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Ma gene vectors ochizira pulmonary cystic fibrosis ayenera kulunjika kumayendedwe oyendetsa mpweya, popeza kutulutsa m'mapapo kulibe mphamvu zochiritsira.Kuchita bwino kwa mavairasi kumagwirizana mwachindunji ndi nthawi yokhalamo wonyamulirayo.Komabe, madzi operekera monga zonyamulira majini mwachibadwa amafalikira mu alveoli panthawi yopuma, ndipo tizigawo tating'onoting'ono tamtundu uliwonse timachotsedwa mofulumira ndi mucociliary transport.Kukulitsa nthawi yokhalamo kwa onyamula majini mumayendedwe opumira ndikofunikira koma kovuta kukwaniritsa.Maginito onyamulira-conjugated particles omwe amatha kulunjika pamwamba pa thirakiti la kupuma amatha kuwongolera kumadera.Chifukwa cha zovuta zamaganizidwe a mu vivo, machitidwe a tinthu tating'onoting'ono totere ta maginito pamtunda wamlengalenga pamaso pa mphamvu ya maginito yogwiritsidwa ntchito sikumveka bwino.Cholinga cha phunziroli chinali kugwiritsa ntchito kujambula kwa synchrotron kuti muwone mu vivo kuyenda kwa maginito angapo a maginito mu trachea ya makoswe a anesthetized kuti aphunzire mphamvu ndi machitidwe a khalidwe la tinthu tating'ono ndi tambirimbiri mu vivo.Tidawonanso ngati kubweretsa maginito a lentiviral pamaso pa maginito kungawonjezeke kutulutsa mphamvu mu trachea ya makoswe.Kujambula kwa Synchrotron X-ray kumawonetsa machitidwe a tinthu tating'ono ta maginito m'malo osunthika komanso osuntha maginito mu vitro ndi mu vivo.Tinthu tating'onoting'ono sitingakokedwe mosavuta pamtunda wa mayendedwe amoyo pogwiritsa ntchito maginito, koma panthawi yoyendetsa, ma depositi amakhazikika pakuwona, komwe mphamvu yamaginito imakhala yamphamvu kwambiri.Kuchita bwino kwa transduction kunawonjezekanso kasanu ndi kamodzi pamene maginito a lentiviral maginito anaperekedwa pamaso pa maginito.Kutengera pamodzi, zotsatirazi zikuwonetsa kuti ma lentiviral maginito particles ndi maginito atha kukhala njira zabwino zopititsira patsogolo kulunjika kwa ma gene vector ndi ma transduction mumayendedwe a mpweya mu vivo.
Cystic fibrosis (CF) imayamba chifukwa cha kusiyana 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 mpweya, malo akuluakulu mu pathogenesis ya cystic fibrosis.Zowonongeka mu CFTR zimatsogolera kumayendedwe osadziwika bwino amadzi, kuchepa kwa madzi am'mlengalenga, komanso kutsika kwakuya kwamadzimadzi (ASL).Zimasokonezanso mphamvu ya kayendedwe ka mucociliary (MCT) kuchotsa mpweya wa particles ndi tizilombo toyambitsa matenda.Cholinga chathu ndi kupanga mankhwala amtundu wa lentiviral (LV) kuti apereke mtundu wolondola wa jini ya CFTR ndikuwongolera thanzi la ASL, MCT, ndi mapapu, ndikupitiliza kupanga matekinoloje atsopano omwe amatha kuyeza magawowa mu vivo1.
Ma vector a LV ndi amodzi mwa omwe amatsogolera ku cystic fibrosis gene therapy, makamaka chifukwa amatha kuphatikiza jini yochizira kukhala ma cell a airway basal cell (ma cell a airway stem).Izi ndi zofunika chifukwa amatha kubwezeretsa hydration ndi ntchofu chilolezo mwa kusiyanitsa ma jini owongolera ma cell apamlengalenga okhudzana ndi cystic fibrosis, zomwe zimabweretsa phindu kwa moyo wonse.Ma vector a LV amayenera kulunjikitsidwa motsutsana ndi ma conductive airways, popeza apa ndipamene kukhudzidwa kwa mapapo mu CF kumayambira.Kutumiza kwa vector mozama kwambiri m'mapapo kungapangitse kuti alveolar transduction, koma izi zilibe chithandizo cha cystic fibrosis.Komabe, zamadzimadzi monga zonyamulira majini mwachibadwa zimasamukira ku alveoli pamene zimakokedwa pambuyo pobereka3,4 ndi particles zochiritsira zimathamangitsidwa mofulumira m'kamwa ndi MCTs.Kuchita bwino kwa kutulutsa kwa LV kumagwirizana mwachindunji ndi kutalika kwa nthawi yomwe vekitala imakhalabe pafupi ndi maselo omwe akuwafunira kuti alole kutengeka kwa ma cell - "nthawi yokhalamo" 5 yomwe imafupikitsidwa mosavuta ndi mpweya wa m'deralo komanso kugwirizanitsa kwa ntchentche ndi MCT particles.Kwa cystic fibrosis, kuthekera kotalikitsa nthawi yakukhala kwa LV mumayendedwe am'mlengalenga ndikofunikira kuti mukwaniritse kusintha kwakukulu m'derali, koma kwakhala kovuta.
Kuti tithane ndi vutoli, tikuganiza kuti maginito a LV (MPs) atha kuthandiza m'njira ziwiri.Choyamba, amatha kutsogozedwa ndi maginito kupita kumtunda wopita kumlengalenga kuti apititse patsogolo kulunjika ndikuthandizira kuti tinthu tating'onoting'ono ta jini tikhale m'dera loyenera la mlengalenga;ndi ASL) kusunthira mu cell wosanjikiza 6. MPs chimagwiritsidwa ntchito ngati chandamale operekera mankhwala magalimoto pamene amamanga kwa ma antibodies, chemotherapy mankhwala, kapena mamolekyu ena ang'onoang'ono kuti amamatira ku nembanemba selo kapena kumangiriza awo ankalemekeza pamwamba zolandilira ndi kudziunjikira pa malo chotupa mu kukhalapo kwa static magetsi.Magnetic fields for cancer therapy 7. Njira zina za "hyperthermic" ndi cholinga chopha maselo a chotupa potenthetsa ma MP akakhala ndi maginito oscillating.Mfundo ya maginito transfection, momwe mphamvu ya maginito imagwiritsidwa ntchito ngati njira yopititsira patsogolo kusamutsidwa kwa DNA m'maselo, imagwiritsidwa ntchito kwambiri mu vitro pogwiritsa ntchito mitundu yosiyanasiyana ya ma gene osagwirizana ndi ma virus ndi ma gene omwe amavuta kutulutsa ma cell. ..Kuchita bwino kwa LV magnetotransfection ndi kutumiza kwa LV MP mu m'galasi mu cell mzere wa bronchial epithelium pamaso pa malo amodzi maginito unakhazikitsidwa, kuonjezera mphamvu ya transduction ndi 186 nthawi poyerekeza ndi LV vekitala yokha.LV MT idagwiritsidwanso ntchito ku in vitro model ya cystic fibrosis, pomwe maginito transfection adachulukitsa kutengera kwa LV mu zikhalidwe za mawonekedwe a air-liquid ndi gawo la 20 pamaso pa cystic fibrosis sputum10.Komabe, mu vivo organ magnetotransfection yalandira chidwi chochepa ndipo idangowunikidwa m'maphunziro ochepa a nyama11,12,13,14,15, makamaka m'mapapo16,17.Komabe, kuthekera kwa maginito transfection mu chithandizo chamapapu mu cystic fibrosis ndi zomveka.Tan et al.(2020) inanena kuti "kafukufuku wotsimikizika wokhudza kutulutsa kwamphamvu kwa m'mapapo kwa maginito a nanoparticles adzatsegula njira yamtsogolo ya CFTR inhalation njira zowongolera zotsatira zachipatala kwa odwala omwe ali ndi cystic fibrosis"6.
