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Ma gene vectors ochizira matenda a cystic fibrosis m'mapapo ayenera kuyang'ana njira zoyendetsera mpweya chifukwa zotumphukira zam'mapapo sizipereka chithandizo chamankhwala. Ma virus amatha kulumikizana mwachindunji ndi nthawi yokhala ndi vector. Nthawi ya ma gene onyamula ma gene mumsewu ndi yofunika koma yovuta kukwaniritsa. Maginito onyamula maginito amtundu wa gene omwe amatha kulunjikitsidwa kumtunda kwa mayendedwe amlengalenga amatha kuwongolera kuwongolera kwa dera. Tinthu tating'onoting'ono ta makoswe opangidwa ndi anesthetized kuti tiwunikire mphamvu ndi machitidwe a munthu ndi kuchuluka kwa tinthu tambiri mu vivo.Tinawonanso ngati kuperekedwa kwa tinthu tating'onoting'ono ta lentiviral pamaso pa maginito kungawonjezeke kutulutsa mphamvu mu trachea ya Synchrotron. kukokera pamwamba pa mpweya wamoyo ndi maginito, koma pa zoyendera, madipoziti ndi anaikira m'munda wa maganizo kumene maginito mphamvu kwambiri.Transduction Mwachangu analinso chinawonjezeka kasanu ndi kamodzi pamene lentiviral maginito particles anaperekedwa pamaso pa maginito field.Together, zotsatirazi zikusonyeza kuti lentiviral maginito particles ndi maginito akhoza kukhala njira zofunika kwambiri chandamale chandamale mu kupititsa patsogolo jini maginito maginito.
Cystic fibrosis (CF) imayamba chifukwa cha kusiyanasiyana kwa jini imodzi yotchedwa CF transmembrane conductance regulator (CFTR) .Protein ya CFTR ndi njira ya ion yomwe imapezeka m'maselo ambiri a epithelial m'thupi lonse, kuphatikizapo ma airways oyendetsa ndege, malo akuluakulu a CF pathogenesis.Zilema za CFTR zimatsogolera kumayendedwe osadziwika bwino a madzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzimadzi, kuchepetsa mpweya wamadzi, kuchepetsa mpweya wamadzi ndi kutsika kwa mpweya. wosanjikiza.Izi zimasokonezanso mphamvu ya kayendedwe ka mucociliary (MCT) kuchotsa particles zokoka mpweya ndi tizilombo toyambitsa matenda kuchokera ku airways.Cholinga chathu ndi kupanga lentiviral (LV) gene therapy kuti apereke buku lolondola la jini la CFTR ndikusintha ASL, MCT, ndi thanzi la m'mapapo, ndi kupitiriza kupanga matekinoloje atsopano omwe amatha kugwiritsira ntchito meseji.
Ma vectors a LV ndi amodzi mwa omwe amatsogolera ku CF airway gene therapy, makamaka chifukwa amatha kuphatikizira jini yochizira m'maselo a airway basal cell (ma cell a airway stem) . Izi ndizofunikira chifukwa zimatha kubwezeretsa kutulutsa kwabwinobwino komanso kutulutsa kwa ntchentche mwa kusiyanitsa kukhala magwiridwe antchito amtundu wa CF-ogwirizana ndi mayendedwe apamlengalenga ogwirizana ndi ma cell, zomwe zimapangitsa kuti ma cell a airway ayende molunjika, zomwe zimapangitsa kuti ma cell amoyo azikhala molunjika. CF matenda a m'mapapo amayamba.Kupereka kwa vector mozama kwambiri m'mapapo kungachititse kuti alveolar transduction, koma izi zilibe phindu lachirengedwe mu CF.Komabe, madzi monga onyamula ma jini amasamukira ku alveoli pa kudzoza pambuyo pobereka3,4 ndipo tinthu tating'onoting'ono timathamangitsidwa mofulumira muzitsulo zapakamwa za LV kufupikitsa kufupi ndi nthawi yokhudzana ndi ma cell a MCT. kulola kutengeka kwa ma cell - "nthawi yogona" 5 - yomwe imachepetsedwa mosavuta ndi kayendedwe ka mpweya wa m'deralo komanso kugwirizanitsa tinthu tating'onoting'ono tating'onoting'ono ndi MCT.For CF, kuthekera kotalikitsa nthawi yokhalamo ya LV mkati mwa mlengalenga ndikofunika kuti tikwaniritse maulendo apamwamba a transduction m'dera lino, koma mpaka pano akhala ovuta.
