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Ma gene vectors ochizira matenda a m'mapapo a cystic fibrosis ayenera kuyang'ana njira zoyendetsera mpweya chifukwa kuperekedwa kwa mapapo m'mphepete mwa mpweya sikupereka phindu la chithandizo. Kugwira ntchito bwino kwa ma virus kumagwirizana mwachindunji ndi nthawi yokhala ma ventricle. Komabe, madzi operekera monga onyamula majini amafalikira mwachilengedwe mu alveoli panthawi yopumira, ndipo tinthu tating'onoting'ono tochiritsira ta mtundu uliwonse timachotsedwa mwachangu ndi kuyenda kwa mucociliary. Kutalikitsa nthawi yokhala kwa onyamula majini m'njira zoyendetsera mpweya ndikofunikira koma kovuta kukwaniritsa. Tinthu tating'onoting'ono tomwe timalumikizidwa ndi gene carrier tomwe tingawongolere pamwamba pa njira zoyendetsera mpweya tingathandize kuwunikira madera. Chifukwa cha zovuta za kuwonetsa mu vivo, machitidwe a tinthu tating'onoting'ono totere ta maginito pamwamba pa njira zoyendetsera mpweya pamaso pa mphamvu ya maginito yomwe imagwiritsidwa ntchito sakumveka bwino. Cholinga cha kafukufukuyu chinali kugwiritsa ntchito synchrotron imaging kuti tiwone mayendedwe a tinthu tating'onoting'ono ta maginito mu trachea ya makoswe ogonetsedwa kuti tiwone momwe zinthu zilili komanso momwe zinthu zilili mu vivo. Kenako tinayang'ananso ngati kuperekedwa kwa tinthu ta maginito ta lentiviral pamaso pa mphamvu ya maginito kungawonjezere kugwira ntchito kwa transduction mu khoswe. trachea. Kujambula kwa X-ray kwa Synchrotron kukuwonetsa momwe tinthu ta maginito timagwirira ntchito m'minda yamaginito yosasuntha komanso yoyenda mu vitro ndi mu vivo. Tinthu tating'onoting'ono sitingakokedwe mosavuta pamwamba pa mpweya wamoyo ndi maginito, koma panthawi yoyendera, malo osungiramo zinthu amawunjikana m'munda wowonera pomwe mphamvu yamaginito ndi yamphamvu kwambiri. Kugwira ntchito bwino kwa transduction kunawonjezekanso kasanu ndi kamodzi pamene tinthu ta maginito ta lentiviral tinaperekedwa pamaso pa mphamvu yamaginito. Zonsezi pamodzi zikusonyeza kuti tinthu ta maginito ta lentiviral ndi minda yamaginito zitha kukhala njira zothandiza pakukweza jini vector targeting ndikuwonjezera kuchuluka kwa transduction pakuyendetsa mpweya mu vivo.
Cystic fibrosis (CF) imayamba chifukwa cha kusintha kwa jini imodzi yotchedwa CF transmembrane conductance regulator (CFTR). Puloteni ya CFTR ndi njira ya ion yomwe imapezeka m'maselo ambiri a epithelial m'thupi lonse, kuphatikizapo njira zoyendetsera mpweya, malo akuluakulu omwe amachititsa kuti CF iyambe kufalikira. Zolakwika za CFTR zimapangitsa kuti madzi asayende bwino, zomwe zimapangitsa kuti pamwamba pa mpweya pakhale madzi komanso kuchepetsa kuya kwa gawo la madzi pamwamba pa mpweya (ASL). Izi zimalepheretsanso mphamvu ya mucociliary transport (MCT) system kuchotsa tinthu tating'onoting'ono tomwe timapuma ndi tizilombo toyambitsa matenda m'njira zoyendetsera mpweya. Cholinga chathu ndikupanga njira yochizira majini ya lentiviral (LV) kuti ipereke kopi yolondola ya jini ya CFTR ndikukweza thanzi la ASL, MCT, ndi mapapo, komanso kupitiliza kupanga matekinoloje atsopano omwe amatha kuyeza izi mu vivo1.
Ma vector a LV ndi amodzi mwa omwe akutsogolera pa chithandizo cha majini a CF airway, makamaka chifukwa amatha kuphatikiza jini yochiritsira mu maselo oyambira a airway (maselo oyambira a airway). Izi ndizofunikira chifukwa amatha kubwezeretsa madzi abwinobwino komanso kuchotsedwa kwa ma mucus mwa kusiyanitsa kukhala maselo ogwirira ntchito a CF omwe amagwirizana ndi majini, zomwe zimapangitsa kuti pakhale phindu la moyo wonse. Ma vector a LV ayenera kulunjika ku njira yoyendetsera mpweya, chifukwa apa ndi pomwe matenda a CF m'mapapo amayamba. Kutumiza kwa vector mkati mwa mapapo kungayambitse kulowetsedwa kwa alveolar, koma izi sizili ndi phindu la chithandizo mu CF. Komabe, madzi monga onyamula majini amasamukira ku alveoli akapumidwa pambuyo pobereka3,4 ndipo tinthu tating'onoting'ono tochiritsira timachotsedwa mwachangu mkamwa ndi MCT. Kugwira ntchito bwino kwa kulowetsedwa kwa LV kumagwirizana mwachindunji ndi nthawi yomwe vector imakhala pafupi ndi maselo omwe akufuna kuti maselo azitha kutenga - "nthawi yokhala"5 - yomwe imachepetsedwa mosavuta ndi kayendedwe ka mpweya wamba wa m'deralo komanso kugwidwa kwa tinthu tating'onoting'ono ndi MCT. Kwa CF, kuthekera kowonjezera nthawi yokhala ya LV mkati mwa airway ndikofunikira kuti pakhale kuchuluka kwa kulowetsedwa m'derali, koma kwakhala kotere. zakhala zovuta kwambiri.
