, eSydney, e-Australia) 22. Lapho kuthwetshulwa izithombe Lapho indawo evalekile ingafinyeleleki, amakhamera amabili e-IP (Panasonic BB-SC382) abekwa cishe kuma-90° ukuze futhi zasetshenziswa ukuqapha indawo yamagnethi maqondana ne-trachea ngesikhathi sokuthwebula izithombe (Isithombe 2b, c). Ukuze kuncishiswe izinto zokunyakaza, kwatholakala isithombe esisodwa ngokuphefumula ngesikhathi sokugeleza kokugcina kolwandle.
Imagnethi ixhunywe esigabeni sesibili esingatholakala kude ngaphandle kwendlu yokuthwebula izithombe. Kuhlolwe izikhundla ezahlukene zemagnethi kanye nokucushwa, okuhlanganisa: Kufakwe nge-engeli engaba ngu-30° ngaphezu kwe-trachea (ukucushwa okuboniswe kuZithombe 2a no-3a); imagnethi eyodwa ngaphezu kwesilwane kanye nenye ngezansi, nezingongolo ezibekwe ukuheha (Isithombe 3b); imagnethi eyodwa ngaphezu kwesilwane kanti enye ngezansi, nezingongolo ezibekwe ukuxosha (Isithombe 3c); kanye nemagnethi eyodwa ngaphezulu futhi iqonde ngqo ku-trachea (Isithombe 3d). Uma isilwane kanye nemagnethi sezilungisiwe futhi i-MP ezohlolwa isilayishiwe epompo yesirinji, nikeza umthamo we-50 μl ngesilinganiso esingu-4 μl/sec ngenkathi uthola izithombe. Imagnethi ibe isihanjiswa emuva naphambili noma eceleni ngaphesheya kwe-trachea ngenkathi iqhubeka nokuthola izithombe.
Ukumiswa kwemagnethi yokuthwebula izithombe ze-in vivo (a) imagnethi eyodwa ngaphezu kwe-trachea nge-engeli engaba ngu-30°, (b) omamagnethi ababili ababekwe ukuheha, (c) omamagnethi ababili ababekwe ukuxosha, (d) imagnethi eyodwa ngaphezulu futhi eqondile ku-trachea. Umqapheli wabheka phansi kusukela emlonyeni kuya emaphashini nge-trachea, futhi ugongolo lwe-X-ray lwadlula ohlangothini lwesobunxele lwegundane lwaphuma ohlangothini lwesokudla. Imagnethi ihanjiswa ngobude bomgudu womoya noma ngakwesobunxele nangakwesokudla ngaphezu kwe-trachea ngakwesokunene sogongolo lwe-X-ray.
Siphinde safuna ukuthola ukubonakala kanye nokuziphatha kwezinhlayiya emigwaqweni yomoya ngaphandle kokuphefumula okuphazamisayo kanye nokunyakaza kwenhliziyo. Ngakho-ke, ekupheleni kwesikhathi sokuthwebula izithombe, izilwane zabulawa ngobuntu ngenxa yokudlula i-pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). Ezinye izilwane zashiywa epulatifomu yokuthwebula izithombe, futhi lapho ukuphefumula nokushaya kwenhliziyo sekumile, inqubo yokuthwebula izithombe yaphindwa, kwengezwa umthamo owengeziwe we-MP uma kungekho MP obonakalayo ebusweni bomoya.
Izithombe ezitholiwe zalungiswa nge-flat-field kanye ne-dark-field zabe sezihlanganiswa zaba yi-movie (amafreyimu angu-20 ngomzuzwana; isivinini esijwayelekile esingu-15-25 × kuye ngesivinini sokuphefumula) kusetshenziswa iskripthi esenziwe ngokwezifiso esibhalwe ku-MATLAB (R2020a, The Mathworks).
Zonke izifundo zokulethwa kwe-vector yezakhi zofuzo ze-LV zenziwa eLaboratory Animal Research Facility eNyuvesi yase-Adelaide futhi zazihlose ukusebenzisa imiphumela yokuhlolwa kwe-SPring-8 ukuhlola ukuthi ukulethwa kwe-LV-MP lapho kukhona insimu yamagnetic kungathuthukisa yini ukudluliselwa kwezakhi zofuzo emzimbeni. Ukuze kuhlolwe imiphumela ye-MP kanye nensimu yamagnetic, amaqembu amabili ezilwane aphathwa: iqembu elilodwa lanikezwa i-LV-MP lapho kufakwe khona i-magnet, kanti elinye iqembu lathola iqembu lokulawula eline-LV-MP ngaphandle kwe-magnet.