Kuti tithane ndi chopingachi, tikuwonetsa kuti ma LV maginito particles (MPs) angathandize m'njira ziwiri zowonjezera.Choyamba, amatha kutsogoleredwa ndi maginito kumalo okwera ndege kuti apititse patsogolo kuwongolera ndikuthandizira kuti tizilombo toyambitsa matenda tizikhala m'dera lomwe mukufuna; ndi ASL) kuti apite ku cell layer 6.MPs akhala akugwiritsidwa ntchito kwambiri ngati magalimoto operekera mankhwala osokoneza bongo pamene amangirira ku ma antibodies, chemotherapeutic mankhwala, kapena mamolekyu ena ang'onoang'ono omwe amamangiriza ku maselo a cell kapena kumangirira ku maselo okhudzana ndi maselo okhudzana ndi maselo ndikudziunjikira pa malo otupa pamaso pa magetsi osasunthika. Magnetic Fields for Cancer Treatment 7. Njira zina za "hyperthermal" zimafuna kutenthetsa aphungu pamene akukumana ndi oscillating magnetic fields, motero amawononga maselo a chotupa. Mfundo ya magnetic transfection, yomwe maginito amagwiritsidwa ntchito ngati transfection agent kuti apititse patsogolo kusamutsidwa kwa DNA ku maselo, amagwiritsidwa ntchito kwambiri mu vitro pogwiritsa ntchito mitundu yosiyanasiyana ya mavairasi omwe sali mavairasi ndi mavairasi amtundu wa LV. magnetotransfection wakhazikitsidwa, ndi m'galasi yobereka LV-MPs kwa munthu bronchial epithelial selo mzere pamaso pa malo amodzi maginito, kuonjezera transduction dzuwa ndi 186-kuyerekeza ndi LV vekitala yekha. sputum. nanoparticle pulmonary delivery idzatsegulira njira zamtsogolo za CFTR inhalation njira zopititsa patsogolo zotsatira zachipatala mwa odwala CF "6.
Makhalidwe a tinthu tating'onoting'ono ta maginito pamayendedwe a mpweya pamaso pa maginito ogwiritsidwa ntchito ndizovuta kuwona ndikuwerenga, motero sitimvetsetseka bwino. Zomwe zimagwiritsidwa ntchito ngati chisonyezero choyambirira cha chithandizo chamankhwala.Kuonjezera apo, njira yathu yowunikira MCT imagwiritsa ntchito tinthu tating'onoting'ono ta 10-35 µm chopangidwa ndi alumina kapena galasi lapamwamba la refractive index monga zizindikiro za MCT zowonekera pogwiritsa ntchito PB-PCXI21.Njira zonse ziwirizi ndizoyenera kuwonetseratu mitundu yosiyanasiyana ya tinthu, kuphatikizapo MP.
Chifukwa cha kusinthasintha kwake kwanthawi yayitali komanso kwakanthawi, njira zathu zowunikira za PB-PCXI zochokera ku ASL ndi MCT ndizoyeneranso kuyesa mphamvu ndi machitidwe amtundu umodzi komanso wochuluka wa tinthu mu vivo kutithandiza kumvetsetsa ndi kukhathamiritsa njira zoperekera majini a MP. Mpweya wa m'mphuno ndi m'mapapo wa mbewa kuti uthandize kufotokozera machitidwe athu osakhala ofanana a jini omwe amawonedwa mu maphunziro athu amtundu wa gene carrier 3,4.
Cholinga cha phunziroli chinali kugwiritsa ntchito synchrotron PB-PCXI kuti muwone momwe ma vivo akuyendera pamagulu a MP mu trachea ya makoswe amoyo. Mu kafukufuku wachiwiri, tidafuna kugwiritsa ntchito kasinthidwe koyenera kameneka kuti tiwonetse mawonekedwe a transduction omwe amachokera ku vivo kutumiza ma LV-MPs kupita kunjira ya makoswe, kutengera lingaliro lakuti kutumiza kwa LV-MPs kungapangitse kusintha kwa kayendedwe ka mpweya.