Kuti tithetse vutoli, tikupangira kuti tinthu ta LV magnetic (MPs) titha kuthandiza m'njira ziwiri zogwirizana. Choyamba, tingathe kutsogoleredwa ndi maginito kupita pamwamba pa mpweya kuti tiwongolere njira yolowera mpweya ndikuthandiza tinthu ta jini kukhala m'dera lomwe mpweya ukuyenda; ndi ASL) kuti tisunthire ku gawo la selo 6. Ma MP akhala akugwiritsidwa ntchito kwambiri ngati njira zoperekera mankhwala pamene amagwirizana ndi ma antibodies, mankhwala a chemotherapeutic, kapena mamolekyu ena ang'onoang'ono omwe amalumikizana ndi nembanemba ya selo kapena kumangirira ku ma receptor oyenera a pamwamba pa selo ndikusonkhana pamalo otupa pamaso pa magetsi osasinthasintha. Magawo a Magnetic Othandizira Khansa 7. Njira zina "zotentha kwambiri" cholinga chake ndi kutentha ma MP akakumana ndi maginito ozungulira, motero amawononga maselo otupa. Mfundo ya transfection ya maginito, momwe maginito amagwiritsidwira ntchito ngati transfection kuti apititse patsogolo kusamutsa kwa DNA kupita ku maselo, imagwiritsidwa ntchito kwambiri mu vitro pogwiritsa ntchito mitundu yosiyanasiyana ya majini osakhala ma virus ndi ma virus pa mizere ya maselo ovuta kugawa. Kugwira ntchito kwa LV magnetotransfection kwakhazikitsidwa, ndi kuperekedwa kwa LV-MPs mu vitro ku mzere wa maselo a epithelial a munthu mukakhala ndi mphamvu ya maginito yokhazikika, zomwe zimawonjezera mphamvu ya transduction ndi nthawi 186 poyerekeza ndi LV vector yokha. LV-MP yagwiritsidwanso ntchito ku chitsanzo cha CF mu vitro, komwe transfection ya maginito inawonjezera transduction ya LV mu cultures ya air-liquid interface ndi nthawi 20 mukakhala ndi sputum ya CF10. Komabe, in vivo magnetotransfection ya ziwalo yalandira chisamaliro chochepa ndipo yangoyesedwa mwa nyama zochepa chabe. maphunziro11,12,13,14,15, makamaka m'mapapo16,17. Komabe, mwayi wopereka maginito mu chithandizo cha mapapo cha CF ndi womveka bwino. Tan et al. (2020) adati "kafukufuku wotsimikizira lingaliro la kupereka bwino kwa maginito nanoparticle pulmonary kudzatsegula njira zamtsogolo zopumira za CFTR kuti ziwongolere zotsatira zachipatala mwa odwala a CF"6.
Khalidwe la tinthu tating'onoting'ono ta maginito pa malo opumira mpweya pamaso pa mphamvu ya maginito yogwiritsidwa ntchito ndi lovuta kuliganizira ndi kuliphunzira, motero silikumveka bwino. Mu maphunziro ena, tinapanga njira ya synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) kuti tiwone mopanda kuwononga ndikuwerengera kusintha kwa mphindi mu vivo mu kuya kwa ASL18 ndi khalidwe la MCT19,20 kuti tiyese mwachindunji mpweya wa Canal surface hydration ndipo tidagwiritsa ntchito ngati chizindikiro choyambirira cha mphamvu ya chithandizo. Kuphatikiza apo, njira yathu yowunikira MCT imagwiritsa ntchito tinthu tating'onoting'ono ta 10–35 µm tomwe timapangidwa ndi alumina kapena galasi la refractive index lalikulu ngati zizindikiro za MCT zomwe zimawonekera pogwiritsa ntchito PB-PCXI21. Njira zonsezi ndizoyenera kuwonetsa mitundu yosiyanasiyana ya tinthu, kuphatikiza MP.
Chifukwa cha kutalika kwake kwa malo ndi nthawi, njira zathu zowunikira za ASL ndi MCT zochokera ku PB-PCXI ndizoyenera kwambiri pofufuza momwe zinthu zilili komanso momwe zinthu zilili mu thupi kuti zitithandize kumvetsetsa ndikuwongolera njira zoperekera majini a MP. Njira yomwe timagwiritsa ntchito pano imachokera ku maphunziro athu pogwiritsa ntchito SPring-8 BL20B2 beamline, momwe tidawonera kayendedwe ka madzi pambuyo popereka mlingo wa vector m'mphuno ndi m'mapapo a mbewa kuti tithandizire kufotokoza njira zathu zowonetsera majini zomwe sizili zofanana zomwe zawonedwa mu maphunziro athu a ziweto a mlingo wa majini 3,4.
Cholinga cha kafukufukuyu chinali kugwiritsa ntchito synchrotron PB-PCXI kuti tiwone mayendedwe a MPs mu trachea ya makoswe amoyo. Maphunziro awa a PB-PCXI adapangidwa kuti ayesere mitundu yosiyanasiyana ya MPs, mphamvu za mphamvu ya maginito, ndi malo kuti adziwe momwe zimakhudzira kuyenda kwa MP. Tinaganiza kuti mphamvu ya maginito yogwiritsidwa ntchito kunja ingathandize MP yoperekedwayo kukhalabe kapena kusamukira kudera lomwe ikufunidwa. Maphunziro awa adatithandizanso kuzindikira mawonekedwe a maginito omwe amawonjezera kuchuluka kwa tinthu tating'onoting'ono tomwe timasungidwa mu trachea titayikidwa. Mu mndandanda wachiwiri wa maphunziro, tidafuna kugwiritsa ntchito mawonekedwe abwino kwambiri awa kuti tiwonetse mawonekedwe a transduction omwe amachokera ku kutumiza kwa LV-MPs mu vivo kupita ku airway ya makoswe, kutengera lingaliro lakuti kutumiza kwa LV-MPs potengera njira yowunikira airway kungapangitse kuti LV-MPs ikhale bwino.
Maphunziro onse a nyama adachitika motsatira malamulo ovomerezedwa ndi University of Adelaide (M-2019-060 ndi M-2020-022) ndi Komiti ya SPring-8 Synchrotron Animal Ethics. Mayeso adachitika motsatira malangizo a ARRIVE.
Kujambula zithunzi zonse za X-ray kunachitika pa mzere wa BL20XU pa synchrotron ya SPring-8 ku Japan, pogwiritsa ntchito njira yofanana ndi yomwe yafotokozedwa kale. Mwachidule, bokosi loyesera linali pamtunda wa mamita 245 kuchokera ku mphete yosungira synchrotron. Mtunda wa 0.6 m kuchokera ku chowunikira umagwiritsidwa ntchito pa kafukufuku wa tinthu tating'onoting'ono ndi mamita 0.3 pa kafukufuku wa in vivo kuti apange zotsatira zosiyana. Mphamvu ya monochromatic beam ya 25 keV inagwiritsidwa ntchito. Zithunzi zinajambulidwa pogwiritsa ntchito chosinthira cha X-ray chapamwamba (SPring-8 BM3) cholumikizidwa ku chowunikira cha sCMOS. Chosinthiracho chimasintha ma X-ray kukhala kuwala kowoneka pogwiritsa ntchito scintillator ya 10 µm wandiweyani (Gd3Al2Ga3O12), yomwe kenako imatsogozedwa ku sensa ya sCMOS pogwiritsa ntchito × 10 microscope object (NA 0.3). Chowunikira cha sCMOS chinali Orca-Flash4.0 (Hamamatsu Photonics, Japan) yokhala ndi kukula kwa array Ma pixel a 2048 × 2048 ndi kukula kwa pixel ya 6.5 × 6.5 µm. Kukhazikitsa kumeneku kumapereka kukula kwa pixel ya isotropic yogwira ntchito ya 0.51 µm ndi malo owonera pafupifupi 1.1 mm × 1.1 mm. Kutalika kwa 100 ms kunasankhidwa kuti kuwonjezere chiŵerengero cha signal-to-noise cha tinthu ta maginito mkati ndi kunja kwa njira yopumira pomwe kumachepetsa zinthu zoyendetsedwa ndi kupuma. Pa maphunziro a in vivo, shutter ya X-ray yofulumira idayikidwa mu njira ya X-ray kuti ichepetse kuchuluka kwa ma radiation potseka kuwala kwa X-ray pakati pa kuwonekera.