Makhalidwe a tinthu tating'onoting'ono ta maginito pamwamba pa thirakiti la kupuma pamaso pa mphamvu ya maginito yogwiritsidwa ntchito ndizovuta kuziwona ndi kuziphunzira, choncho sizikumveka bwino.M'maphunziro ena, tapanga njira ya Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) yowonera mosasokoneza komanso kuchuluka kwa mphindi mu vivo kusintha kwakuya kwa ASL18 ndi machitidwe a MCT19, 20 kuyeza mwachindunji njira ya gasi pamwamba pamadzi. ndipo imagwiritsidwa ntchito ngati chithandizo choyambirira chamankhwala.Kuphatikiza apo, njira yathu yowerengera ya MCT imagwiritsa ntchito tinthu tating'onoting'ono ta 10-35 µm chopangidwa ndi aluminiyamu kapena magalasi apamwamba a refractive index monga zolembera za MCT zowoneka ndi PB-PCXI21.Njira zonsezi ndizoyenera kujambula mitundu yosiyanasiyana ya tinthu, kuphatikiza a MP.
Chifukwa cha kusamvana kwapang'onopang'ono komanso kwakanthawi, zoyeserera zathu za PB-PCXI zochokera ku ASL ndi MCT ndizoyenera kuphunzira momwe zimakhalira komanso machitidwe a tinthu tating'onoting'ono komanso tambiri mu vivo kutithandiza kumvetsetsa ndi kukhathamiritsa njira zoperekera jini za MP.Njira yomwe timagwiritsa ntchito pano idatengera maphunziro athu pogwiritsa ntchito chingwe cha SPring-8 BL20B2, momwe tidawonera kusuntha kwamadzimadzi pambuyo popereka mlingo wa vector ya dummy mumpweya wammphuno ndi m'mapapo wa mbewa kuti tithandizire kufotokozera mawonekedwe athu amitundu yosiyanasiyana. mu jini yathu.maphunziro a zinyama ndi mlingo wonyamulira wa 3.4.
Cholinga cha phunziroli chinali kugwiritsa ntchito PB-PCXI synchrotron kuti muwone mwatsatanetsatane mayendedwe a vivo angapo a MP mu trachea ya makoswe amoyo.Maphunziro oyerekeza a PB-PCXI awa adapangidwa kuti ayese mndandanda wa MP, mphamvu ya maginito, ndi malo kuti adziwe momwe amakhudzira kayendetsedwe ka MP.Tinkaganiza kuti mphamvu ya maginito yakunja ingathandize kuti MF yoperekedwa ikhalebe kapena kusunthira kumalo omwe mukufuna.Maphunzirowa adatipatsanso mwayi wodziwa masanjidwe a maginito omwe amakulitsa kuchuluka kwa tinthu tating'onoting'ono totsalira mu trachea pambuyo poyika.M'kafukufuku wachiwiri, tidafuna kugwiritsa ntchito masinthidwe abwinowa kuti tiwonetse mawonekedwe osinthika omwe amabwera chifukwa chotumiza ma LV-MPs kupita ku makoswe, poganiza kuti kubweretsa ma LV-MPs poyang'ana njira ya ndege. pakuwonjezeka kwa kutulutsa kwa LV..
Maphunziro onse a nyama adachitidwa motsatira ndondomeko zovomerezeka ndi University of Adelaide (M-2019-060 ndi M-2020-022) ndi SPring-8 Synchrotron Animal Ethics Committee.Zoyesererazo zidachitika motsatira malingaliro a ARRIVE.
Zithunzi zonse za x-ray zidajambulidwa pamzere wa BL20XU pa synchrotron ya SPring-8 ku Japan pogwiritsa ntchito khwekhwe lofanana ndi lomwe lafotokozedwa kale21,22.Mwachidule, bokosi loyesera linali 245 m kuchokera ku mphete yosungiramo synchrotron.Kutalikirana kwachitsanzo kupita ku detector kwa 0.6 m kumagwiritsidwa ntchito pofufuza tinthu tating'onoting'ono ndi 0.3m pamaphunziro a kujambula mu vivo kuti apange zotsatira zosiyana.Mtengo wa monochromatic wokhala ndi mphamvu ya 25 keV unagwiritsidwa ntchito.Zithunzizi zidapezedwa pogwiritsa ntchito chosinthira chapamwamba cha X-ray (SPring-8 BM3) chophatikizidwa ndi chowunikira cha sCMOS.Transducer imatembenuza ma X-ray kukhala kuwala kowonekera pogwiritsa ntchito 10 µm thick scintillator (Gd3Al2Ga3O12), yomwe imalunjikitsidwa ku sensa ya sCMOS pogwiritsa ntchito × 10 (NA 0.3) microscope cholinga.Chojambulira cha sCMOS chinali Orca-Flash4.0 (Hamamatsu Photonics, Japan) yokhala ndi mapikisi a 2048 × 2048 ndi kukula kwa pixel yaiwisi ya 6.5 × 6.5 µm.Kukonzekera kumeneku kumapereka kukula kwa pixel ya isotropic ya 0.51 µm ndi malo owonera pafupifupi 1.1 mm × 1.1 mm.Kutalika kwa nthawi ya 100 ms kusankhidwa kuti kukulitsa chiŵerengero cha maginito ndi phokoso la maginito mkati ndi kunja kwa mpweya ndikuchepetsa zoyenda zomwe zimachitika chifukwa cha kupuma.Kwa maphunziro a vivo, chotsekera chothamanga cha X-ray chinayikidwa munjira ya X-ray kuti achepetse mlingo wa radiation poletsa mtengo wa X-ray pakati pa zowonekera.