Maphunziro onse a zinyama anachitidwa motsatira ndondomeko zovomerezeka ndi University of Adelaide (M-2019-060 ndi M-2020-022) ndi SPring-8 Synchrotron Animal Ethics Committee.Kuyesera kunachitika motsatira malangizo a ARRIVE.
Zithunzi zonse za X-ray zinkachitidwa pa BL20XU beamline pa SPring-8 synchrotron ku Japan, pogwiritsa ntchito khwekhwe lofanana ndi lomwe linafotokozedwa kale21,22.Mwachidule, bokosi loyesera linali 245 mamita kuchokera ku mphete yosungiramo synchrotron.Chitsanzo-to-detector mtunda wa 0.6 m amagwiritsidwa ntchito pa maphunziro a particle imaging ya 0vo3 mA kuti apange maphunziro a particle imaging. Mphamvu ya mtengo wa monochromatic ya 25 keV inagwiritsidwa ntchito. Zithunzi zinajambulidwa pogwiritsa ntchito chosinthira chapamwamba kwambiri cha X-ray (SPring-8 BM3) chophatikizidwa ndi chowunikira cha sCMOS. Chosinthiracho chimatembenuza ma X-ray kukhala kuwala kowoneka bwino pogwiritsa ntchito 10 µm thick scintillator (Gd3Al2Ga3C3C yolunjika ku microscope × MOS1) pogwiritsa ntchito microscope × MOS12 (NA 0.3) .Chowunikira cha sCMOS chinali Orca-Flash4.0 (Hamamatsu Photonics, Japan) chokhala ndi kukula kwa mapikiselo 2048 × 2048 ndi kukula kwa pixel yaiwisi ya 6.5 × 6.5 µm. Kukonzekera uku kumapereka kukula kwa pixel ya isotropic ya 0.51 × pafupifupi 0.51 × 1 mm. mm.Utali wowonekera wa 100 ms unasankhidwa kuti uwonjezere chiŵerengero cha maginito ndi phokoso la maginito a maginito mkati ndi kunja kwa njira ya mpweya pamene kuchepetsa kusuntha kwa mpweya wopangidwa ndi kupuma.
Chonyamulira cha LV sichinagwiritsidwe ntchito mu maphunziro aliwonse oyerekeza a SPring-8 PB-PCXI chifukwa chipinda chojambulira cha BL20XU sichiri chovomerezeka cha Biosafety Level 2. M'malo mwake, tinasankha aphungu odziwika bwino kuchokera kwa ogulitsa awiri ogulitsa malonda-omwe amaphimba kukula kwake, zipangizo, kuyika kwachitsulo, ndi kugwiritsa ntchito mkati mwa magalasi oyendayenda, magalasi a MP ndi magalasi amakhudza momwe maginito amoyo amakhudzidwira. ma airways. pamwamba.MPs osiyanasiyana kukula kwa 0,25 kuti 18 μm ndipo anapangidwa kuchokera zosiyanasiyana zipangizo (onani Table 1), koma zikuchokera chitsanzo aliyense, kuphatikizapo kukula kwa maginito particles mkati MP, sichidziwika.Kuchokera pa maphunziro athu ochuluka a MCT 19, 20, 21, 23, 24, tikuyembekeza kuti MP 5 panjira yowoneka ngati yang'ono kuchotsa mafelemu otsatizana kuti muwone kuwonjezereka kwa kayendetsedwe ka MP. MP imodzi yokha ya 0.25 μm ndi yaying'ono kusiyana ndi momwe chipangizo chojambula chimapangidwira, koma PB-PCXI ikuyembekezeka kuzindikira kusiyana kwake kwa voliyumu ndi kayendedwe ka madzi amadzimadzi omwe amaikidwa pambuyo poyikira.