Chonyamulira cha LV sichinagwiritsidwe ntchito mu maphunziro aliwonse a kujambula zithunzi a SPring-8 PB-PCXI chifukwa chipinda chojambulira zithunzi cha BL20XU sichinavomerezedwe ndi Biosafety Level 2. M'malo mwake, tinasankha mitundu yosiyanasiyana ya ma MP odziwika bwino kuchokera kwa ogulitsa awiri amalonda—omwe ali ndi kukula kosiyanasiyana, zipangizo, kuchuluka kwa chitsulo, ndi ntchito—choyamba kuti timvetse momwe mphamvu zamaginito zimakhudzira kayendedwe ka MP mkati mwa mitsempha yagalasi, kenako m'njira zamoyo zopumira. pamwamba. Ma MP ali ndi kukula kuyambira 0.25 mpaka 18 μm ndipo amapangidwa kuchokera ku zinthu zosiyanasiyana (onani Gome 1), koma kapangidwe ka chitsanzo chilichonse, kuphatikizapo kukula kwa tinthu ta maginito mkati mwa MP, sikudziwika. Kutengera ndi maphunziro athu a MCT 19, 20, 21, 23, 24, tikuyembekeza kuti ma MP ang'onoang'ono ngati 5 μm amatha kuwoneka pamwamba pa tracheal airway, mwachitsanzo pochotsa mafelemu otsatizana kuti muwone kuwoneka bwino kwa kayendedwe ka MP. MP imodzi ya 0.25 μm ndi yaying'ono kuposa resolution ya chipangizo chojambulira, koma PB-PCXI ikuyembekezeka kuzindikira kusiyana kwawo kwa voliyumu ndi mayendedwe a madzi a pamwamba omwe amaikidwapo atayikidwa.
Zitsanzo za MP iliyonse mu Table 1 zinakonzedwa mu capillaries yagalasi ya 20 μl (Drummond Microcaps, PA, USA) yokhala ndi mainchesi amkati a 0.63 mm. Tinthu tating'onoting'ono timapezeka m'madzi, pomwe tinthu ta CombiMag timapezeka mumadzimadzi a wopanga. Chubu chilichonse chimadzazidwa ndi madzi pang'ono (pafupifupi 11 μl) ndikuyikidwa pa chogwirira cha chitsanzo (onani Chithunzi 1). Ma capillaries agalasi adayikidwa mopingasa pagawo la chitsanzo mu bokosi lojambulira, motsatana, ndikuyika m'mphepete mwa madziwo. Magnetti ya nickel shell ya 19 mm (28 mm kutalika) ya rare earth neodymium iron boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Australia) yokhala ndi maginito otsalira a 1.17 Tesla idalumikizidwa pagawo losiyana lomasulira kuti ikwaniritse Kusintha malo ake patali panthawi yojambulira. Kupeza zithunzi za X-ray kumayamba pamene maginito ayikidwa pafupifupi 30 mm pamwamba pa chitsanzo, ndipo zithunzi zimapezeka pa liwiro la mafelemu 4 pa sekondi. Pakujambula, maginito adabweretsedwa pafupi ndi galasi. chubu cha capillary (pafupifupi 1 mm kutali) kenako nkusinthidwa m'mbali mwa chubu kuti muwone zotsatira za mphamvu ya munda ndi malo ake.
Kukhazikitsa zithunzi za mu vitro zomwe zili ndi zitsanzo za MP m'mitsempha yagalasi pa gawo losinthira la chitsanzo cha xy. Njira ya kuwala kwa X-ray imalembedwa ndi mzere wofiira wokhala ndi mipata.
Atangozindikira kuti MPs akuwoneka bwino m'thupi, gulu la iwo linayesedwa m'thupi mwa makoswe achikazi a mtundu wa Wistar (omwe ali ndi zaka zoposa 12, ~ 200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Japan) ndi 4 mg/kg butorphanol (Vetorphale®, Meiji Seika) makoswe anapatsidwa mankhwala oletsa ululu ndi mankhwala osakaniza a Pharma), Japan) mwa kubayidwa mkati mwa peritoneal. Pambuyo pa mankhwala oletsa ululu, anakonzedwa kuti azitha kujambula zithunzi pochotsa ubweya wozungulira trachea, kuyika chubu cha endotracheal (ET; 16 Ga iv cannula, Terumo BCT) ndikuziletsa kuyenda chagada pa mbale yojambula zithunzi yopangidwa mwapadera yokhala ndi thumba lotentha kuti thupi lisunge kutentha kwa thupi 22. Kenako mbale yojambulira zithunzi inalumikizidwa ku gawo losinthira chitsanzo m'bokosi lojambulira zithunzi pa ngodya pang'ono kuti igwirizane trachea molunjika pachithunzi cha X-ray, monga momwe zasonyezedwera pa Chithunzi 2a.
(a) Kukhazikitsa zithunzi za mu vivo mu bokosi la zithunzi za SPring-8, njira ya kuwala kwa X-ray imalembedwa ndi mzere wofiira wokhala ndi mipata.(b,c) Kuzindikira malo a maginito pa trachea kunachitika patali pogwiritsa ntchito makamera awiri a IP omwe ali ndi orthogonally. Kumanzere kwa chithunzi cha chinsalu, chingwe cha waya chomwe chikugwira mutu chikuwoneka, ndipo cannula yotumizira ili pamalo ake mkati mwa chubu cha ET.
Dongosolo la pampu ya syringe yolamulidwa ndi kutali (UMP2, World Precision Instruments, Sarasota, FL) pogwiritsa ntchito syringe yagalasi ya 100 μl linalumikizidwa ku chubu cha PE10 (OD 0.61 mm, ID 0.28 mm) kudzera mu singano ya 30 Ga. Ikani chizindikiro pa chubu kuti muwonetsetse kuti nsonga ili pamalo oyenera mu trachea mukayika chubu cha ET. Pogwiritsa ntchito micropump, chopukutira cha syringe chinachotsedwa pomwe nsonga ya chubuyo inamizidwa mu chitsanzo cha MP kuti chiperekedwe. Chubu chotumizira chodzaza chinayikidwa mu chubu cha endotracheal, ndikuyika nsonga mkati mwa gawo lamphamvu kwambiri la mphamvu yathu yamaginito yomwe tinkayembekezera. Kupeza zithunzi kunayang'aniridwa pogwiritsa ntchito chowunikira kupuma cholumikizidwa ku bokosi lathu la nthawi lozikidwa pa Arduino, ndipo zizindikiro zonse (monga kutentha, kupuma, kutsegula/kutseka kwa shutter ndi kupeza zithunzi) zinajambulidwa pogwiritsa ntchito Powerlab ndi LabChart (AD Instruments, Sydney, Australia) 22. Pojambula Pamene chotchingacho chinali chosafikirika, makamera awiri a IP (Panasonic BB-SC382) adayikidwa pa madigiri pafupifupi 90 kuti Zinagwiritsidwa ntchito poyang'anira malo a maginito poyerekeza ndi trachea panthawi yojambula zithunzi (Chithunzi 2b, c). Pofuna kuchepetsa zinthu zoyenda, chithunzi chimodzi chinapezeka pa mpweya uliwonse panthawi ya mtsinje wotuluka m'madzi.