Ma media a LV sanagwiritsidwe ntchito mu maphunziro aliwonse oyerekeza a SPring-8 PB-PCXI chifukwa chipinda chojambulira cha BL20XU sichinatsimikizidwe ndi Biosafety Level 2.M'malo mwake, tinasankha aphungu ambiri odziwika bwino kuchokera kwa ogulitsa awiri ogulitsa malonda omwe ali ndi kukula kwake, zipangizo, zitsulo zachitsulo, ndi ntchito, - choyamba kuti timvetsetse momwe maginito amakhudzira kayendedwe ka a MP mu ma capillaries a galasi, ndiyeno mu moyo airways.pamwamba.Kukula kwa MP kumasiyanasiyana kuchokera ku 0.25 mpaka 18 µm ndipo amapangidwa kuchokera ku zipangizo zosiyanasiyana (onani Table 1), koma mapangidwe a chitsanzo chilichonse, kuphatikizapo kukula kwa maginito a MP, sichidziwika.Kutengera ndi maphunziro athu ochulukirapo a MCT 19, 20, 21, 23, 24, tikuyembekeza kuti a MP mpaka 5 µm amatha kuwoneka panjira yodutsa mpweya, mwachitsanzo, pochotsa mafelemu otsatizana kuti awone kuwoneka bwino kwa kayendetsedwe ka MP.MP imodzi yokha ya 0.25 µm ndi yaying'ono kuposa momwe chipangizocho chimapangidwira, koma PB-PCXI ikuyembekezeka kuzindikira kusiyana kwawo kwa volumetric ndi kayendedwe ka madzi apamtunda omwe amayikidwa atayikidwa.
Zitsanzo za MP aliyense pa tebulo.1 inakonzedwa mu 20 μl galasi capillaries (Drummond Microcaps, PA, USA) ndi m'mimba mwake wa 0.63 mm.Tinthu tating'onoting'ono timapezeka m'madzi, pomwe tinthu tating'ono ta CombiMag timapezeka mumadzi omwe amapanga.Chubu chilichonse chimadzazidwa theka ndi madzi (pafupifupi 11 µl) ndikuyikidwa pachosungira (onani Chithunzi 1).Ma capillaries a galasi adayikidwa mopingasa pa siteji mu chipinda chojambula, motsatira, ndikuyika m'mphepete mwa madzi.Maginito a 19 mm (utali wa 28 mm) wa nickel-shell maginito opangidwa ndi nthaka yosowa kwambiri, neodymium, iron ndi boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Australia) yokhala ndi 1.17 T yotsalira patulani tebulo losinthira kuti mukwaniritse Kusintha kwakutali kwanu popereka.Kujambula kwa X-ray kumayamba pamene maginito ali pa 30 mm pamwamba pa chitsanzo ndipo zithunzi zimapezedwa pa mafelemu 4 pamphindi.Pa kujambula, maginito anabweretsedwa pafupi galasi capillary chubu (pa mtunda wa pafupifupi 1 mm) ndiyeno anasuntha pamodzi chubu kuwunika zotsatira za munda mphamvu ndi udindo.
Kukonzekera kwa kujambula kwa in vitro komwe kumakhala ndi zitsanzo za MP mu ma capillaries agalasi panthawi yomasulira sampuli ya xy.Njira ya X-ray mtengo imayikidwa ndi mzere wofiira wa madontho.
Mawonekedwe a in vitro a MP atakhazikitsidwa, kagawo kakang'ono ka iwo adayesedwa mu vivo pa makoswe amtchire a Wistar albino (~ masabata 12, ~ 200 g).Medetomidine 0.24 mg/kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg/kg (Dormicum®, Astellas Pharma, Japan) ndi butorphanol 4 mg/kg (Vetorphale®, Meiji Seika).Makoswe adawotchedwa ndi Pharma (Japan) osakaniza ndi jekeseni wa intraperitoneal.Pambuyo pa opaleshoni, adakonzekera kujambula mwa kuchotsa ubweya wozungulira trachea, kuyika chubu cha endotracheal (ET; 16 Ga intravenous cannula, Terumo BCT), ndi kuwapangitsa kuti asasunthike pamtunda pa mbale yojambula yomwe ili ndi thumba lotentha. kusunga kutentha kwa thupi.22. Chojambula chojambulacho chinalumikizidwa ku siteji yachitsanzo mu bokosi lojambula pamakona pang'ono kuti agwirizane ndi trachea molunjika pa chithunzi cha x-ray monga momwe tawonetsera pa Chithunzi 2a.
(a) Kukhazikitsa kwa zithunzithunzi za vivo mu gawo loyerekeza la SPring-8, njira ya X-ray yojambulidwa ndi mzere wamadontho ofiira.(b,c) Kusintha kwa maginito a tracheal kunachitika patali pogwiritsa ntchito makamera awiri a IP okhala ndi orthogonally.Kumanzere kwa chithunzicho pazenera, mutha kuwona chingwe cha waya chikugwira mutu ndi cannula yobweretsera yoyikidwa mkati mwa chubu cha ET.
Dongosolo la pampu la syringe loyang'anira kutali (UMP2, World Precision Instruments, Sarasota, FL) pogwiritsa ntchito syringe yagalasi ya 100 µl idalumikizidwa ndi PE10 chubu (0.61 mm OD, 0.28 mm ID) pogwiritsa ntchito singano ya 30 Ga.Lembani chubu kuti muwonetsetse kuti nsongayo ili pamalo oyenera mu trachea pamene mukulowetsa endotracheal chubu.Pogwiritsa ntchito micropump, syringe plunger inachotsedwa ndipo nsonga ya chubu inamizidwa mu chitsanzo cha MP kuti chiperekedwe.Chubu chobweretsera chodzaza chinalowetsedwa mu chubu cha endotracheal, ndikuyika nsonga pa gawo lamphamvu kwambiri lomwe timayembekezera kuti tigwiritse ntchito maginito.Kupeza zithunzi kunkayendetsedwa pogwiritsa ntchito chojambulira mpweya cholumikizidwa ndi bokosi lathu la Arduino, ndipo zizindikiro zonse (mwachitsanzo, kutentha, kupuma, kutsegula / kutseka, ndi kupeza zithunzi) zinajambulidwa pogwiritsa ntchito Powerlab ndi LabChart (AD Instruments, Sydney, Australia) 22 Poyerekeza Pamene nyumbayo inalibe, makamera awiri a IP (Panasonic BB-SC382) adayikidwa pafupifupi 90 ° wina ndi mzake ndipo amagwiritsidwa ntchito kulamulira malo a maginito okhudzana ndi trachea panthawi yojambula (Chithunzi 2b, c).Pofuna kuchepetsa zinthu zoyenda, chithunzi chimodzi pa mpweya chinapezedwa panthawi ya malo opumira.
Maginito amamangiriridwa ku gawo lachiwiri, lomwe lingakhale patali kunja kwa thupi lojambula.Maudindo osiyanasiyana ndi masinthidwe a maginito adayesedwa, kuphatikizapo: anaika pa ngodya ya pafupifupi 30 ° pamwamba pa trachea (makonzedwe akuwonetsedwa mu Zithunzi 2a ndi 3a);maginito imodzi pamwamba pa nyama ndi ina pansi, ndi mitengo yoyikidwa kuti ikopeke (Chithunzi 3b)., maginito amodzi pamwamba pa nyama ndi m'munsimu, ndi mitengo yomwe imayikidwa kuti iwonongeke (Chithunzi 3c), ndi maginito amodzi pamwamba ndi perpendicular to trachea (Chithunzi 3d).Mukayika chinyama ndi maginito ndikuyika MP yoyesedwa mu pampu ya syringe, perekani mlingo wa 50 µl pamlingo wa 4 µl/sekondi mutapeza zithunzi.Maginito amasunthidwa mmbuyo ndi mtsogolo motsatira kapena kudutsa trachea pamene akupitiriza kupeza zithunzi.