Zitsanzo za MP aliyense mu Table 1 zinakonzedwa mu 20 μl galasi capillaries (Drummond Microcaps, PA, USA) ndi m'mimba mwake wa 0.63 mm. Corpuscular particles akupezeka m'madzi, pamene CombiMag particles akupezeka mu eni ake madzimadzi. Chubu chilichonse ndi theka lodzaza ndi madzi (pafupifupi 1 μ) 1) .The galasi capillaries anaikidwa horizontally pa chitsanzo siteji mu chithunzi bokosi, motero, ndi pabwino m'mbali mwa madzimadzi.A 19 mm awiri (28 mm kutalika) faifi tambala chipolopolo osowa dziko lapansi neodymium chitsulo boron (NdFeB) magnet (N35, mphaka no. LM1652, Australia1 magnetization a Electronics). adalumikizidwa ku gawo lomasulira losiyana kuti akwaniritse Sinthani malo ake patali panthawi yojambula.Kupeza chithunzi cha X-ray kumayamba pomwe maginito ayikidwa pafupifupi 30 mm pamwamba pa chitsanzo, ndipo zithunzi zimapezedwa pamlingo wa mafelemu a 4 pa sekondi imodzi. Panthawi yojambula, maginito adabweretsedwa pafupi ndi chubu cha capillary (pafupifupi 1 mm kutali) ndiyeno kumasuliridwa motsatira chubu mpaka kumunda.
Kukonzekera kwa zithunzithunzi zokhala ndi zitsanzo za MP m'makapilari agalasi pa siteji yomasulira ya xy. Njira ya mtengo wa X-ray imakhala ndi mzere wofiira wodumphira.
Pamene maonekedwe a in vitro a MPs adakhazikitsidwa, gawo laling'ono la iwo linayesedwa mu vivo mu makoswe amtundu wa albino Wistar (~ masabata a 12, ~ 200 g) .0.24 mg / kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg / kg midazolam (Dormicum®, mg4, Astella Phaorpha, Japan) buts (Vetorphale®, Meiji Seika) Makoswe adagwidwa ndi mankhwala osakaniza a Pharma), Japan) ndi jekeseni wa intraperitoneal. 22 .Chojambula chojambulacho chinalumikizidwa ku gawo lomasulira lachitsanzo mu bokosi lojambula pamakona pang'ono kuti agwirizane ndi trachea molunjika mu chithunzi cha X-ray, monga momwe tawonetsera pa Chithunzi 2a.
(a) Kukonzekera kwa maginito a vivo mu bokosi lojambula la SPring-8, njira ya X-ray imayikidwa ndi mzere wofiira wofiira.
Dongosolo la pampu la syringe lakutali (UMP2, World Precision Instruments, Sarasota, FL) pogwiritsa ntchito syringe ya galasi ya 100 μl inagwirizanitsidwa ndi PE10 tubing (OD 0.61 mm, ID 0.28 mm) kudzera mu singano ya 30 Ga. Lembani chubu kuti muwonetsetse kuti nsonga ili mu malo olondola. Plunger ya syringe inachotsedwa pamene nsonga ya chubu inamizidwa mu chitsanzo cha MP kuti chiperekedwe. Chubu chobweretsera chodzaza chinayikidwa mu chubu cha endotracheal, ndikuyika nsonga mkati mwa gawo lamphamvu kwambiri la magnetic field yomwe timayembekezera. kupeza) zinalembedwa pogwiritsa ntchito Powerlab ndi LabChart (AD Instruments, Sydney, Australia) 22. Pojambula Pamene mpanda unali wosafikirika, makamera awiri a IP (Panasonic BB-SC382) adayikidwa pafupifupi 90 ° kwa wina ndi mzake ndipo ankagwiritsidwa ntchito kuyang'anira malo a maginito okhudzana ndi trachea, Ficmize (miniging. zojambula, chithunzi chimodzi chinapezedwa pa mpweya uliwonse panthawi ya mapeto a mafunde othamanga.
Maginito amamangiriridwa ku gawo lachiwiri lomwe lingathe kupezeka kutali ndi nyumba yojambula zithunzi.Malo osiyanasiyana a maginito ndi masinthidwe anayesedwa, kuphatikizapo: Wokwera pamtunda wa pafupifupi 30 ° pamwamba pa trachea (masinthidwe akuwonetsedwa mu Zithunzi 2a ndi 3a); maginito imodzi pamwamba pa nyama ndi ina pansi, ndi mitengo yoyikidwa kuti ikope (Chithunzi 3b); maginito imodzi pamwamba pa nyama ndi ina pansi, ndi mitengo yoyikidwa kuti ithamangitse (Chithunzi 3c); ndi maginito amodzi pamwamba ndi perpendicular ku trachea (Chithunzi 3d) Chinyama ndi maginito zikakonzedwa ndipo MP kuti iyesedwe imalowetsedwa mu mpope wa syringe, perekani mlingo wa 50 μl pa mlingo wa 4 μl / sec pamene mukupeza zithunzi.