Maginito amamangiriridwa ku gawo lachiwiri lomwe lingapezeke kutali kuchokera kunja kwa nyumba yojambulira zithunzi. Malo osiyanasiyana a maginito ndi mawonekedwe ake adayesedwa, kuphatikizapo: Anayikidwa pa ngodya ya pafupifupi 30° pamwamba pa trachea (mawonekedwe omwe akuwonetsedwa mu Zithunzi 2a ndi 3a); maginito imodzi pamwamba pa nyama ndi ina pansi, ndi mitengo yokonzedwa kuti ikoke (Chithunzi 3b); maginito imodzi pamwamba pa nyama ndi ina pansi, ndi mitengo yokonzedwa kuti ibwezeretse (Chithunzi 3c); ndi maginito imodzi pamwamba ndipo yolunjika ku trachea (Chithunzi 3d). Nyama ndi maginito zikakonzedwa ndipo MP yoti iyesedwe ikalowetsedwa mu syringe pump, perekani mlingo wa 50 μl pamlingo wa 4 μl/sekondi pamene mukupeza zithunzi. Maginitowo amasunthidwa kumbuyo ndi kutsogolo motsatira kapena mozungulira trachea pamene akupitiriza kupeza zithunzi.
Kapangidwe ka maginito ka kujambula mu vivo (a) maginito amodzi pamwamba pa trachea pa ngodya ya pafupifupi 30°, (b) maginito awiri okonzedwa kuti akoke, (c) maginito awiri okonzedwa kuti abwezeretse, (d) maginito amodzi pamwamba ndipo olunjika mu trachea. Woyang'anitsitsa anayang'ana pansi kuchokera pakamwa kupita ku mapapo kudzera mu trachea, ndipo kuwala kwa X-ray kunadutsa mbali yakumanzere ya khoswe ndikutuluka mbali yakumanja. Maginito amasunthidwa kutalika kwa njira yolowera mpweya kapena kumanzere ndi kumanja pamwamba pa trachea molunjika ku kuwala kwa X-ray.
Tinayesetsanso kudziwa momwe tinthu tating'onoting'ono timaonekera komanso momwe timachitira m'njira zopumira popanda kupuma kosokoneza komanso kuyenda kwa mtima. Chifukwa chake, kumapeto kwa nthawi yojambula zithunzi, nyama zinaphedwa mwankhanza chifukwa cha kumwa mankhwala osokoneza bongo a pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). Nyama zina zinasiyidwa papulatifomu yojambula zithunzi, ndipo kupuma ndi kugunda kwa mtima zikasiya, njira yojambula zithunzi inabwerezedwanso, kuwonjezera mlingo wina wa MP ngati palibe MP yomwe inkaoneka pamwamba pa njira yopumira.
Zithunzi zomwe zidapezedwa zidakonzedwa ngati bwalo lathyathyathya ndi lamdima kenako nkusonkhanitsidwa kukhala filimu (mafelemu 20 pa sekondi; liwiro la 15-25 × lachizolowezi kutengera kuthamanga kwa mpweya) pogwiritsa ntchito script yolembedwa mu MATLAB (R2020a, The Mathworks).
Kafukufuku wonse wokhudza kuperekedwa kwa majini a LV adachitika ku Laboratory Animal Research Facility ku University of Adelaide ndipo cholinga chake chinali kugwiritsa ntchito zotsatira za kuyesera kwa SPring-8 kuti awone ngati kuperekedwa kwa LV-MP pamaso pa mphamvu ya maginito kungathandize kusamutsa majini m'thupi. Kuti awone zotsatira za MP ndi mphamvu ya maginito, magulu awiri a nyama adachiritsidwa: gulu limodzi linapatsidwa LV-MP ndi maginito atayikidwa, ndipo gulu lina linalandira gulu lolamulira ndi LV-MP yopanda maginito.
Ma vector a majini a LV adapangidwa pogwiritsa ntchito njira zomwe zafotokozedwa kale 25, 26. Vector ya LacZ imasonyeza jini ya beta-galactosidase ya nuclear-localized yoyendetsedwa ndi constitutive MPSV promoter (LV-LacZ), yomwe imapanga mankhwala a blue reaction m'maselo osinthidwa, omwe amawoneka m'maselo a minofu ya m'mapapo ndi m'magawo a minofu. Kuchuluka kwa titration kunachitika m'maselo powerengera pamanja kuchuluka kwa maselo abwino a LacZ ndi hemocytometer kuti awerengere titer mu TU/ml. Zonyamula zimasungidwa pa -80 °C, zimasungunuka musanagwiritse ntchito, ndikumangiriridwa ku CombiMag posakaniza pa chiŵerengero cha 1:1 ndikuyika pa ayezi kwa mphindi zosachepera 30 musanaperekedwe.
Makoswe a Sprague Dawley wamba (n = 3/gulu, ~2-3 adapatsidwa mankhwala oletsa ululu mkati mwa chiberekero ndi chisakanizo cha 0.4 mg/kg medetomidine (Domitor, Ilium, Australia) ndi jakisoni wa 60 mg/kg ketamine (Ilium, Australia) wa mwezi umodzi) ip) ndi kulowetsedwa pakamwa popanda opaleshoni ndi 16 Ga iv cannula. Kuti zitsimikizire kuti minofu ya tracheal airway ilandila LV transduction, idakonzedwa pogwiritsa ntchito njira yathu yodziwira kale zamakina, momwe pamwamba pa tracheal airway idapakidwa mozungulira ndi waya (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. Kuperekedwa kwa LV-MP mu tracheal kenako kudachitika mu kabati yotetezeka yachilengedwe pafupifupi mphindi 10 pambuyo pa kusokonezeka.
Mphamvu ya maginito yomwe idagwiritsidwa ntchito mu kuyeseraku idapangidwa mofanana ndi kafukufuku wa X-ray wa in vivo, ndi maginito omwewo omwe adasungidwa pamwamba pa trachea pogwiritsa ntchito distillation stent clips (Chithunzi 4). Voliyumu ya 50 μl (2 × 25 μl aliquots) ya LV-MP idaperekedwa mu trachea (n = nyama 3) pogwiritsa ntchito pipette yokhala ndi nsonga ya gel monga tafotokozera kale. Gulu lolamulira (n = nyama 3) lidalandira ma LV-MP omwewo popanda kugwiritsa ntchito maginito. Pambuyo poti kulowetsedwa kwatha, cannula imachotsedwa mu chubu cha ET ndipo nyamayo imachotsedwa. Maginito amakhalabe pamalopo kwa mphindi 10, kenako imachotsedwa. Makoswe adalandira mlingo wa meloxicam (1 ml/kg) (Ilium, Australia) kutsatiridwa ndi kubweza mankhwala oletsa ululu pobayira 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia). Makoswe adasungidwa ofunda ndikuyang'aniridwa mpaka atachira kwathunthu kuchokera ku mankhwala oletsa ululu.
Chipangizo choperekera LV-MP mu kabati yotetezera yachilengedwe. Chipinda chopepuka cha Luer cha chubu cha ET chikuwoneka chikutuluka pakamwa ndipo nsonga ya gel ya pipette yomwe ili pachithunzichi imayikidwa kudzera mu chubu cha ET mpaka kuzama komwe mukufuna kulowa mu trachea.