Kukonzekera kwa maginito kwa kujambula kwa vivo (a) maginito amodzi pamwamba pa trachea pamtunda wa pafupifupi 30 °, (b) maginito awiri opangidwa kuti akopeke, (c) maginito awiri opangidwira kuti agwedeze, (d) maginito amodzi pamwamba ndi perpendicular to the trachea.Woyang'anirayo adayang'ana pansi kuchokera mkamwa kupita ku mapapo kudzera mu trachea ndipo mtanda wa X-ray unadutsa kumanzere kwa khoswe ndikutuluka kumanja.Maginito amasunthidwa motalika kwa msewu kapena kumanzere ndi kumanja pamwamba pa trachea molunjika ku mtengo wa X-ray.
Tinayesetsanso kudziwa maonekedwe ndi khalidwe la tinthu tating'onoting'ono tomwe timadutsa mumlengalenga popanda kusakaniza kupuma ndi kugunda kwa mtima.Choncho, kumapeto kwa nthawi yojambula zithunzi, zinyama zinagwidwa mwaumunthu chifukwa cha kuwonjezereka kwa pentobarbital (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg ip).Zinyama zina zinasiyidwa pa nsanja yojambula zithunzi, ndipo pambuyo pa kutha kwa kupuma ndi kugunda kwa mtima, ndondomeko yojambula zithunzi inabwerezedwa, kuwonjezera mlingo wowonjezera wa MP ngati palibe MP yomwe ikuwonekera pamtunda.
Zithunzi zotsatiridwazo zidawongoleredwa pagawo lathyathyathya ndi lamdima kenako ndikusonkhanitsidwa kukhala kanema (mafelemu 20 pamphindikati; 15–25 × liwiro labwinobwino kutengera kupuma) pogwiritsa ntchito script yolembedwa mu MATLAB (R2020a, The Mathworks).
Maphunziro onse okhudzana ndi kutumiza ma gene vector a LV adachitika ku University of Adelaide Laboratory Animal Research Center ndipo cholinga chake chinali kugwiritsa ntchito zotsatira za kuyesa kwa SPring-8 kuti awone ngati kutumiza kwa LV-MP pamaso pa mphamvu ya maginito kungalimbikitse kusamutsa kwa majini mu vivo. .Kuti muwone zotsatira za MF ndi maginito, magulu awiri a zinyama adachiritsidwa: gulu limodzi linabayidwa ndi LV MF ndi maginito a maginito, ndipo gulu lina linabayidwa ndi gulu lolamulira ndi LV MF popanda maginito.
Ma LV gene vectors apangidwa pogwiritsa ntchito njira zomwe zafotokozedwa kale 25, 26.Vector ya LacZ imawonetsa jini ya nyukiliya ya beta-galactosidase yoyendetsedwa ndi MPSV constitutive promotioner (LV-LacZ), yomwe imapanga chinthu chopangidwa ndi buluu m'maselo osinthidwa, owonekera kumaso ndi zigawo za mapapu.Titration inkachitidwa mu zikhalidwe zama cell powerengera pamanja kuchuluka kwa maselo a LacZ-positive pogwiritsa ntchito hemocytometer kuti awerengere gawo mu TU/ml.Zonyamulira ndi cryopreserved pa -80 ° C, thawed asanagwiritse ntchito, ndipo amangiriridwa ku CombiMag posakaniza 1: 1 ndi incubating pa ayezi kwa mphindi zosachepera 30 isanakwane.
Normal Sprague Dawley makoswe (n = 3 / gulu, ~ 2-3 anesthetized ip ndi chisakanizo cha 0.4mg / kg medetomidine (Domitor, Ilium, Australia) ndi 60mg / kg ketamine (Ilium, Australia) pa 1 mwezi wa zaka) ip ) jakisoni komanso kulowetsedwa kwapakamwa kosapanga opaleshoni ndi 16 Ga mtsempha wamagazi.Kuonetsetsa kuti minofu ya tracheal airway imalandira kutembenuzidwa kwa LV, idakonzedwa pogwiritsa ntchito ndondomeko yathu yosokoneza makina yomwe tafotokozera kale momwe msewu wapamtunda wa tracheal unagwedezeka ndi dengu la waya (N-Circle, nitinol stone extractor popanda nsonga NTSE-022115 ) -UDH , Cook Medical, USA) 30 p28.Kenako, pafupifupi mphindi 10 pambuyo pa kusokonezeka mu nduna yachitetezo chachilengedwe, kayendetsedwe ka tracheal ka LV-MP kunachitika.
Mphamvu ya maginito yomwe imagwiritsidwa ntchito poyeserayi idapangidwa mofanana ndi kafukufuku wa mu vivo x-ray, ndi maginito omwewo omwe amagwiridwa pa trachea ndi distillation stent clamps (Chithunzi 4).Voliyumu ya 50 µl (2 x 25 µl aliquots) ya LV-MP inaperekedwa ku trachea (n = 3 nyama) pogwiritsa ntchito pipette ya gel-nsonga monga tafotokozera kale.Gulu lolamulira (n = 3 nyama) lidalandira LV-MP yomweyo popanda kugwiritsa ntchito maginito.Akamaliza kulowetsedwa, cannula imachotsedwa mu chubu cha endotracheal ndipo nyama imatulutsidwa.Maginito amakhalapo kwa mphindi 10 asanachotsedwe.Makoswe anapatsidwa mlingo wa meloxicam (1 ml/kg) (Ilium, Australia) motsatiridwa ndi kuchotsedwa kwa anesthesia ndi jekeseni wa intraperitoneal wa 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia).Makoswe anali kutenthedwa ndipo amawonedwa mpaka kuchira kwathunthu kwa anesthesia.
Chipangizo chotumizira cha LV-MP mu kabati yoteteza zachilengedwe.Mutha kuona kuti manja otuwa a Luer-lock a chubu cha ET amatuluka mkamwa, ndipo nsonga ya pipette ya gel yomwe ikuwonetsedwa pachithunzicho imayikidwa kudzera mu chubu cha ET mpaka kuya komwe mukufuna mu trachea.