Amavekhtha ezakhi zofuzo ze-LV akhiqizwa kusetshenziswa izindlela ezichazwe ngaphambilini 25, 26. Ivekhtha ye-LacZ iveza i-nuclear-located beta-galactosidase gene eqhutshwa yi-constitutive MPSV promoter (LV-LacZ), ekhiqiza umkhiqizo wokusabela okuluhlaza okwesibhakabhaka kumaseli ashintshiwe, abonakala emaphethelweni ezicubu zamaphaphu kanye nezingxenye zezicubu. I-Titration yenziwa kuma-cell cultures ngokubala ngesandla inani lamaseli e-LacZ positive nge-hemocytometer ukuze kubalwe i-titer ku-TU/ml. Abathwali bayagcinwa ku--80 °C, bancibilikiswe ngaphambi kokusetshenziswa, bese beboshwa ku-CombiMag ngokuxuba ngesilinganiso esingu-1:1 bese befakwa eqhweni okungenani imizuzu engama-30 ngaphambi kokulethwa.
Amagundane ajwayelekile e-Sprague Dawley (n = 3/iqembu, ~2-3 ahlinzwa ngaphakathi kwesisu ngengxube ye-0.4 mg/kg medetomidine (Domitor, Ilium, Australia) kanye nomjovo we-60 mg/kg ketamine (Ilium, Australia) onenyanga) ip) kanye nokufakwa komlomo okungewona ukuhlinzwa nge-16 Ga iv cannula. Ukuqinisekisa ukuthi izicubu zomoya we-tracheal zithola ukudluliselwa kwe-LV, kwalungiswa kusetshenziswa inqubo yethu yokuphazamiseka komshini echazwe ngaphambilini, lapho ubuso bomoya we-tracheal bahlikihlwa khona ngebhasikidi yocingo (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. Ukuphathwa kwe-LV-MP nge-tracheal kwabe sekwenziwa kwikhabhinethi yokuphepha kwezinto eziphilayo cishe imizuzu eyi-10 ngemva kokuphazamiseka.
Insimu yamagnetic esetshenziswe kulolu vivinyo yahlelwa ngendlela efanayo nesifundo se-X-ray se-in vivo, kanye nama-magnet afanayo abanjwe ngaphezu kwe-trachea kusetshenziswa iziqeshana ze-distillation stent (Isithombe 4). Umthamo we-50 μl (2 × 25 μl aliquots) we-LV-MP wafakwa ku-trachea (n = 3 izilwane) kusetshenziswa i-pipette equkethe ithiphu lejeli njengoba kuchaziwe ngaphambilini. Iqembu lokulawula (n = 3 izilwane) lathola ama-LV-MP afanayo ngaphandle kokusebenzisa i-magnet. Ngemva kokuthi ukufakwa sekuqediwe, i-cannula iyasuswa epayipini le-ET bese isilwane siyakhishwa. I-magnet ihlala endaweni yayo imizuzu eyi-10, bese isuswa. Amagundane athole umthamo we-meloxicam ongaphansi kwesikhumba (1 ml/kg) (Ilium, Australia) olandelwa ukuguqulwa kwe-anesthesia ngokujova i-1 mg/kg ye-atipamazole hydrochloride (Antisedan, Zoetis, Australia). Amagundane agcinwa efudumele futhi eqashwe kuze kube yilapho elulama ngokuphelele ekuhlaselweni yi-anesthesia.
Idivayisi yokulethwa ye-LV-MP ekhabetheni lokuphepha lebhayoloji. Ihabhu le-Luer elimpunga elikhanyayo le-ET tube lingabonakala liphuma emlonyeni futhi isihloko sejeli se-pipette esiboniswe esithombeni sifakwa nge-ET tube kuze kube sekujuleni okufunwayo ku-trachea.