Sabata imodzi pambuyo pa njira yoperekera mankhwala a LV-MP, nyama zinaphedwa mwankhanza ndi mpweya wa CO2 100% ndipo LacZ inayesedwa pogwiritsa ntchito mankhwala athu a X-gal. Mphete zitatu za cartilaginous kwambiri zinachotsedwa kuti zitsimikizire kuti kuwonongeka kulikonse kwa makina kapena kusungidwa kwa madzi kuchokera ku malo olowera mu chubu cha endotracheal sikunaphatikizidwe mu kusanthula. Trachea iliyonse inadulidwa motalikirapo kuti ipange magawo awiri kuti iwunikidwe, ndipo inayikidwa mu mbale yokhala ndi rabara ya silicone (Sylgard, Dow Inc) pogwiritsa ntchito singano ya Minutien (Fine Science Tools) kuti iwonetse pamwamba pa kuwala. Kugawa ndi mawonekedwe a maselo osinthidwa zinatsimikiziridwa ndi kujambula kutsogolo pogwiritsa ntchito maikulosikopu ya Nikon (SMZ1500) ndi kamera ya DigiLite ndi pulogalamu ya TCapture (Tucsen Photonics, China). Zithunzi zinapezeka pa kukula kwa 20x (kuphatikiza malo apamwamba kwambiri a m'lifupi lonse la trachea), ndi kutalika konse kwa trachea kujambulidwa pang'onopang'ono, kuonetsetsa kuti chithunzi chilichonse chikufanana mokwanira kuti chithunzi "chisokedwe". Zithunzi kuchokera pa chilichonse Kenako ma trachea anasonkhanitsidwa kukhala chithunzi chimodzi chophatikizika pogwiritsa ntchito Image Composite Editor v2.0.3 (Microsoft Research) pogwiritsa ntchito njira yoyendetsera kayendedwe ka planar. Madera owonetsera a LacZ m'zithunzi zophatikizika za trachea kuchokera ku nyama iliyonse adayesedwa pogwiritsa ntchito script ya MATLAB yokha (R2020a, MathWorks) monga momwe tafotokozera kale, pogwiritsa ntchito makonda a 0.35 < Hue < 0.58, Saturation > 0.15, ndi Value < 0.7. Potsatira mawonekedwe a minofu, chigoba chinapangidwa pamanja mu GIMP v2.10.24 pa chithunzi chilichonse chophatikizika kuti adziwe malo a minofu ndikuletsa kupezeka kulikonse kolakwika kuchokera kunja kwa minofu ya trachea. Madera ophatikizika ochokera ku zithunzi zonse zophatikizika kuchokera ku nyama iliyonse adaphatikizidwa kuti apange malo onse ophatikizika a nyama imeneyo. Malo ophatikizika adagawidwa ndi malo onse ophatikizika kuti apange malo abwinobwino.
Chigawo chilichonse cha trachea chinaikidwa mu paraffin ndipo zigawo za 5 μm zinadulidwa. Zigawo zinapakidwa utoto wofiira wosasunthika kwa mphindi 5 ndipo zithunzi zinapezedwa pogwiritsa ntchito maikulosikopu ya Nikon Eclipse E400, kamera ya DS-Fi3 ndi pulogalamu yojambulira zinthu ya NIS (mtundu 5.20.00).
Kusanthula konse kwa ziwerengero kunachitika mu GraphPad Prism v9 (GraphPad Software, Inc.). Kufunika kwa ziwerengero kunayikidwa pa p ≤ 0.05. Kukhazikika kunatsimikiziridwa pogwiritsa ntchito mayeso a Shapiro-Wilk, ndipo kusiyana kwa utoto wa LacZ kunayesedwa pogwiritsa ntchito mayeso a t osaphatikizidwa.
Ma MP asanu ndi limodzi omwe afotokozedwa mu Table 1 adawunikidwa pogwiritsa ntchito PCXI, ndipo mawonekedwe ake afotokozedwa mu Table 2. Ma MP awiri a polystyrene (MP1 ndi MP2; 18 μm ndi 0.25 μm, motsatana) sanawonekere pansi pa PCXI, koma zitsanzo zina zonse zinali zodziwika (zitsanzo zikuwonetsedwa mu Chithunzi 5). MP3 ndi MP4 (10-15% Fe3O4; 0.25 μm ndi 0.9 μm, motsatana) zikuwoneka pang'ono. Ngakhale kuti zili ndi tinthu tating'ono kwambiri tomwe tinayesedwa, MP5 (98% Fe3O4; 0.25 μm) ndiyo inali yodziwika kwambiri. Chogulitsa cha CombiMag MP6 n'chovuta kuchiwona. Nthawi zonse, luso lathu lozindikira MP linakulitsidwa kwambiri pomasulira maginito kumbuyo ndi kumbuyo mofanana ndi capillary. Pamene maginito ankachoka pa capillary, tinthu tating'onoting'ono tinatambasulidwa mu zingwe zazitali, koma pamene maginito ankayandikira ndipo mphamvu ya maginito inkawonjezeka, zingwe za tinthu tating'onoting'ono zinafupika pamene tinthu tating'onoting'ono tinkasamukira pamwamba pa capillary. (onani Kanema Wowonjezera S1: MP4), kuonjezera kuchuluka kwa tinthu tating'onoting'ono pamwamba. Mosiyana ndi zimenezi, maginito akachotsedwa pa capillary, mphamvu ya munda imachepa ndipo ma MP amakonzedwanso kukhala zingwe zazitali zomwe zimatambasuka kuchokera pamwamba pa capillary (onani Kanema Wowonjezera S2:MP4). Maginito akasiya kuyenda, tinthu tating'onoting'ono timapitiriza kuyenda kwa kanthawi kochepa titafika pamalo ofanana. Pamene MP ikupita patsogolo ndi kutali ndi pamwamba pa capillary, tinthu tating'onoting'ono timene timagwiritsa ntchito maginito nthawi zambiri timakoka zinyalala kudzera mumadzimadzi.
Kuwoneka kwa MP pansi pa PCXI kumasiyana kwambiri pakati pa zitsanzo.(a) MP3, (b) MP4, (c) MP5 ndi (d) MP6.Zithunzi zonse zomwe zawonetsedwa pano zajambulidwa ndi maginito omwe ali pafupifupi 10 mm mwachindunji pamwamba pa capillary. Mabwalo akuluakulu omwe akuwoneka ndi thovu la mpweya lomwe lagwidwa mu capillaries, zomwe zikuwonetsa bwino mawonekedwe akuda ndi oyera m'mphepete mwa chithunzi cha phase contrast. Bokosi lofiira lili ndi kukula kowonjezera contrast.Dziwani kuti ma diameter a maginito schematics mu ziwerengero zonse si a kukula kwenikweni ndipo ndi pafupifupi nthawi 100 kuposa zomwe zawonetsedwa.