Patatha sabata imodzi pambuyo pa kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe ka kayendetsedwe kake, nyama zinaperekedwa nsembe mwaumunthu ndi mpweya wa 100% CO2 ndipo mawu a LacZ adayesedwa pogwiritsa ntchito chithandizo chathu cha X-gal.Mphete zitatu za caudal cartilage zidachotsedwa kuti zitsimikizire kuti kuwonongeka kwa makina kapena kusungidwa kwamadzimadzi chifukwa cha endotracheal tube placement sikungaphatikizidwe pakuwunika.Thupi lililonse limadulidwa motalika kuti lipeze magawo awiri oti aunike ndikuyikidwa m'kapu yomwe ili ndi rabara ya silikoni (Sylgard, Dow Inc) pogwiritsa ntchito singano ya Minutien (Zida Zasayansi Zabwino) kuti muwone m'maso momwe kuwala kumawonekera.Kagawidwe ndi mawonekedwe a maselo osinthidwa adatsimikiziridwa ndi kujambula kutsogolo pogwiritsa ntchito maikulosikopu ya Nikon (SMZ1500) yokhala ndi kamera ya DigiLite ndi pulogalamu ya TCapture (Tucsen Photonics, China).Zithunzi zinapezedwa pa kukula kwa 20x (kuphatikizapo kukhazikika kwakukulu kwa m'lifupi lonse la trachea), ndi kutalika konse kwa trachea kumasonyezedwa pang'onopang'ono, kupereka kukwanira kokwanira pakati pa chithunzi chilichonse kuti zithunzizo zikhale "zosokedwa".Zithunzi zochokera ku trachea iliyonse zidaphatikizidwa kukhala chithunzi chimodzi chophatikizika pogwiritsa ntchito mtundu wa Composite Image Editor 2.0.3 (Microsoft Research) pogwiritsa ntchito planar motion algorithm. Dera la mawu a LacZ mkati mwa zithunzi zophatikizika zamtundu uliwonse kuchokera ku nyama iliyonse zidawerengedwa pogwiritsa ntchito cholembera cha MATLAB (R2020a, MathWorks) monga tafotokozera kale28, pogwiritsa ntchito makonda a 0.35 <Hue <0.58, Saturation> 0.15, ndi Value <0.7. Dera la mawu a LacZ mkati mwa zithunzi zophatikizika zamtundu uliwonse kuchokera ku nyama iliyonse zidawerengedwa pogwiritsa ntchito cholembera cha MATLAB (R2020a, MathWorks) monga tafotokozera kale28, pogwiritsa ntchito makonda a 0.35 <Hue <0.58, Saturation> 0.15, ndi Value <0.7. LacZ imagwira ntchito popanga makina opangira zida zamagetsi zamagetsi zamagetsi zamagetsi zamagetsi, zamagetsi zamagetsi, zamagetsi, ndi zida zopangira zida zamagetsi zamagetsi zamagetsi, zamagetsi zamagetsi, zamagetsi, zamagetsi, ndi zida zamagetsi zamagetsi zamagetsi, zamagetsi zamagetsi, zamagetsi, zamagetsi, ndi zida zamagetsi, zamagetsi, zamagetsi, zamagetsi, ndi zida zamagetsi zamagetsi, zamagetsi, zamagetsi, zamagetsi, ndi zamagetsi, ndi zida zamagetsi zamagetsi zamagetsi, zamagetsi zamagetsi, zamagetsi, zamagetsi, zamagetsi, ndi zamagetsi, ndi zida zamagetsi zamagetsi zamagetsi, zamagetsi zamagetsi, zamagetsi, zamagetsi, ndi zida, ndi zida zopangira zida zamagetsi thWorks), monga описано ранее28, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 ndi значение <0 , 7. Dera la mawu a LacZ pazithunzi zophatikizika za tracheal kuchokera ku nyama iliyonse adawerengedwa pogwiritsa ntchito cholembera cha MATLAB (R2020a, MathWorks) monga tafotokozera kale28 pogwiritsa ntchito makonda a 0.350.15 ndi mtengo <0 .7.如前所述，使用自动MATLAB 脚本（R2020a，MathWorks) 0.58, 饱和度> 0.15 ndi 值<0.7 的设置.還 前所述 , 自动自动 Matlab 脚本（（r2020a ， Mathworks） 來自每 只 的气管合 图像 用的 只 . 5 <色调 <0.58,> 0.15 ndi <0.7 的。。. .................. Zolemba za LacZ pamasamba ochezera a pa Intaneti ndi zokopa alendo, mwachitsanzo, определяли с использованием автоматистар, 2 к описано ранее, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 ndi значение <0,7 . Madera omwe LacZ amafotokozera pazithunzi zophatikizika za trachea ya nyama iliyonse adawerengedwa pogwiritsa ntchito cholembera cha MATLAB (R2020a, MathWorks) monga tafotokozera kale pogwiritsa ntchito zoikamo za 0.35 <hue <0.58, saturation> 0.15 ndi mtengo <0.7.Potsata ma contours a minofu mu GIMP v2.10.24, chigoba chinapangidwa pamanja pa chithunzi chilichonse chophatikizika kuti chizindikire chigawo cha minofu ndikuletsa kudziwika kulikonse kwabodza kunja kwa minyewa ya tracheal.Madera odetsedwa kuchokera pazithunzi zonse zamagulu osiyanasiyana a nyama iliyonse amawerengedwa kuti apereke malo onse odetsedwa a nyamayo.Malo opaka utoto adagawidwa ndi gawo lonse la chigoba kuti apeze malo okhazikika.
Trachea iliyonse idayikidwa mu parafini ndikuyika 5 µm wandiweyani.Magawo adatsutsidwa ndi kufiira kosalowerera ndale kwa mphindi 5 ndipo zithunzi zidapezedwa pogwiritsa ntchito maikulosikopu ya Nikon Eclipse E400, kamera ya DS-Fi3 ndi pulogalamu yamtundu wa NIS (mtundu wa 5.20.00).
Kusanthula konse kwa ziwerengero kunachitika mu GraphPad Prism v9 (GraphPad Software, Inc.).Kufunika kwachiwerengero kudakhazikitsidwa pa p ≤ 0.05.Chizoloŵezi chinayesedwa pogwiritsa ntchito mayeso a Shapiro-Wilk ndipo kusiyana kwa madontho a LacZ kunayesedwa pogwiritsa ntchito t-test yopanda pake.
Aphungu asanu ndi limodzi omwe akufotokozedwa mu Table 1 adawunikidwa ndi PCXI, ndipo maonekedwe akufotokozedwa mu Table 2. Ma MP1 awiri a polystyrene (MP1 ndi MP2; 18 µm ndi 0.25 µm, motsatira) sanawonekere ndi PCXI, koma zitsanzo zotsalira zikhoza kudziwika. (zitsanzo zikuwonetsedwa mu Chithunzi 5).MP3 ndi MP4 zimawoneka mofooka (10-15% Fe3O4; 0.25 µm ndi 0.9 µm, motsatana).Ngakhale MP5 (98% Fe3O4; 0.25 µm) inali ndi tinthu tating'ono tating'ono toyesedwa, inali yodziwika kwambiri.CombiMag MP6 mankhwala ndizovuta kusiyanitsa.Nthawi zonse, luso lathu lozindikira ma MFs lidapititsidwa bwino kwambiri posuntha maginito mmbuyo ndi kutsogolo molingana ndi capillary.Pamene maginito amachoka ku capillary, tinthu tating'onoting'ono timatulutsidwa mu unyolo wautali, koma pamene maginito amayandikira ndipo mphamvu ya maginito ikuwonjezeka, tinthu tating'onoting'ono timafupikitsa pamene tinthu tating'onoting'ono timasamukira kumtunda wa capillary (onani Supplemental Video S1). : MP4), kukulitsa kachulukidwe ka tinthu pamwamba.Mosiyana ndi zimenezi, maginito akachotsedwa ku capillary, mphamvu ya kumunda imachepa ndipo aphungu amakonzekeranso unyolo wautali kuchokera kumtunda wa capillary (onani Supplementary Video S2: MP4).Maginito akasiya kusuntha, tinthu tating'onoting'ono timapitirizabe kuyenda kwa nthawi ndithu titafika pa malo ofanana.Pamene MP ikupita kutali ndi kumtunda kwa capillary, maginito a maginito amatha kujambula zinyalala kudzera mumadzimadzi.