Kukonzekera kwa maginito kwa chithunzi cha vivo (a) maginito amodzi pamwamba pa trachea pamtunda wa pafupifupi 30 °, (b) maginito awiri omwe amaikidwa kuti akope, (c) maginito awiri omwe amaikidwa kuti athamangitse, (d) maginito amodzi pamwamba ndi perpendicular mu trachea. Mbali.Maginito amasunthidwa motalika kwa njira yodutsa mpweya kapena kumanzere ndi kumanja pamwamba pa trachea molunjika ku mtengo wa X-ray.
Tinafunanso kudziwa kuwonekera ndi khalidwe la tinthu tating'onoting'ono mu mpweya popanda kupuma kosokoneza komanso kuyenda kwa mtima.Choncho, kumapeto kwa nthawi yojambula zithunzi, nyama zinaphedwa mwaumunthu chifukwa cha pentobarbital overdose (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg / kg, ip inasiyidwa pa nsanja yamtima ndi kamodzi, nyama zojambulidwa zinasiyidwa pa nsanja yojambula). Kujambula kunabwerezedwa, kuwonjezera mlingo wowonjezera wa MP ngati palibe MP yomwe imawoneka pamtunda.
Zithunzi zomwe zidapezedwazo zinali zathyathyathya komanso zamdima zomwe zidakonzedwa kenako zidasonkhanitsidwa kukhala kanema (mafelemu 20 pamphindikati; 15-25 × liwiro labwinobwino kutengera kupuma) pogwiritsa ntchito script yolembedwa mu MATLAB (R2020a, The Mathworks).
Maphunziro onse operekera ma gene vector a LV anachitika ku Laboratory Animal Research Facility ku Yunivesite ya Adelaide ndipo cholinga chake chinali kugwiritsa ntchito zotsatira za kuyesa kwa SPring-8 kuti awone ngati kuperekedwa kwa LV-MP pamaso pa maginito kumatha kupititsa patsogolo kusamutsidwa kwa majini mu vivo. maginito.
Ma gene vectors a LV anapangidwa pogwiritsa ntchito njira zomwe zafotokozedwa kale 25, 26. Vector ya LacZ imasonyeza jini ya nyukiliya ya beta-galactosidase yomwe imayendetsedwa ndi constitutive MPSV promoter (LV-LacZ), yomwe imapanga mankhwala opangidwa ndi buluu m'maselo osinthidwa, omwe amawonekera m'mapapo am'mapapo ndi zigawo za minofu. hemocytometer kuwerengera titer mu TU / ml. Onyamula ndi cryopreserved pa -80 ° C, thawed isanayambe kugwiritsidwa ntchito, ndi kumangirizidwa ku CombiMag mwa kusakaniza pa 1: 1 chiŵerengero ndi incubating pa ayezi kwa osachepera mphindi 30 isanafike yobereka.
Normal Sprague Dawley makoswe (n = 3 / gulu, ~ 2-3 anali anesthetized intraperitoneally ndi chisakanizo cha 0.4 mg / kg medetomidine (Domitor, Ilium, Australia) ndi 60 mg / kg ketamine (Ilium, Australia) mwezi wazaka) ip) jekeseni ndi osapanga opaleshoni oral cannulation 16 Kuonetsetsa kuti mpweya wodutsa m'kamwa ungathe. imalandira LV transduction, idakonzedwa pogwiritsa ntchito protocol yathu yomwe tafotokozera kale yosokoneza, momwe njira yodutsa mpweya imagwedezeka ndi waya wa waya (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Tracheal kabati pambuyo pa chitetezo cha LV-MPological pambuyo pa maminiti a LV-MPological.