Isonto elilodwa ngemuva kwenqubo yokulinganisa i-LV-MP, izilwane zabulawa ngobuntu ngokuphefumula i-CO2 engu-100% futhi ukubonakaliswa kwe-LacZ kwahlolwa kusetshenziswa ukwelashwa kwethu okujwayelekile kwe-X-gal. Amasongo amathathu e-caudal amaningi kakhulu e-cartilaginous asusiwe ukuqinisekisa ukuthi noma yimuphi umonakalo womshini noma ukugcinwa koketshezi kusuka ekubekweni kwe-endotracheal tube kwakungafakwanga ekuhlaziyweni. I-trachea ngayinye yasikwa ngobude ukuze kudalwe izingxenye ezimbili zokuhlaziywa, futhi zafakwa esitsheni esinerabha ye-silicone (Sylgard, Dow Inc) kusetshenziswa inaliti ye-Minutien (Fine Science Tools) ukuze kubonwe ubuso be-luminal. Ukusatshalaliswa kanye nephethini yamaseli ashintshiwe kwaqinisekiswa ngezithombe zangaphambili kusetshenziswa i-Nikon microscope (SMZ1500) ngekhamera ye-DigiLite kanye nesofthiwe ye-TCapture (Tucsen Photonics, China). Izithombe zatholakala ngokukhulisa okungu-20x (kufaka phakathi indawo ephezulu kakhulu yobubanzi obugcwele be-trachea), kanye nobude bonke be-trachea obuthathwe isinyathelo ngesinyathelo, ukuqinisekisa ukufana okwanele phakathi kwesithombe ngasinye ukuvumela isithombe "ukuthunga". Izithombe ezivela kumuntu ngamunye Ama-trachea abe esehlanganiswa abe isithombe esisodwa esihlanganisiwe kusetshenziswa i-Image Composite Editor v2.0.3 (Microsoft Research) kusetshenziswa i-algorithm yokunyakaza okuhleliwe. Izindawo zokubonakaliswa kwe-LacZ ezithombeni ezihlanganisiwe ze-trachea ezivela esilwaneni ngasinye zalinganiswa kusetshenziswa iskripthi se-MATLAB esisebenzisa ngokuzenzakalelayo (R2020a, MathWorks) njengoba kuchaziwe ngaphambilini, kusetshenziswa izilungiselelo ezingu-0.35 < Hue < 0.58, Saturation > 0.15, kanye ne-Value < 0.7. Ngokulandela imidwebo yezicubu, imaski yakhiqizwa ngesandla ku-GIMP v2.10.24 yesithombe ngasinye esihlanganisiwe ukuze kutholakale indawo yezicubu futhi kuvinjelwe noma yikuphi ukutholwa okungamanga okuvela ngaphandle kwezicubu ze-tracheal. Izindawo ezinemibala kuzo zonke izithombe ezihlanganisiwe ezivela esilwaneni ngasinye zahlanganiswa ukuze kukhiqizwe indawo ephelele enemibala yaleso silwane. Indawo enemibala yabe isihlukaniswa yindawo ephelele yemaski ukuze kukhiqizwe indawo evamile.
I-trachea ngayinye yafakwa ku-paraffin kwathi izingxenye ezingu-5 μm zasikwa. Izingxenye zabekwa uphawu olubomvu olusheshayo olungathathi hlangothi imizuzu emi-5 kwathi izithombe zatholakala kusetshenziswa imakroskopu ye-Nikon Eclipse E400, ikhamera ye-DS-Fi3 kanye nesofthiwe yokubamba izinto ze-NIS (inguqulo 5.20.00).
Konke ukuhlaziywa kwezibalo kwenziwe ku-GraphPad Prism v9 (GraphPad Software, Inc.). Ukubaluleka kwezibalo kubekwe ku-p ≤ 0.05. Ukuvamile kuqinisekiswe kusetshenziswa isivivinyo se-Shapiro-Wilk, futhi umehluko ekudayini kwe-LacZ uhlolwe kusetshenziswa isivivinyo se-t esingabhangqiwe.
Ama-MP ayisithupha achazwe kuThebula 1 ahlolwe kusetshenziswa i-PCXI, futhi ukubonakala kuchazwe kuThebula 2. Ama-MP amabili e-polystyrene (MP1 kanye ne-MP2; 18 μm kanye no-0.25 μm, ngokulandelana) awazange abonakale ngaphansi kwe-PCXI, kodwa amanye amasampula ayebonakala (izibonelo ziboniswe kuMfanekiso 5). I-MP3 kanye ne-MP4 (10-15% Fe3O4; 0.25 μm kanye no-0.9 μm, ngokulandelana) abonakala kancane. Nakuba equkethe ezinye zezinhlayiya ezincane kakhulu ezihlolwe, i-MP5 (98% Fe3O4; 0.25 μm) iyona eyayivelele kakhulu. Umkhiqizo we-CombiMag MP6 kunzima ukuwubona. Kuzo zonke izimo, ikhono lethu lokuthola i-MP lathuthukiswa kakhulu ngokuhumusha i-magnet emuva naphambili ngokuhambisana ne-capillary. Lapho omantshi besuka ku-capillary, izinhlayiya zanda ngezintambo ezinde, kodwa njengoba omantshi besondela futhi amandla e-magnetic field ekhuphuka, izintambo zezinhlayiya zafinyezwa njengoba izinhlayiya zithuthela phezulu kwe-capillary. (bheka i-Sepplementary Video S1: MP4), okwandisa ubuningi bezinhlayiya zobuso. Ngokuphambene nalokho, lapho i-magnet isuswa ku-capillary, amandla ensimu ayancipha futhi ama-MP ahlela kabusha abe yizintambo ezinde ezisuka ebusweni obungaphezulu be-capillary (bheka i-Sepplementary Video S2: MP4). Ngemva kokuba i-magnet iyeke ukuhamba, izinhlayiya ziyaqhubeka nokuhamba isikhashana ngemva kokufinyelela isikhundla sokulingana. Njengoba i-MP iqhubekela phambili futhi isuka ebusweni obuphezulu be-capillary, izinhlayiya zamagnetic zivame ukuhudula udoti oketshezini.