Mawonekedwe a MP pansi pa PCXI amasiyana kwambiri pakati pa zitsanzo.(a) MP3, (b) MP4, (c) MP5 ndi (d) MP6.Zithunzi zonse zomwe zikuwonetsedwa apa zidajambulidwa ndi maginito omwe ali pafupifupi 10 mm pamwamba pa capillary.Zozungulira zowoneka ngati zazikuluzikulu ndi thovu la mpweya lomwe limatsekeredwa m'ma capillaries, kuwonetsa momveka bwino mawonekedwe amtundu wakuda ndi woyera wa chithunzi chosiyana.Bokosi lofiira limasonyeza kukulitsa komwe kumawonjezera kusiyana.Dziwani kuti ma diameter a mabwalo a maginito pazithunzi zonse sayenera kukula ndipo ndi akulu kuwirikiza ka 100 kuposa momwe asonyezedwera.
Pamene maginito akuyenda kumanzere ndi kumanja pamwamba pa capillary, ngodya ya chingwe cha MP imasintha kuti igwirizane ndi maginito (onani Chithunzi 6), motero kulongosola mizere ya maginito.Kwa MP3-5, chord ikafika pachimake, tinthu tating'onoting'ono timakokera kumtunda kwa capillary.Izi nthawi zambiri zimapangitsa kuti aphungu azisonkhana m'magulu akuluakulu pafupi ndi mphamvu ya maginito (onani Supplementary Video S3: MP5).Izi zimawonekeranso makamaka pojambula pafupi ndi mapeto a capillary, zomwe zimapangitsa MP kusonkhanitsa ndi kuyang'ana pa mawonekedwe a mpweya wamadzimadzi.The particles mu MP6, amene anali ovuta kusiyanitsa kuposa MP3-5, sanali kukoka pamene maginito anasuntha pamodzi capillary, koma MP zingwe dissociated, kusiya particles mu view (onani Supplementary Video S4: MP6).Nthawi zina, mphamvu ya maginito itachepetsedwa posuntha maginito kutali ndi malo ojambulira, aphungu onse otsala amatsika pang'onopang'ono mpaka pansi pa chubu ndi mphamvu yokoka, kutsalira mu chingwe (onani Supplementary Video S5: MP3) .
Mbali ya chingwe cha MP imasintha pamene maginito amasunthira kumanja pamwamba pa capillary.(a) MP3, (b) MP4, (c) MP5 ndi (d) MP6.Bokosi lofiira limasonyeza kukulitsa komwe kumawonjezera kusiyana.Chonde dziwani kuti mavidiyo owonjezerawa ndi odziwitsa zambiri chifukwa amawulula zofunikira za tinthu tating'onoting'ono komanso chidziwitso champhamvu chomwe sichingawonekere muzithunzi zosasunthika.
Mayesero athu asonyeza kuti kusuntha maginito kumbuyo ndi kutsogolo pang'onopang'ono pa trachea kumathandizira kuwonetsera kwa MF muzochitika zovuta kuyenda mu vivo.Palibe mayeso a vivo omwe adachitidwa chifukwa mikanda ya polystyrene (MP1 ndi MP2) sinawonekere mu capillary.Iliyonse mwa ma MF anayi otsalawo idayesedwa mu vivo ndi olamulira atali a maginito omwe adayikidwa pamwamba pa trachea pamtunda wa pafupifupi 30 ° kupita ku ofukula (onani Zithunzi 2b ndi 3a), chifukwa izi zidapangitsa kuti unyolo wautali wa MF ukhale wothandiza kwambiri. kuposa maginito..kasinthidwe anathetsedwa.MP3, MP4 ndi MP6 sizinapezeke mu trachea ya nyama iliyonse yamoyo.Poyang'ana kupuma kwa makoswe pambuyo popha nyama mwaumunthu, tinthu tating'onoting'ono tinakhalabe tosaoneka ngakhale pamene voliyumu yowonjezera idawonjezeredwa pogwiritsa ntchito pampu ya syringe.MP5 inali ndi iron oxide yapamwamba kwambiri ndipo inali gawo lokhalo lowoneka, motero idagwiritsidwa ntchito kuyesa ndikuwonetsa machitidwe a MP mu vivo.
Kuyika kwa maginito pa trachea panthawi yoyika MF kunapangitsa kuti ma MF ambiri, koma osati onse, akhazikike m'malo owonera.Kulowa kwa tinthu tating'onoting'ono kumawonedwa bwino mu nyama zophedwa mwaumunthu.Chithunzi 7 ndi Supplementary Video S6: MP5 ikuwonetsa kuthamanga kwa maginito ndi kuyanjanitsa kwa tinthu tating'ono pamwamba pa trachea ya ventral, kusonyeza kuti aphungu amatha kulunjika kumadera omwe akufunidwa a trachea.Pofufuza kutali kwambiri ndi trachea pambuyo pa kubereka kwa MF, ma MF ena adapezeka pafupi ndi carina, zomwe zikuwonetsa mphamvu zamaginito zosakwanira kuti zitolere ndikugwira ma MF onse, popeza zidaperekedwa kudera lamphamvu kwambiri maginito pa nthawi yamadzimadzi.ndondomeko.Komabe, kuchulukira kwa MP pambuyo pobereka kunali kokulirapo kuzungulira malo azithunzi, kutanthauza kuti a MP ambiri adatsalira m'malo oyenda mpweya pomwe mphamvu ya maginito inali yokwera kwambiri.
Zithunzi za (a) zisanachitike ndi (b) pambuyo poperekedwa kwa MP5 mu trachea ya khoswe yemwe wavulazidwa posachedwa wokhala ndi maginito omwe adayikidwa pamwamba pa malo ojambulira.Malo omwe akujambulidwawo ali pakati pa mphete ziwiri za cartilaginous.Pali madzimadzi mumsewu wa mpweya asanaperekedwe kwa MP.Bokosi lofiira limasonyeza kukulitsa komwe kumawonjezera kusiyana.Zithunzizi zatengedwa kuchokera muvidiyo yomwe ili mu S6: MP5 Supplementary Video.
Kusuntha maginito pa trachea mu vivo kunapangitsa kusintha kwa ngodya ya MP unyolo pa airway pamwamba, mofanana ndi zomwe zimawonedwa mu capillaries (onani Chithunzi 8 ndi Supplementary Video S7: MP5).Komabe, mu kafukufuku wathu, a MP sakanatha kukokedwa pamwamba pa mathirakiti amoyo, monga momwe ma capillaries amatha.Nthawi zina, unyolo wa MP umatalika pomwe maginito akuyenda kumanzere ndi kumanja.Chochititsa chidwi, tidapezanso kuti tinthu tating'onoting'ono timasintha kuya kwamadzimadzi amadzimadzi pamene maginito amasunthidwa motalika motsatira trachea, ndipo amakula pamene maginito amasunthidwa pamwamba pamutu ndipo tinthu tating'onoting'ono tating'ono tating'ono timazungulira (onani . Kanema Wowonjezera S7).: MP5 pa 0:09, pansi kumanja).Mayendedwe ake anasintha pamene maginito anasunthidwa chatsamira pamwamba pa trachea (mwachitsanzo, kumanzere kapena kumanja kwa nyamayo, osati kutalika kwa trachea).Tinthu tating'onoting'ono tinkawoneka bwino panthawi yoyendayenda, koma maginito atachotsedwa mu trachea, nsonga za zingwe za tinthu zinayamba kuonekera (onani Supplementary Video S8: MP5, kuyambira pa 0:08).Izi zimagwirizana ndi machitidwe owonedwa a mphamvu ya maginito pansi pa mphamvu ya maginito yogwiritsidwa ntchito mu capillary ya galasi.