Pamene maginito amatembenuzidwa kumanzere ndi kumanja pamwamba pa capillary, ngodya ya chingwe cha MP imasintha kuti igwirizane ndi maginito (onani Chithunzi 6), motero kufotokoza mizere ya mphamvu ya maginito. Pa MP3-5, chord ikafika pa ngodya yolowera, tinthu tating'onoting'ono timakokedwa pamwamba pa capillary. Izi nthawi zambiri zimapangitsa kuti ma MP azisonkhana m'magulu akuluakulu pafupi ndi komwe mphamvu ya maginito ili yamphamvu kwambiri (onani Supplementary Video S3:MP5). Izi zimawonekeranso makamaka pojambula pafupi ndi mapeto a capillary, zomwe zimapangitsa kuti ma MP azisonkhana ndikukhazikika pa mawonekedwe amadzimadzi ndi mpweya. Tinthu tating'onoting'ono mu MP6, tomwe tinali ovuta kuzindikira kuposa MP3-5, sitinakokedwe pamene maginito ankayenda motsatira capillary, koma zingwe za MP zinalekanitsidwa, zomwe zinasiya tinthu tating'onoting'ono m'munda wowonera (onani Supplementary Video S4:MP6). Nthawi zina, pamene mphamvu ya maginito yogwiritsidwa ntchito inachepetsedwa posuntha maginito mtunda wautali kuchokera pamalo ojambulira, ma MP otsalawo ankatsika pang'onopang'ono pansi pa chubu ndi mphamvu yokoka pamene anali mu chingwe (onani Supplementary Video S5: MP3).
Ngodya ya chingwe cha MP imasintha pamene maginito akutembenuzidwa kumanja pamwamba pa capillary.(a) MP3, (b) MP4, (c) MP5 ndi (d) MP6. Bokosi lofiira lili ndi kukula kowonjezera kusiyana. Dziwani kuti makanema owonjezera ndi othandiza chifukwa amavumbula kapangidwe ka tinthu tofunikira komanso chidziwitso chosinthika chomwe sichingawonekere pazithunzi izi zosasunthika.
Mayeso athu adawonetsa kuti kusuntha maginito pang'onopang'ono kumbuyo ndi kumbuyo kudzera mu trachea kumathandiza kuwona MP potengera kuyenda kovuta mu vivo. Kuyesa mu vivo sikunachitike chifukwa mikanda ya polystyrene (MP1 ndi MP2) sinawonekere mu capillary. MP iliyonse yotsala inayesedwa mu vivo ndi maginito long axis yokhazikitsidwa pamwamba pa trachea pa ngodya ya pafupifupi 30° mpaka yowongoka (onani Zithunzi 2b ndi 3a), chifukwa izi zidapangitsa kuti pakhale maunyolo ataliatali a MP ndipo anali othandiza kwambiri kuposa momwe maginito adakhazikitsidwira. MP3, MP4 ndi MP6 sizinapezeke mu trachea ya nyama zamoyo zilizonse. Pamene njira zopumira za makoswe zidajambulidwa nyama zitaphedwa mwankhanza, tinthu tating'onoting'ono timakhalabe tosaoneka ngakhale pamene voliyumu yowonjezera idawonjezedwa pogwiritsa ntchito syringe pump. MP5 inali ndi iron oxide yambiri ndipo inali tinthu tokha tomwe timawoneka, ndipo chifukwa chake idagwiritsidwa ntchito kuwunika ndikuwonetsa khalidwe la MP mu vivo.
Kuyika maginito pamwamba pa trachea panthawi yopereka MP kunapangitsa kuti ma MP ambiri, koma osati onse, azisonkhana m'munda wowonera. Tinthu tomwe timalowa mu trachea timawonedwa bwino kwambiri m'zinyama zomwe zimaperekedwa nsembe mwaumunthu. Chithunzi 7 ndi Kanema Wowonjezera S6: MP5 ikuwonetsa kugwidwa mwachangu kwa maginito ndi kulumikizana kwa tinthu pamwamba pa trachea ya ventral, zomwe zikusonyeza kuti ma MP amatha kutumizidwa kumadera omwe amafunidwa a trachea. Pofufuza kutali kwambiri m'mphepete mwa trachea pambuyo popereka MP, ma MP ena adapezeka pafupi ndi carina, zomwe zikusonyeza kuti mphamvu ya maginito sinali yokwanira kusonkhanitsa ndi kusunga ma MP onse, chifukwa adaperekedwa kudzera m'dera lomwe mphamvu ya maginito inali yayikulu panthawi ya madzi. Komabe, kuchuluka kwa ma MP pambuyo pa kubereka kunali kwakukulu kuzungulira malo omwe adajambulidwa, zomwe zikusonyeza kuti ma MP ambiri adatsala m'madera opumira mpweya komwe mphamvu ya maginito yogwiritsidwa ntchito inali yayikulu kwambiri.
Zithunzi kuchokera ku (a) asanayambe ndi (b) atapereka MP5 mu trachea ya mbewa yomwe yangophedwa kumene ndi maginito atayikidwa pamwamba pa malo ojambulira. Malo ojambulirawo ali pakati pa mphete ziwiri za cartilage. MP isanaperekedwe, pali madzi m'njira yolowera mpweya. Bokosi lofiira lili ndi kukula kowonjezera. Zithunzi izi zikuchokera mu kanema wowonetsedwa mu Supplementary Video S6:MP5.
Kumasulira maginito m'mbali mwa trachea mu vivo kunapangitsa kuti unyolo wa MP usinthe ngodya mkati mwa pamwamba pa mpweya m'njira yofanana ndi yomwe imawonedwa m'mitsempha yamagazi (onani Chithunzi 8 ndi Kanema Wowonjezera S7: MP5). Komabe, mu kafukufuku wathu, ma MP sakanatha kukokedwa pamwamba pa mpweya wamoyo monga momwe angachitire ndi mitsempha yamagazi. Nthawi zina, unyolo wa MP umakhala wautali pamene maginito akuyenda kumanzere ndi kumanja. Chosangalatsa n'chakuti, tinapezanso kuti chingwe cha tinthu chikuwoneka kuti chikusintha kuya kwa gawo lamadzimadzi pamwamba pamene maginito asunthidwa motalikirapo m'mbali mwa trachea, ndikukulirakulira pamene maginito asunthidwa mwachindunji pamwamba ndipo chingwe cha tinthu chimazunguliridwa pamalo oyima (onani Kanema Wowonjezera S7). : MP5 pa 0:09, pansi kumanja). Kachitidwe ka kayendedwe kake kanasintha pamene maginito ankasunthidwa pamwamba pa trachea mozungulira (ndiko kuti, kumanzere kapena kumanja kwa nyamayo osati kutalika kwa trachea). Tinthu tinali kuwoneka bwino pamene tinkayenda, koma maginito atachotsedwa mu trachea, nsonga za tinthu tinayamba kuwoneka (onani Kanema Wowonjezera S8:MP5, kuyambira pa 0:08). Izi zikugwirizana ndi khalidwe la MP lomwe tidawona pansi pa mphamvu ya maginito yogwiritsidwa ntchito mu capillary yagalasi.
Zitsanzo za zithunzi zomwe zikuwonetsa MP5 mu trachea ya khoswe wamoyo wogonekedwa ndi mankhwala oletsa ululu.(a) Maginito amagwiritsidwa ntchito kupeza zithunzi pamwambapa ndi kumanzere kwa trachea, kenako (b) maginito atasunthidwa kupita kumanja. Bokosi lofiira lili ndi kukula kowonjezera kusiyana. Zithunzi izi ndi za kanema yemwe wawonetsedwa mu Supplementary Video S7:MP5.