Mphamvu ya maginito yomwe imagwiritsidwa ntchito poyeserayi inakonzedwa mofanana ndi kafukufuku wojambula zithunzi wa X-ray mu vivo, ndi maginito omwewo omwe anali pamwamba pa trachea pogwiritsa ntchito distillation stent clips (Chithunzi 4) .Voliyumu ya 50 μl (2 × 25 μl aliquots) ya LV-MP inaperekedwa mu trachea (n gel tiptte gulu lofotokozedwa kale) pogwiritsa ntchito gel tiptte gulu (tip 3). = Zinyama za 3) zinalandira ma LV-MPs omwewo popanda kugwiritsa ntchito maginito.Pambuyo kulowetsedwa kwatha, cannula imachotsedwa ku chubu cha ET ndipo nyamayo imachotsedwa.Maginito amakhalapo kwa mphindi 10, kenako amachotsedwa.Makhoswe adalandira mlingo wochepa wa meloxicam (1 ml / kg) (Ilium, Australia jekeseni ya atimaversp ya anspassia ya 1 mg / kg). hydrochloride (Antisedan, Zoetis, Australia) . Makoswe ankasungidwa kutentha ndi kuyang'aniridwa mpaka kuchira kwathunthu kuchokera ku opaleshoni.
Chida choperekera LV-MP mu kabati yachitetezo chachilengedwe.Chingwe chotuwa cha Luer chopepuka cha chubu cha ET chikhoza kuwonedwa chikukwera kuchokera pakamwa ndipo nsonga ya gel ya pipette yomwe ikuwonetsedwa pachithunzichi imayikidwa kudzera mu chubu cha ET mpaka kuya kofunidwa mu trachea.
Patangotha ​​​​sabata imodzi pambuyo pa ndondomeko ya dosing ya LV-MP, nyama zinaphedwa mwaumunthu ndi 100% CO2 inhalation ndi LacZ kufotokozera kunayesedwa pogwiritsa ntchito mankhwala athu ovomerezeka a X-gal. Mphete zitatu za caudal kwambiri za cartilaginous zinachotsedwa kuti zitsimikizire kuti kuwonongeka kwa makina kapena kusungirako madzi kuchokera ku endotracheal tube placement sikunaphatikizidwe mu kusanthula. mbale yokhala ndi mphira wa silikoni (Sylgard, Dow Inc) pogwiritsa ntchito singano ya Minutien (Fine Science Tools) kuti muone momwe kuwala kumawonekera.Kugawa ndi mawonekedwe a maselo osinthidwa kunatsimikiziridwa ndi kujambula kutsogolo pogwiritsa ntchito maikulosikopu ya Nikon (SMZ1500) ndi DigiLite kamera ndi pulogalamu ya TCapture (Tucsen Photonics, China). m'lifupi mwa trachea), ndi utali wonse wa trachea chithunzi pang'onopang'ono, kuonetsetsa palinso kokwanira pakati pa chithunzi chilichonse kulola fano "kusoka".Zithunzi kuchokera pa trachea iliyonse kenako anasonkhanitsidwa mu gulu limodzi chifaniziro ntchito Image Composite Editor v2.0.3 (Microsoft Research) utilizing planar zoyenda aligorivimu. automated MATLAB script (R2020a, MathWorks) monga tafotokozera kale, pogwiritsa ntchito zoikamo za 0.35 0.15, ndi Value <0.7.Potsata mikombero ya minofu, chigoba chinapangidwa pamanja mu GIMP v2.10.24 kuti azindikire mawonekedwe akunja amtundu uliwonse kuti azindikire mawonekedwe akunja amtundu uliwonse kuti azindikire chigawo chilichonse chabodza kuti azindikire chigawo chilichonse chabodza pamtundu uliwonse. Tizilombo totchedwa tracheal tissue
Trachea iliyonse idayikidwa mu parafini ndipo magawo a 5 μm adadulidwa. Zigawo zidatsutsidwa ndi zofiira zosalowerera ndale kwa mphindi 5 ndipo zithunzi zidapezedwa pogwiritsa ntchito microscope ya Nikon Eclipse E400, kamera ya DS-Fi3 ndi pulogalamu yamtundu wa NIS (mtundu wa 5.20.00).