Ukubonakala kwe-MP ngaphansi kwe-PCXI kuyahlukahluka kakhulu phakathi kwamasampula.(a) MP3, (b) MP4, (c) MP5 kanye (d) MP6.Zonke izithombe eziboniswe lapha zithathwe ngemagnethi etholakala cishe ngamamilimitha ayi-10 ngqo ngaphezu kwe-capillary. Izindilinga ezinkulu ezibonakalayo zingamabhamuza omoya abanjwe kuma-capillary, okubonisa ngokusobala izici zomphetho omnyama nomhlophe zesithombe sokungafani kwesigaba.Ibhokisi elibomvu liqukethe ukukhulisa okukhulisa umehluko.Qaphela ukuthi ububanzi be-magnet schematics kuzo zonke izibalo abulingani futhi cishe bukhulu ngokuphindwe kayi-100 kunalokho okubonisiwe.
Njengoba i-magnet ihunyushwa ngakwesobunxele nangakwesokudla phezulu kwe-capillary, i-engeli yentambo ye-MP iyashintsha ukuze ivumelane ne-magnet (bheka Umfanekiso 6), ngaleyo ndlela ichaza imigqa yensimu yamagnetic. Ku-MP3-5, ngemva kokuba i-chord ifinyelele i-engeli yomkhawulo, izinhlayiya zidonswa phezu kobuso be-capillary. Lokhu kuvame ukuholela ekutheni ama-MP aqoqeke abe amaqembu amakhulu eduze nalapho insimu yamagnetic inamandla khona (bheka i-Supplementary Video S3:MP5). Lokhu kubonakala kakhulu lapho kuthwetshulwa eduze kokuphela kwe-capillary, okubangela ukuthi ama-MP ahlangane futhi agxile esibonakalayo somoya-uketshezi. Izinhlayiya ku-MP6, ezazinzima kakhulu ukuzibona kune-MP3-5, azidonswanga njengoba i-magnet ihamba nge-capillary, kodwa izintambo ze-MP zahlukana, zishiya izinhlayiya ensimini yokubuka (bheka i-Supplementary Video S4:MP6). Kwezinye izimo, lapho insimu yamagnetic esetshenzisiwe incishiswa ngokuhambisa i-magnet ibanga elide ukusuka endaweni yokuthwebula izithombe, noma yimaphi ama-MP asele ayehla kancane kancane aye phansi kobuso bepayipi ngamandla adonsela phansi ngenkathi ehlala entanjeni (bheka i-Supplementary Video S5: MP3).
I-engeli yentambo ye-MP iyashintsha njengoba i-magnet ihunyushwa ngakwesokudla ngaphezu kwe-capillary.(a) MP3, (b) MP4, (c) MP5 kanye (d) MP6. Ibhokisi elibomvu liqukethe ukukhulisa okuthuthukisa umehluko.Qaphela ukuthi amavidiyo engeziwe ayafundisa njengoba embula isakhiwo sezinhlayiya ezibalulekile kanye nolwazi oluguquguqukayo olungenakubonwa kulezi zithombe ezimile.
Ukuhlolwa kwethu kukhombisile ukuthi ukuhambisa i-magnet kancane emuva naphambili ku-trachea kwenza kube lula ukubona i-MP ngomongo wokunyakaza okuyinkimbinkimbi ku-vivo. Ukuhlolwa kwe-in vivo akwenziwanga njengoba ubuhlalu be-polystyrene (MP1 kanye ne-MP2) babungabonakali ku-capillary. I-MP ngayinye kwezine ezisele yahlolwa ku-vivo nge-magnet long axis ehlelwe ngaphezu kwe-trachea nge-engeli engaba ngu-30° kuya phezulu (bheka Izibalo 2b kanye no-3a), njengoba lokhu kwaholela ezintanjeni ze-MP ezinde futhi kwasebenza kangcono kunokumiswa kwe-magnet okuphelile. I-MP3, i-MP4 kanye ne-MP6 azitholakalanga ku-trachea yanoma yiziphi izilwane eziphilayo. Lapho izindlela zomoya zamagundane zithathwa isithombe ngemva kokuba izilwane zibulewe ngokobuntu, izinhlayiya zahlala zingabonakali ngisho nalapho ivolumu eyengeziwe yengezwa kusetshenziswa ipompo yesirinji. I-MP5 yayinokuqukethwe okuphezulu kakhulu kwe-iron oxide futhi yayiyiyona kuphela inhlayiya ebonakalayo, ngakho-ke yasetshenziswa ukuhlola nokuchaza ukuziphatha kwe-MP ku-vivo.