Pamene ma poles awiriwa adakonzedwa molunjika kumpoto ndi kum'mwera pamwamba ndi pansi pa trachea (monga kukopa; Chithunzi 3b), ma chords a MP adawonekera motalika ndipo anali m'mbali mwa trachea osati pamwamba pa trachea (onani Kanema Wowonjezera S9: MP5). Komabe, kuchuluka kwa tinthu tating'onoting'ono pamalo amodzi (monga pamwamba pa trachea) sikunazindikirike pambuyo popereka madzi pamene chipangizo cha maginito awiri chikugwiritsidwa ntchito, zomwe nthawi zambiri zimachitika pamene chipangizo cha maginito amodzi chikugwiritsidwa ntchito. Kenako maginito amodzi akakonzedwa kuti abwezere ma poles omwe adasinthidwa (Chithunzi 3c), chiwerengero cha tinthu tating'onoting'ono tomwe timawoneka m'munda wowonera sichinawonekere kuwonjezeka pambuyo popereka. Kukhazikitsa kwa maginito awiriwa kumakhala kovuta chifukwa cha mphamvu yayikulu ya maginito yomwe imakoka kapena kukankhira maginito, motsatana. Kukhazikitsako kunasinthidwa kukhala maginito amodzi ofanana ndi njira yolowera mpweya koma kudutsa munjira yolowera mpweya pa madigiri 90 kotero kuti mizere yamunda idadutsa khoma la trachea molunjika (Chithunzi 3d), njira yowunikira yomwe idapangidwa kuti idziwe ngati kuphatikiza tinthu kumbali. khoma linkaoneka. Komabe, mu kasinthidwe aka, panalibe kuyenda kodziwika kwa MP accumulation kapena maginito movement. Kutengera zotsatira zonsezi, kasinthidwe ka single-magnet, 30-degree orientation (Chithunzi 3a) kanasankhidwa kuti kagwiritsidwe ntchito pa maphunziro a in vivo gene carrier.
Pamene nyamayo inkajambulidwa mobwerezabwereza nthawi yomweyo ataphedwa ndi munthu, kusowa kwa kuyenda kosokoneza kwa minofu kunatanthauza kuti mizere yopyapyala komanso yayifupi ya tinthu tating'onoting'ono ingathe kuoneka m'munda womveka bwino wa pakati pa chondral, "wogwedezeka" mogwirizana ndi kuyenda kwa maginito. Komabe, sitingathe kuona bwino kukhalapo ndi kuyenda kwa tinthu ta MP6.
Mlingo wa LV-LacZ unali 1.8 × 108 TU/ml, ndipo pambuyo pa 1:1 kusakaniza ndi CombiMag MP (MP6), nyama zinalandira mlingo wa 50 μl tracheal wa 9 × 107 TU/ml LV vehicle (monga 4.5 × 106 TU/rat). Mu maphunziro awa, m'malo momasulira maginito panthawi yobereka, tinakhazikitsa maginito pamalo amodzi kuti tidziwe ngati LV transduction (a) ingakhale yabwino poyerekeza ndi venctor delivery popanda mphamvu ya maginito, ndipo (b) ikhoza kukhala yolunjika. Maselo a Airway amasamutsidwa kupita kumadera omwe ali ndi maginito a pamwamba pa mpweya.
Kupezeka kwa maginito ndi kugwiritsa ntchito CombiMag pamodzi ndi ma vector a LV sikunawonekere kukhala ndi zotsatirapo zoipa pa thanzi la nyama, monga momwe zinalili ndi njira yathu yodziwika bwino yoperekera ma vector a LV. Zithunzi zakutsogolo za dera la tracheal lomwe linakhudzidwa ndi kusokonezeka kwa makina (Chithunzi Chowonjezera 1) zinasonyeza kuti panali kuchuluka kwakukulu kwa transduction mu gulu la nyama zomwe zinapatsidwa LV-MP pomwe maginito analipo (Chithunzi 9a). Kuchepa kochepa kwa utoto wa buluu wa LacZ kunalipo mu gulu lolamulira (Chithunzi 9b). Kuyeza kwa malo odetsedwa a X-Gal kunawonetsa kuti kupereka LV-MP pamaso pa mphamvu ya maginito kunapangitsa kuti pakhale kusintha pafupifupi kasanu ndi kamodzi (Chithunzi 9c).
Zitsanzo za zithunzi zophatikizika zomwe zikuwonetsa tracheal transduction ndi LV-MP (a) pamaso pa mphamvu ya maginito ndi (b) popanda maginito.(c) Kusintha kwakukulu kwa ziwerengero mu dera lokhazikika la LacZ transduction mkati mwa trachea pogwiritsa ntchito maginito (*p = 0.029, t-test, n = 3 pagulu lililonse, amatanthauza ± SEM).
Magawo ofiira ofulumira osalowerera (chitsanzo chomwe chawonetsedwa mu Chithunzi Chowonjezera 2) adawonetsa maselo ofiira a LacZ omwe ali ndi mawonekedwe ofanana ndi malo omwe adanenedwa kale.
Vuto lalikulu pa chithandizo cha majini a mpweya ndilo kudziwika bwino kwa tinthu tating'onoting'ono tomwe timanyamula mpweya m'malo ofunikira komanso kukwaniritsa mphamvu zambiri zotumizira mpweya m'mapapo oyenda pomwe mpweya ukuyenda bwino komanso kutuluka kwa mamina. Kwa anthu onyamula mpweya wa LV omwe adapangidwa kuti azitha kuchiza matenda a CF airway, kuwonjezera nthawi yokhala ya tinthu tating'onoting'ono tomwe timanyamula mpweya m'njira zoyendetsera mpweya kwakhala cholinga chomwe sichinatheke mpaka pano. Monga momwe Castellani et al. adanenera, kugwiritsa ntchito mphamvu zamaginito kuti ziwongolere kutumizira mpweya kuli ndi ubwino poyerekeza ndi njira zina zotumizira majini monga electroporation, chifukwa zimatha kuphatikiza kusavuta, kugwiritsa ntchito ndalama moyenera, malo otumizira, kugwira ntchito bwino, komanso nthawi yochepa yoperekera mpweya, komanso mwina mlingo wocheperako wa wonyamula mpweya. Komabe, kuyika kwa tinthu ta maginito m'njira zoyendetsera mpweya motsogozedwa ndi mphamvu zamaginito zakunja sikunafotokozedwepo, komanso kuthekera kwa njira iyi sikunawonetsedwe munjira zoyendetsera mpweya kuti kuwonjezere kuchuluka kwa majini m'njira zoyendetsera mpweya zomwe zili bwino.