Kusanthula kwa chiwerengero chonse kunachitika mu GraphPad Prism v9 (GraphPad Software, Inc.) .Kufunika kwachiwerengero kunayikidwa pa p ≤ 0.05.Normality inatsimikiziridwa 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 anayesedwa pogwiritsa ntchito PCXI, ndipo maonekedwe akufotokozedwa mu Table 2. Ma MP1 awiri a polystyrene (MP1 ndi MP2; 18 μm ndi 0.25 μm, motsatira) sizinawoneke pansi pa PCXI, koma zina zonsezo zinali zodziwika (zitsanzo zikuwonetsedwa mu 5% Fegure; 0,25 μm ndi 0.9 μm, motero) ndi zooneka mozizirira. Pamene maginito anachoka ku capillary, ndi particles anawonjezera mu zingwe yaitali, koma maginito anayandikira ndi maginito mphamvu kuchuluka, ndi tinthu zingwe adzafupikitsidwa monga particles anasamukira kumtunda pamwamba pa capillary (onani Supplementary Video S1: MP4), kuwonjezeka The tinthu osalimba Connsiversely wa kuchotsedwa kwa maginito kumunda, pamene mphamvu ya capillary imachotsedwa pamtunda. amachepetsa ndipo aphungu amasintha kukhala zingwe zazitali zomwe zimachokera kumtunda kwa capillary (onani Supplementary Video S2: MP4). Maginito akasiya kuyenda, tinthu tating'onoting'ono timapitiriza kuyenda kwa kanthawi kochepa titafika pa malo ogwirizana.
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 zinatengedwa ndi maginito omwe ali pafupifupi 10 mm pamwamba pa capillary.Zozungulira zazikulu zowoneka ndi ming'oma ya mpweya yomwe imatsekeredwa m'kati mwa capillaries, kusonyeza momveka bwino kusiyana kwakuda ndi koyera komwe kuli ndi mbali zofiira za bokosi lofiira. Dziwani kuti ma diameter a maginito schematics muzithunzi zonse sayenera kukula ndipo ndi pafupifupi 100 kuwirikiza kuposa momwe zikuwonetsedwa.
Monga maginito amamasuliridwa kumanzere ndi kumanja pamwamba pa capillary, ngodya ya MP chingwe kusintha kuti agwirizane ndi maginito (onani Chithunzi 6), motero delineating maginito munda mizere.Pa MP3-5, pambuyo chord kufika pa ngodya, ndi particles amakokera pamwamba pamwamba pa capillary. Supplementary Video S3: MP5). Izi zimawonekeranso makamaka pojambula pafupi ndi kumapeto kwa capillary, zomwe zimapangitsa MPs kuti aziphatikizana ndikuyang'ana pa mawonekedwe amadzimadzi-mpweya. Tinthu ta MP6, zomwe zinali zovuta kuzizindikira kuposa MP3-5, sizinakokedwe pamene maginito anasuntha pamodzi ndi capillary, koma MP, kusiya mawonedwe a chigawo cha Video. S4: MP6) .Nthawi zina, mphamvu ya maginito yogwiritsidwa ntchito itachepetsedwa posuntha maginito kutali kwambiri ndi malo ojambulira, aphungu onse otsala amatsika pang'onopang'ono pansi pa chubu ndi mphamvu yokoka pamene akukhalabe mu chingwe (onani Supplementary Video S5: MP3).
Mbali ya chingwe cha MP imasintha pamene maginito amamasuliridwa kumanja pamwamba pa capillary.(a) MP3, (b) MP4, (c) MP5 ndi (d) MP6. Bokosi lofiira lili ndi kukweza kosiyana kosiyana.Dziwani kuti mavidiyo owonjezera ndi odziwitsa pamene amavumbulutsa zofunikira za tinthu tating'ono ndi chidziwitso champhamvu chomwe sichingawonekere muzithunzi zosasunthika.