Ukubeka i-magnet phezu kwe-trachea ngesikhathi sokulethwa kwe-MP kwaholela ekutheni ama-MP amaningi, kodwa hhayi wonke, agxile emkhakheni wokubuka. Izinhlayiya ezingena ku-trachea zibonakala kahle ezilwaneni ezihlatshelwe abantu. Isithombe 7 kanye nevidiyo eyengeziwe S6: I-MP5 ikhombisa ukubanjwa okusheshayo kwe-magnetic kanye nokuqondaniswa kwezinhlayiya ebusweni be-ventral trachea, okubonisa ukuthi ama-MP angaqondiswa ezindaweni ezifiselekayo ze-trachea. Lapho kuseshwa kude kakhulu ku-trachea ngemva kokulethwa kwe-MP, amanye ama-MP atholakale eduze kwe-carina, okuphakamisa ukuthi amandla e-magnetic field ayenganele ukuqoqa nokugcina wonke ama-MP, njengoba ayelethwa esifundeni samandla amakhulu e-magnetic field ngesikhathi senqubo yoketshezi. Noma kunjalo, amazinga e-MP ngemuva kokubeletha ayephezulu endaweni ethathwe isithombe, okuphakamisa ukuthi ama-MP amaningi ahlala ezindaweni zomoya lapho amandla e-magnetic field asetshenziswayo ayephezulu kakhulu.
Izithombe ezivela ku-(a) ngaphambi kanye no-(b) ngemuva kokulethwa kwe-MP5 ku-trachea yegundane elisanda kubulawa kanye nemagnethi ebekwe ngqo ngaphezu kwendawo yesithombe. Indawo ethathwe esithombeni itholakala phakathi kwamasongo amabili e-cartilage. Ngaphambi kokulethwa kwe-MP, kukhona uketshezi oluthile endleleni yokuphefumula. Ibhokisi elibomvu liqukethe ukukhulisa okuthuthukisa umehluko. Lezi zithombe zivela kuvidiyo eboniswe ku-Supplementary Video S6:MP5.
Ukuhumusha i-magnet eduze kwe-trachea in vivo kubangele ukuthi i-MP chain ishintshe i-engeli ngaphakathi kobuso bomoya ngendlela efana naleyo ebonwa kuma-capillary (bheka uMfanekiso 8 kanye ne-Supplementary Video S7:MP5). Kodwa-ke, ocwaningweni lwethu, ama-MP awakwazanga ukuhudulwa ebusweni bomoya ophilayo njengoba ayengenza ngama-capillary. Kwezinye izimo, i-MP chain izokhula isikhathi eside njengoba i-magnet ihamba kwesobunxele nakwesokudla. Ngokuthakazelisayo, sithole nokuthi i-particle string ibonakala ishintsha ukujula kwengqimba yoketshezi olungaphezulu lapho i-magnet ihanjiswa ngobude eceleni kwe-trachea, futhi iyanda lapho i-magnet ihanjiswa ngqo phezulu futhi i-particle string ijikeleziswa endaweni eqondile (bheka i-Supplementary Video S7). : MP5 ngo-0:09, phansi kwesokudla). Iphethini yesici sokunyakaza yashintsha lapho i-magnet idluliselwa phezulu kwe-trachea eceleni (okungukuthi, ngakwesobunxele noma kwesokudla sesilwane kunokuba ihambisane nobude be-trachea). Izinhlayiya zazisabonakala ngokucacile njengoba zihamba, kodwa lapho i-magnet isuswa ku-trachea, izihloko zezintambo zezinhlayiya zabonakala (bheka i-Supplementary Video S8:MP5, kusukela ngo-0:08). Lokhu kuhambisana nokuziphatha kwe-MP esikubonile ngaphansi kwensimu yamagnetic esetshenzisiwe ku-capillary yengilazi.
Izithombe zesibonelo ezibonisa i-MP5 ku-trachea yegundane eliphilayo elinobuhlungu bokulala.(a) I-magnet isetshenziselwa ukuthola izithombe ezingenhla nangakwesobunxele se-trachea, bese kuba (b) ngemva kokuba i-magnet ithuthelwe kwesokudla. Ibhokisi elibomvu liqukethe ukukhulisa okuthuthukisa umehluko. Lezi zithombe zivela kuvidiyo eboniswe ku-Supplementary Video S7:MP5.