Mayeso athu a in vitro synchrotron PCXI adawonetsa kuti tinthu tonse timene tidayesa, kupatula polystyrene MP, tidawoneka mu seti yojambulira yomwe tidagwiritsa ntchito. Pamaso pa mphamvu ya maginito, ma MP amapanga zingwe zomwe kutalika kwake kumakhudzana ndi mtundu wa tinthu ndi mphamvu ya mphamvu ya maginito (monga kuyandikira ndi kuyenda kwa maginito). Monga momwe zasonyezedwera pa Chithunzi 10, zingwe zomwe timaziona zimapangidwa chifukwa cha tinthu tating'onoting'ono tomwe timapangidwa ndi maginito ndikuyambitsa mphamvu yake ya maginito. Minda yosiyana iyi imapangitsa tinthu tina tofanana kuti tigwirizane ndikulumikizana, ndi mayendedwe ofanana ndi zingwe chifukwa cha mphamvu zakomweko kuchokera ku mphamvu zakomweko zokopa komanso zonyansa za tinthu tina.
Kuwonetsa mwachidule (a,b) tinthu tating'onoting'ono tomwe timapangidwa mkati mwa mitsempha yodzaza madzi ndi (c,d) trachea yodzazidwa ndi mpweya. Dziwani kuti mitsempha yamagazi ndi trachea sizimakokedwa kuti zigwirizane ndi kukula. Gulu (a) lilinso ndi kufotokozera kwa MP, komwe kuli tinthu ta Fe3O4 tokonzedwa mu zingwe.
Pamene maginito anasunthidwa pamwamba pa capillary, ngodya ya chingwe cha tinthu inafika pamlingo wofunikira wa MP3-5 wokhala ndi Fe3O4, pambuyo pake chingwe cha tinthu sichinakhalenso pamalo oyamba, koma chinasunthira pamwamba kupita pamalo atsopano. Izi zitha kuchitika chifukwa pamwamba pa capillary yagalasi ndi yosalala mokwanira kuti izi zitheke. Chochititsa chidwi n'chakuti, MP6 (CombiMag) sinachite izi, mwina chifukwa tinthu tinali tating'ono, tinali ndi zokutira zosiyana kapena zolipiritsa pamwamba, kapena madzi onyamula omwe ali ndi mphamvu adakhudza kuthekera kwawo kosuntha. Kusiyana kwa chithunzi cha tinthu ta CombiMag nakonso ndi kofooka, zomwe zikusonyeza kuti madzi ndi tinthu tating'onoting'ono tingakhale ndi kuchulukana kofanana ndipo motero sitingasunthirena mosavuta. Tinthu tating'onoting'ono tingathenso kuima ngati maginito ayenda mofulumira kwambiri, zomwe zikusonyeza kuti mphamvu ya maginito singathe nthawi zonse kuthana ndi kukangana pakati pa tinthu tating'onoting'ono mumadzimadzi, zomwe zikusonyeza kuti mwina sizodabwitsa kuti mphamvu ya maginito ndi mtunda pakati pa maginito ndi malo omwe akufunidwa. Zofunika kwambiri. Potengera pamodzi, zotsatirazi zikusonyezanso kuti, ngakhale maginito amatha kugwira ma MP ambiri omwe amayenda m'dera lomwe akufunidwa, sizingatheke kuti maginito azitha kudalirika. kuti tisunthire tinthu ta CombiMag pamwamba pa trachea. Chifukwa chake, tatsimikiza kuti maphunziro a in vivo LV-MP ayenera kugwiritsa ntchito mphamvu zamagetsi zosasunthika kuti zigwire madera enaake a mtengo wa mpweya.
Tinthu timeneti tikaperekedwa m'thupi, zimakhala zovuta kuzizindikira pogwiritsa ntchito minofu yovuta ya thupi, koma luso lozizindikira linakulitsidwa pomasulira maginito mopingasa pamwamba pa trachea kuti "asunthe" zingwe za MP. Ngakhale kuti kujambula zithunzi zamoyo n'kotheka, n'kosavuta kuzindikira kayendedwe ka tinthu timeneti nyama ikaphedwa mwachibadwa. Kuchuluka kwa MP nthawi zambiri kunali kwakukulu pamalo awa pamene maginito anali pamwamba pa malo ojambulira, ngakhale kuti tinthu tina timapezeka nthawi zambiri m'mbali mwa trachea. Mosiyana ndi maphunziro a in vitro, tinthuti sitingakokedwe m'mbali mwa trachea pomasulira maginito. Kupeza kumeneku kukugwirizana ndi momwe ntchofu yomwe imaphimba pamwamba pa trachea nthawi zambiri imagwirira tinthu tomwe timapumidwa, kuwagwira mu ntchofu kenako nkuchotsedwa ndi njira yochotsera mucociliary.
Tinaganiza kuti kugwiritsa ntchito maginito pokopa pamwamba ndi pansi pa trachea (Chithunzi 3b) kungapangitse kuti mphamvu ya maginito ikhale yofanana, osati mphamvu ya maginito yomwe imakhala yolimba kwambiri nthawi ina, zomwe zingayambitse kufalikira kwa tinthu tating'onoting'ono mofanana. Komabe, kafukufuku wathu woyamba sanapeze umboni womveka bwino wotsimikizira lingaliro ili. Momwemonso, kukonza maginito awiri kuti abwezeretse (Chithunzi 3c) sikunapangitse kuti tinthu tating'onoting'ono tipezeke m'dera lomwe lajambulidwa. Zomwe zapezekazi zikuwonetsa kuti kukhazikitsidwa kwa maginito awiri sikukuwonjezera kwambiri kuwongolera kwa MP, ndipo mphamvu zamphamvu zamaginito zomwe zimachokera zimakhala zovuta kuzikonza, zomwe zimapangitsa kuti njira iyi ikhale yosavuta. Mofananamo, kuyang'ana maginito pamwamba ndi kudzera mu trachea (Chithunzi 3d) sikunawonjezerenso kuchuluka kwa tinthu tating'onoting'ono tomwe tasungidwa m'dera lomwe lajambulidwa. Zina mwa zosinthazi sizingapambane chifukwa zimapangitsa kuti mphamvu ya maginito ikhale yotsika mkati mwa dera lomwe lajambulidwa. Chifukwa chake, kasinthidwe ka maginito ka angle ya madigiri 30 (Chithunzi 3a) kamaonedwa kuti ndi njira yosavuta komanso yothandiza kwambiri yoyesera mu vivo.
Kafukufuku wa LV-MP adawonetsa kuti pamene ma LV vectors adaphatikizidwa ndi CombiMag ndikuperekedwa pambuyo pa kusokonezeka kwa thupi pamaso pa mphamvu ya maginito, kuchuluka kwa transduction kunawonjezeka kwambiri mu trachea poyerekeza ndi zowongolera. Kutengera maphunziro a synchrotron imaging ndi zotsatira za LacZ, mphamvu ya maginito inali yokhoza kusunga LV mkati mwa trachea ndikuchepetsa kuchuluka kwa tinthu ta vector tomwe timalowa nthawi yomweyo m'mapapo. Kusintha kotereku kungapangitse kuti pakhale magwiridwe antchito apamwamba pomwe kumachepetsa ma titers operekedwa, transduction yosakhala yolunjika, zotsatira zoyipa zotupa komanso chitetezo chamthupi, komanso ndalama zonyamula majini. Chofunika kwambiri, malinga ndi wopanga, CombiMag ingagwiritsidwe ntchito limodzi ndi njira zina zosamutsira majini, kuphatikiza ndi ma virus ena (monga AAV) ndi nucleic acids.