Zitsanzo za zithunzi zomwe zikuwonetsa MP5 mu trachea ya khoswe wamoyo.(a) Maginito amagwiritsidwa ntchito kupeza zithunzi pamwamba ndi kumanzere kwa trachea, ndiye (b) mutasuntha maginito kumanja.Bokosi lofiira limasonyeza kukulitsa komwe kumawonjezera kusiyana.Zithunzi izi zikuchokera mu kanema wa S7's Supplementary Video: MP5.
Pamene mizati iwiriyi ikuyang'aniridwa kumpoto ndi kum'mwera pamwamba ndi pansi pa trachea (ie, kukopa; Chithunzi 3b), zolembera za MP zinkawoneka motalika ndipo zinali pakhoma lakumbuyo la trachea osati pamtunda wa dorsal. trachea (onani Zowonjezera).Kanema S9:MP5).Komabe, kuchuluka kwa tinthu tating'onoting'ono pamalo amodzi (ie, pamtunda wa trachea) sikunazindikirike pambuyo poyendetsa madzimadzi pogwiritsa ntchito chipangizo chapawiri maginito, chomwe nthawi zambiri chimapezeka ndi chipangizo chimodzi cha maginito.Ndiye, pamene maginito amodzi adakonzedwa kuti athamangitse mizati yosiyana (Chithunzi 3c), chiwerengero cha tinthu tating'ono ting'onoting'ono m'munda sichinawonjezere pambuyo pobereka.Kukhazikitsa maginito onse awiri ndizovuta chifukwa cha mphamvu ya maginito yomwe imakopa kapena kukankha maginito motsatana.Kukonzekerako kunasinthidwa kukhala maginito amodzi ofanana ndi ma airways koma kudutsa mumlengalenga pamtunda wa 90 digiri kuti mizere ya mphamvu idawoloka khoma la tracheal orthogonally (Chithunzi 3d), njira yomwe ikufuna kudziwa kuthekera kwa tinthu tating'onoting'ono. khoma la lateral.kuwonedwa.Komabe, mu kasinthidwe uku, panalibe mayendedwe odziwikiratu a MF kapena kayendedwe ka maginito.Malingana ndi zotsatira zonsezi, kasinthidwe ndi maginito amodzi ndi ma 30-degree orientation anasankhidwa mu vivo maphunziro a jini onyamula (mkuyu 3a).
Nyamayo ikawonetsedwa kangapo pambuyo poperekedwa nsembe mwaumunthu, kusapezeka kwa kusokoneza kwa minofu kumatanthauza kuti mizere yowongoka, yocheperako imatha kuzindikirika pagawo lomveka bwino la intercartilaginous, 'kugwedezeka' molingana ndi kayendedwe ka maginito.onani bwino kukhalapo ndi kuyenda kwa tinthu ta MP6.
Mutu wa LV-LacZ unali 1.8 x 108 IU/mL, ndipo mutasakaniza 1: 1 ndi CombiMag MP (MP6), nyama zinabayidwa ndi 50 μl ya mlingo wa tracheal wa 9 x 107 IU/ml wa galimoto ya LV (ie 4.5) x 106 TU/khoswe).).M'maphunzirowa, m'malo mosuntha maginito panthawi yogwira ntchito, tidayika maginito pamalo amodzi kuti tiwone ngati kusintha kwa LV kungathe (a) kukhala bwino poyerekeza ndi kutumiza kwa vector popanda mphamvu ya maginito, ndi (b) ngati njira yodutsa mpweya ingathe. khalani wolunjika.Maselo omwe amasinthidwa m'madera omwe akukhudzidwa ndi maginito a chapamwamba kupuma thirakiti.
Kukhalapo kwa maginito ndi kugwiritsa ntchito CombiMag kuphatikiza ndi ma vector a LV sikunawonekere kusokoneza thanzi la nyama, monga momwe zidalili dongosolo lathu loperekera ma vector a LV.Zithunzi zam'mbuyo za dera la tracheal zomwe zimasokonezeka ndi makina (Supplementary Fig. 1) zinasonyeza kuti gulu la LV-MP lomwe linagwiritsidwa ntchito linali ndi magawo apamwamba kwambiri a transduction pamaso pa maginito (mkuyu 9a).Kupaka utoto wochepa wa buluu wa LacZ kunalipo mu gulu lolamulira (Chithunzi 9b).Kuwerengera kwa zigawo za X-Gal-stained normalized zimasonyeza kuti kayendetsedwe ka LV-MP pamaso pa maginito kunapangitsa kuti pakhale kusintha kwa 6-fold (mkuyu 9c).
Chitsanzo cha zithunzi zophatikizika zomwe zikuwonetsa kutulutsa kwa tracheal ndi LV-MP (a) pamaso pa maginito ndi (b) popanda maginito.(c) Kuwongolera kwakukulu m'dera lokhazikika la LacZ transduction mu trachea pogwiritsa ntchito maginito (* p = 0.029, t-test, n = 3 pa gulu, zikutanthauza ± zolakwika zamtundu uliwonse).
Magawo osalowerera ndale ofiira ofiira (chitsanzo chowonetsedwa mu Supplementary Fig. 2) adawonetsa kuti ma cell opaka utoto wa LacZ analipo pachitsanzo chomwecho komanso pamalo omwewo monga tafotokozera kale.
Vuto lalikulu mu chithandizo cha jini yapamsewu imakhalabe yodziwika bwino ya tinthu zonyamulira m'malo osangalatsa komanso kukwaniritsidwa kwapamwamba kwambiri pakutulutsa mphamvu m'mapapo am'manja pamaso pa kutuluka kwa mpweya ndi kutulutsa kogwira kwa ntchofu.Kwa onyamula ma LV ochizira matenda opumira mu cystic fibrosis, kukulitsa nthawi yokhala ndi tinthu tating'onoting'ono mumayendedwe oyendetsa mpweya wakhala cholinga chosatheka kufikira pano.Monga adanenera Castellani et al., kugwiritsa ntchito maginito kuti apititse patsogolo kusinthana kuli ndi zabwino kuposa njira zina zoperekera majini monga electroporation chifukwa zimatha kuphatikiza kuphweka, chuma, kutumiza m'malo, kuchulukirachulukira, komanso nthawi yofupikitsa.ndipo mwina mlingo wochepa wa galimoto10.Komabe, mu vivo kuyika ndi khalidwe la maginito particles mu mpweya pansi pa chisonkhezero cha mphamvu ya maginito kunja sikunafotokozedwe, ndipo kwenikweni luso la njira iyi kuonjezera milingo ya ma jini mumayendedwe amoyo osasunthika sikunawonetsedwe mu vivo.