Lapho izigxobo ezimbili zihlelwe ngendlela eqondiswa enyakatho-eningizimu ngaphezulu nangaphansi kwe-trachea (okungukuthi, ukuheha; Umfanekiso 3b), ama-MP chords avele amade futhi atholakala ohlangothini lwe-trachea kunokuba abe phezu kwe-dorsal tracheal surface (bheka i-Supplementary Video S9:MP5). Kodwa-ke, ukugcwala okuphezulu kwezinhlayiya endaweni eyodwa (okungukuthi, ubuso be-dorsal be-trachea) akuzange kutholakale ngemuva kokulethwa koketshezi lapho kusetshenziswa idivayisi ye-dual-magnet, okuvame ukwenzeka lapho kusetshenziswa idivayisi ye-single-magnet. Lapho i-magnet eyodwa ihlelwe ukuze ixoshe izigxobo ezibuyiselwe emuva (Umfanekiso 3c), inani lezinhlayiya ezibonakalayo ensimini yokubuka alibonakalanga likhuphuka ngemva kokulethwa. Ukusethwa kokubili kokulungiselelwa kwe-dual-magnet kuyinselele ngenxa yamandla aphezulu e-magnetic field adonsa noma asunduze oma-magnet, ngokulandelana. Ukusethwa kwabe sekushintshwa kwaba yi-single magnet ehambisana nomoya kodwa idlula emoyeni ngama-degree angu-90 ukuze imigqa yensimu iwele udonga lwe-tracheal ngendlela eqondile (Umfanekiso 3d), indlela eklanyelwe ukunquma ukuthi ukuhlanganiswa kwezinhlayiya ohlangothini udonga lwalungabonakala. Kodwa-ke, kulokhu kulungiselelwa, bekungekho ukunyakaza okubonakalayo kokuqongelela kwe-MP noma ukunyakaza kwemagnet. Ngokusekelwe kuyo yonke le miphumela, ukulungiselelwa kokuqondiswa kwe-single-magnet, okungamadigri angu-30 (Isithombe 3a) kwakhethwa ezifundweni zokuthwala izakhi zofuzo ezi-in vivo.
Lapho lesi silwane sithathwa ngokuphindaphindiwe ngokushesha ngemva kokubulawa komuntu, ukungabikho kokunyakaza kwezicubu okudidayo kwakusho ukuthi imigqa yezinhlayiya ezincane nezimfushane ingabonakala ensimini ecacile ye-interchondral, "enyakazayo" ngokuhambisana nokunyakaza kokuhumusha kwemagnethi. Noma kunjalo, asikakuboni ngokucacile ukuba khona kanye nokunyakaza kwezinhlayiya ze-MP6.
I-LV-LacZ titer yayingu-1.8 × 108 TU/ml, futhi ngemva kokuxubana okungu-1:1 ne-CombiMag MP (MP6), izilwane zathola umthamo we-tracheal ongu-50 μl wemoto ye-LV engu-9 × 107 TU/ml (okungukuthi i-4.5 × 106 TU/rat). ). Kulezi zifundo, esikhundleni sokuhumusha i-magnet ngesikhathi sokubeletha, silungise i-magnet endaweni eyodwa ukuze sinqume ukuthi i-LV transduction (a) ingathuthukiswa yini uma kuqhathaniswa nokulethwa kwe-vector lapho kungekho nsimu yamagnetic, futhi (b) ingagxila. Amaseli e-Airway adluliselwa ezindaweni eziqondiwe zamagnetic zomoya ophezulu.
Ukuba khona kwamagnethi kanye nokusetshenziswa kwe-CombiMag kuhlanganiswe nama-LV vectors akubonakali kunemiphumela emibi empilweni yezilwane, njengoba kwenza inqubo yethu ejwayelekile yokulethwa kwe-LV vector. Izithombe zangaphambili zesifunda se-tracheal ezitholwe ukuphazamiseka komshini (Isithombe Esingeziwe 1) zikhombisile ukuthi kwakukhona amazinga aphezulu kakhulu okudluliselwa kwe-transduction eqenjini lezilwane eziphathwe nge-LV-MP lapho imagnethi ikhona (Isithombe 9a). Inani elincane kuphela le-LacZ staining eluhlaza okwesibhakabhaka elalikhona eqenjini lokulawula (Isithombe 9b). Ukulinganiswa kwezindawo ezinemibala ye-X-Gal ezijwayelekile kubonise ukuthi ukuphathwa kwe-LV-MP lapho kukhona insimu yamagnetic kukhiqize ukuthuthuka okuphindwe kasithupha (Isithombe 9c).
Izithombe eziyisibonelo ezihlanganisiwe ezibonisa ukudluliselwa kwe-tracheal yi-LV-MP (a) lapho kukhona insimu yamagnetic kanye (b) lapho kungekho magnet.(c) Ukuthuthuka okuphawulekayo ngokwezibalo endaweni ejwayelekile yokudluliselwa kwe-LacZ ngaphakathi kwe-trachea lapho kusetshenziswa i-magnet (*p = 0.029, t-test, n = 3 ngeqembu, isilinganiso ± SEM).
Izingxenye ezibomvu ezisheshayo nezingathathi hlangothi (isibonelo esiboniswe ku-Supplementary Fig. 2) zibonise amaseli anombala we-LacZ akhona ngendlela efanayo nendawo njengoba kubikiwe ngaphambilini.
Inselele ebalulekile yokwelashwa kwezakhi zofuzo zomoya isalokhu iwukutholakala okunembile kwezinhlayiya zomthwali ezindaweni ezithakaselwayo kanye nokufinyelela amazinga aphezulu okusebenza kahle kokudluliselwa kwamaphaphu ahambayo lapho kukhona ukugeleza komoya kanye nokuphuma kwe-mucus okusebenzayo. Kwabathwali be-LV abaklanyelwe ukwelapha isifo somoya se-CF, ukwandisa isikhathi sokuhlala kwezinhlayiya zomthwali ngaphakathi kwemigudu yomoya eqhubayo kube umgomo onzima kuze kube manje. Njengoba kuboniswe nguCastellani et al., ukusetshenziswa kwamasimu kazibuthe ukuthuthukisa ukudluliselwa kunezinzuzo uma kuqhathaniswa nezinye izindlela zokulethwa kwezakhi zofuzo njenge-electroporation, njengoba kungahlanganisa ubulula, ukusebenza kahle kwezindleko, indawo yokulethwa, ukusebenza kahle okwandisiwe, kanye nezikhathi ezimfushane zokufakwa, kanye nomthamo omncane womthwali10. Kodwa-ke, ukufakwa kwe-in vivo kanye nokuziphatha kwezinhlayiya zikamazibuthe emigwaqweni yomoya ngaphansi kwethonya lamandla kazibuthe angaphandle akukaze kuchazwe, futhi empeleni ukusebenza kwale ndlela akukabonakali ku-in vivo ukuthuthukisa amazinga okuvezwa kwezakhi zofuzo emigwaqweni yomoya ephilayo engaguquki.
Ukuhlolwa kwethu kwe-in vitro synchrotron PCXI kubonise ukuthi zonke izinhlayiya esizivivile, ngaphandle kwe-polystyrene MP, zazibonakala ekusethweni kwezithombe esizisebenzisile. Lapho kukhona insimu yamagnetic, ama-MP akha izintambo ezinobude obuhlobene nohlobo lwezinhlayiya kanye namandla ensimu yamagnetic (okungukuthi ukusondela kanye nokunyakaza kwemagnet). Njengoba kuboniswe kuMfanekiso 10, izintambo esizibonayo zakhiwa ngenxa yokuthi inhlayiya ngayinye inamandla kamagnethi futhi ibangela insimu yayo yamagnetic yendawo. Lezi zinsimu ezihlukene zibangela ukuthi ezinye izinhlayiya ezifanayo zihlangane futhi zixhumane, ngokunyakaza okufana nentambo yeqembu ngenxa yamandla endawo avela emandleni endawo akhangayo nanyanyekayo ezinye izinhlayiya.
Ukubonisa ngokwesikimu (a,b) izinhlayiya ezilandelanayo ezikhiqizwa ngaphakathi kwama-capillary agcwele uketshezi kanye (c,d) ne-trachea egcwele umoya. Qaphela ukuthi ama-capillary kanye ne-trachea akudonswanga esikalini. Iphaneli (a) liqukethe nencazelo ye-MP, equkethe izinhlayiya ze-Fe3O4 ezihlelwe ngezintambo.
Lapho i-magnet ithuthwa ngaphezu kwe-capillary, i-engeli yentambo yezinhlayiya yafinyelela umkhawulo obalulekile we-MP3-5 equkethe i-Fe3O4, ngemva kwalokho intambo yezinhlayiya ayisahlali endaweni yokuqala, kodwa yahamba phezu kobuso yaya endaweni entsha. i-magnet. Lo mphumela kungenzeka uvele ngoba ubuso be-capillary yengilazi bubushelelezi ngokwanele ukuvumela lokhu kunyakaza kwenzeke. Ngokuthakazelisayo, i-MP6 (CombiMag) ayizange iziphathe ngale ndlela, mhlawumbe ngoba izinhlayiya zazincane, zinezimbozo ezihlukene noma izindleko zobuso, noma uketshezi oluthwalayo oluyimfihlo luthinte ikhono lazo lokunyakaza. Umehluko wesithombe wezinhlayiya ze-CombiMag nawo ubuthakathaka, okuphakamisa ukuthi uketshezi nezinhlayiya zingaba nobukhulu obufanayo futhi ngenxa yalokho zingahambisani kalula. Izinhlayiya zingabhajwa futhi uma i-magnet ihamba ngokushesha kakhulu, okubonisa ukuthi amandla e-magnetic field awakwazi njalo ukunqoba ukungqubuzana phakathi kwezinhlayiya oketshezini, okuphakamisa ukuthi mhlawumbe akumangazi ukuthi amandla e-magnetic field kanye nebanga phakathi kwe-magnet nendawo okuqondiwe Kubaluleke kakhulu. Uma kuhlanganiswa, le miphumela iphinde iphakamise ukuthi, ngenkathi i-magnets ingabamba ama-MP amaningi ageleza endaweni okuqondiwe kuyo, akunakwenzeka ukuthi i-magnets ingathembeka. ukuze kushukunyiswe izinhlayiya ze-CombiMag ebusweni be-trachea. Ngakho-ke, siphetha ngokuthi izifundo ze-LV-MP ze-in vivo kufanele zisebenzise amasimu kazibuthe angaguquki ukuze ziqondise ngokomzimba izindawo ezithile zesihlahla somoya.