Mayesero athu a in vitro pa PCXI synchrotron adawonetsa kuti tinthu tating'onoting'ono tomwe tidayesa, kupatulapo MP polystyrene, zidawoneka pakuyika kwazithunzi zomwe tidagwiritsa ntchito.Pamaso pa mphamvu ya maginito, maginito amapanga zingwe, kutalika kwake kumagwirizana ndi mtundu wa particles ndi mphamvu ya maginito (ie, kuyandikira ndi kuyenda kwa maginito).Monga momwe chithunzi 10 chikusonyezera, zingwe zomwe timaziwona zimapangidwira pamene tinthu tating'onoting'ono timakhala tokhala ndi maginito ndikupangitsa mphamvu yake ya maginito.Minda yosiyanayi imapangitsa kuti tinthu tating'ono tofanana tisonkhanitse ndikulumikizana ndi zingwe zamagulu chifukwa cha mphamvu zakumaloko kuchokera kumphamvu zakumaloko zokopa komanso kuthamangitsidwa kwa tinthu tina.
Chithunzi chosonyeza (a, b) maunyolo a tinthu tomwe timapanga mkati mwa ma capillaries odzaza madzi ndi (c, d) trachea yodzaza mpweya.Dziwani kuti ma capillaries ndi trachea samakokedwa pamlingo.Gulu (a) lilinso ndi kufotokoza kwa MF yomwe ili ndi Fe3O4 particles zokonzedwa mu unyolo.
Pamene maginito anasuntha pa capillary, ngodya ya tinthu chingwe anafika pachimake yovuta kwa MP3-5 munali Fe3O4, kenako tinthu chingwe sichinakhalenso mu malo ake oyambirira, koma anasamukira pamwamba ku malo atsopano.maginito.Izi mwina zimachitika chifukwa pamwamba pa galasi capillary ndi yosalala mokwanira kulola kusunthaku kuchitika.Chochititsa chidwi n'chakuti MP6 (CombiMag) sinachite motere, mwina chifukwa tinthu tating'onoting'ono tating'ono, tinali ndi zokutira zosiyana kapena pamwamba, kapena madzi onyamula katundu adakhudza kusuntha kwawo.Kusiyanitsa kwa chithunzi cha CombiMag ndikocheperako, kutanthauza kuti madzi ndi tinthu tating'onoting'ono titha kukhala ndi kachulukidwe komweko ndipo chifukwa chake sizingasunthike mosavuta.Tinthu tating'onoting'ono tithanso kumamatira ngati maginito ikuyenda mwachangu kwambiri, zomwe zikuwonetsa kuti mphamvu ya maginito sangathe kuthana ndi kukangana komwe kuli pakati pa tinthu tamadzimadzi, zomwe zikutanthauza kuti mphamvu ya maginito ndi mtunda wapakati pa maginito ndi malo omwe mukufuna sayenera kubwera zodabwitsa.zofunika.Zotsatirazi zikuwonetsanso kuti ngakhale maginito amatha kugwira ma microparticles ambiri omwe akuyenda kudera lomwe mukufuna, sizokayikitsa kuti maginito atha kudaliridwa kuti asunthe tinthu tating'ono ta CombiMag pamtunda wa trachea.Chifukwa chake, tidawona kuti mu vivo maphunziro a LV MF akuyenera kugwiritsa ntchito maginito osasunthika kuti alondole madera ena a mtengo wanjira.
Tinthu tating'onoting'ono tomwe taperekedwa m'thupi, zimakhala zovuta kuzizindikira potengera momwe thupi limasinthira, koma kuthekera kwawo kodziwikiratu kwasinthidwa ndikusuntha maginito molunjika pa trachea kuti "agwedeze" zingwe za MP.Ngakhale kujambula zenizeni n'kotheka, n'kosavuta kuzindikira kayendedwe ka tinthu nyamayo itaphedwa mwaumunthu.Kuchulukira kwa MP nthawi zambiri kumakhala kokwezeka kwambiri pamalopo pomwe maginito adayikidwa pamalo ojambulira, ngakhale tinthu tating'onoting'ono tinkapezeka kutsika kwa trachea.Mosiyana ndi maphunziro a in vitro, tinthu tating'onoting'ono sitingathe kukokera pansi pa trachea ndi kuyenda kwa maginito.Izi zikugwirizana ndi momwe ntchofu yomwe imakwirira pamwamba pa trachea imayendetsa tinthu tating'onoting'ono tomwe timakoka mpweya, kuwatsekera m'mamina ndikuwachotsa kudzera muco-ciliary clearance mechanism.
Tinkaganiza kuti kugwiritsa ntchito maginito pamwamba ndi pansi pa trachea kuti akopeke (mkuyu 3b) kungapangitse kuti pakhale maginito ofanana kwambiri, osati mphamvu ya maginito yomwe imakhala yokhazikika kwambiri panthawi imodzi, zomwe zingayambitse kugawidwa kofanana kwa particles..Komabe, phunziro lathu loyambirira silinapeze umboni womveka bwino wotsimikizira lingaliro ili.Mofananamo, kuika maginito awiri kuti athamangitse (mkuyu 3c) sikunapangitse kuti tinthu tambiri tikhazikike m'deralo.Zomwe zapeza ziwirizi zikuwonetsa kuti kukhazikitsidwa kwa maginito apawiri sikuwongolera kwambiri kuwongolera kwanuko kwa MP akuloza, komanso kuti mphamvu zamaginito zomwe zimatsatira zimakhala zovuta kuyimba, zomwe zimapangitsa kuti njirayi ikhale yosathandiza.Mofananamo, kuwongolera maginito pamwamba ndi kudutsa trachea (Chithunzi 3d) sikunawonjezere chiwerengero cha tinthu tating'onoting'ono totsalira m'deralo.Ena mwa masinthidwe enawa sangakhale opambana chifukwa amachepetsa mphamvu ya maginito pamalo oyika.Choncho, kasinthidwe ka maginito kamodzi pa madigiri a 30 (mkuyu 3a) amaonedwa kuti ndi ophweka komanso ogwira mtima kwambiri mu njira yoyesera ya vivo.
Kafukufuku wa LV-MP adawonetsa kuti ma vector a LV ataphatikizidwa ndi CombiMag ndikuperekedwa atasokonezeka ndi maginito, milingo ya transduction idakula kwambiri mu trachea poyerekeza ndi zowongolera.Kutengera maphunziro a synchrotron imaging ndi zotsatira za LacZ, mphamvu yamaginito idawoneka kuti imatha kusunga LV mu trachea ndikuchepetsa kuchuluka kwa tinthu tating'onoting'ono tomwe tidalowa mkati mwa mapapo.Kuwongolera kotereku kumatha kupangitsa kuti pakhale kuchita bwino kwambiri pomwe kuchepetsa ma titers operekedwa, kusamutsa kopanda cholinga, zotupa ndi chitetezo chamthupi, komanso mtengo wosinthira jini.Chofunika kwambiri, malinga ndi wopanga, CombiMag ingagwiritsidwe ntchito pamodzi ndi njira zina zotumizira majini, kuphatikizapo ma vectors ena a ma virus (monga AAV) ndi nucleic acids.