Uma izinhlayiya zilethwa emzimbeni, kunzima ukuzibona ngokwesimo sezicubu zomzimba ezihambayo eziyinkimbinkimbi, kodwa ikhono lokuzibona lathuthukiswa ngokuhumusha i-magnet ngokuvundlile ngaphezu kwe-trachea ukuze "inyakazise" izintambo ze-MP. Nakuba kungenzeka ukuthwebula izithombe bukhoma, kulula ukubona ukunyakaza kwezinhlayiya uma isilwane sesibulewe ngobuntu. Amazinga e-MP ngokuvamile ayephezulu kakhulu kule ndawo lapho i-magnet ibekwe ngaphezu kwendawo yokuthwebula izithombe, yize ezinye izinhlayiya zazivame ukutholakala kude ne-trachea. Ngokungafani nezifundo ze-in vitro, izinhlayiya azikwazi ukuhudulwa ku-trachea ngokuhumusha i-magnet. Lokhu okutholakele kuhambisana nendlela i-mucus emboza ubuso be-trachea evame ukucubungula ngayo izinhlayiya eziphefumulayo, izibambe ku-mucus bese zisuswa yindlela yokususa i-mucociliary.
Sicabange ukuthi ukusetshenziswa kwama-magnets ukuze kukhange ngaphezu nangaphansi kwe-trachea (Umfanekiso 3b) kungabangela insimu yamagnetic efanayo, kunokuba insimu yamagnetic egxile kakhulu endaweni eyodwa, okungenzeka kuholele ekusabalaleni okufanayo kwezinhlayiya. Kodwa-ke, ucwaningo lwethu lokuqala alutholanga ubufakazi obucacile bokusekela lo mbono. Ngokufanayo, ukumisa ama-magnets amabili ukuze axoshe (Umfanekiso 3c) akuzange kubangele ukufakwa kwezinhlayiya eziningi endaweni ethathwe isithombe. Lokhu okutholakele okubili kubonisa ukuthi ukusethwa kwe-dual-magnet akuthuthukisi kakhulu ukulawulwa kwendawo kokuqondiswa kwe-MP, nokuthi amandla anamandla abangelwayo kunzima ukuwamisa, okwenza le ndlela ingasebenzi kahle. Ngokufanayo, ukuqondisisa i-magnet ngenhla nangaphakathi kwe-trachea (Umfanekiso 3d) nakho akuzange kwandise inani lezinhlayiya ezigcinwe endaweni ethathwe isithombe. Ezinye zalezi zilungiselelo ezihlukile zingase zingaphumeleli ngoba ziphumela emandleni aphansi ensimu yamagnetic ngaphakathi kwendawo yokufaka. Ngakho-ke, ukucushwa kwe-single-degree 30-degree magnet (Umfanekiso 3a) kubhekwa njengendlela elula nephumelelayo kakhulu yokuhlola i-in vivo.
Ucwaningo lwe-LV-MP lukhombisile ukuthi lapho ama-vector e-LV ehlanganiswa ne-CombiMag futhi ethunyelwa ngemva kokuphazamiseka ngokomzimba lapho kukhona insimu yamagnetic, amazinga okudluliselwa akhushulwa kakhulu ku-trachea uma kuqhathaniswa nezilawuli. Ngokusekelwe ezifundweni ze-synchrotron imaging kanye nemiphumela ye-LacZ, insimu yamagnetic ngokusobala yayikwazi ukulondoloza i-LV ngaphakathi kwe-trachea futhi inciphise inani lezinhlayiya ze-vector ezangena ngokushesha emaphashini. Ukuthuthukiswa okunjalo kokuqondisa kungaholela ekusebenzeni kahle okuphezulu ngenkathi kunciphisa ama-titers alethiwe, ukudluliselwa okungeyona inhloso, imiphumela emibi yokuvuvukala kanye neyokuzivikela komzimba, kanye nezindleko zokuthwala izakhi zofuzo. Okubalulekile, ngokusho komkhiqizi, i-CombiMag ingasetshenziswa kanye nezinye izindlela zokudlulisa izakhi zofuzo, kufaka phakathi nezinye i-vectors zamagciwane (njenge-AAV) kanye nama-nucleic acid.