Mayesero athu anasonyeza kuti kusuntha maginito pang'onopang'ono mmbuyo ndi mtsogolo pamodzi trachea facilitates kuwonetsera kwa MP mu nkhani ya kuyenda zovuta mu vivo.In vivo kuyezetsa sanachitidwe monga polystyrene mikanda (MP1 ndi MP2) sanali kuonekera mu capillary.Aliyense wa otsala anayi MPs anayesedwa mu vivo ndi maginito yaitali olamulira configured pa ngodya30 pamwamba pa tracheatical ° configured pamwamba pa ngodya30 Zithunzi 2b ndi 3a), chifukwa izi zinapangitsa kuti unyolo wautali wa MP ndipo ukhale wogwira mtima kwambiri kuposa kasinthidwe ka maginito terminated.MP3, MP4 ndi MP6 sanapezeke mu trachea ya nyama iliyonse yamoyo. Pamene makoswe airways ankafaniziridwa nyama zitaphedwa mwaumunthu, tinthu tating'onoting'ono tinakhalabe tosaoneka ngakhale pamene voliyumu yowonjezera idawonjezedwa pogwiritsa ntchito chitsulo chopopera ndi chitsulo chowoneka bwino cha MP5. Chifukwa chake idagwiritsidwa ntchito kuwunika ndikuwonetsa machitidwe a MP.
Kuyika maginito pa trachea pa MP yobereka kunachititsa ambiri, koma si onse, MPs kukhala anaikira m'munda view.Tinthu kulowa mu trachea bwino anaziwona mu umunthu nsembe nyama. Kufufuza kwambiri pa trachea pambuyo pobereka MP, aphungu ena adapezeka pafupi ndi carina, zomwe zimasonyeza kuti mphamvu ya maginito inali yosakwanira kusonkhanitsa ndi kusunga aphungu onse, popeza anaperekedwa kudera la mphamvu ya maginito pa nthawi ya madzi.
Zithunzi zochokera ku (a) zisanachitike ndi (b) pambuyo popereka MP5 mu trachea ya khoswe wogwiriridwa posachedwapa wokhala ndi maginito omwe ali pamwamba pa malo ojambulidwawo.
Kumasulira maginito pamodzi trachea mu vivo kunachititsa MP unyolo kusintha ngodya mkati airway pamwamba m'njira yofanana ndi kuoneka mu capillaries (onani Chithunzi 8 ndi Supplementary Video S7: MP5). Komanso anapeza kuti tinthu chingwe chikuwoneka kusintha kuya kwa pamwamba madzi wosanjikiza wosanjikiza pamene maginito anasuntha longitudinally pamodzi trachea, ndi kumawonjezera pamene maginito anasuntha mwachindunji pamwamba ndi tinthu chingwe ndi zimayenda ndi malo ofukula (onani Supplementary Video S7). : MP5 pa 0:09, pansi kumanja).Mchitidwe wa kusuntha wa khalidwe unasintha pamene maginito anamasuliridwa pamwamba pa trachea mozungulira (ndiko kuti, kumanzere kapena kumanja kwa nyamayo kusiyana ndi kutalika kwa trachea) 0:08).Izi zikugwirizana ndi khalidwe la MP lomwe tidawona pansi pa mphamvu ya maginito yogwiritsidwa ntchito mu capillary yagalasi.
Zitsanzo za zithunzi zosonyeza MP5 mu trachea ya khoswe wogonetsa.(a) Maginito amagwiritsidwa ntchito kupeza zithunzi pamwamba ndi kumanzere kwa trachea, kenako (b) maginito atasunthidwa kumanja.Bokosi lofiira lili ndi kukulitsa kosiyana. Zithunzizi zikuchokera mu kanema wowonetsedwa mu Supplementary Video S7:MP5.
Pamene mizati iwiriyo inakonzedwa kumtunda wa kumpoto ndi kum'mwera pamwamba ndi pansi pa trachea (ie kukopa; Mkuyu 3b), zolembera za MP zinawonekera motalika ndipo zinali pamphepete mwa trachea m'malo mwa dorsal tracheal pamwamba (onani Supplementary Video S9: MP5) . Kutumiza pamene chipangizo chawiri-maginito chinagwiritsidwa ntchito, chomwe chimachitika pamene chipangizo cha maginito chimodzi chimagwiritsidwa ntchito.Kenako pamene maginito amodzi adakonzedwa kuti athamangitse mizati yotembenuzidwa (mkuyu. 3c), chiwerengero cha tinthu tating'ono tating'ono tating'ono tating'ono tating'onoting'ono sichinawonekere kuwonjezeka pambuyo pobereka. limodzi maginito kufanana ndi airway koma kudutsa airway pa 90 madigiri kuti mizere munda anawoloka tracheal khoma orthogonally (mkuyu. 3d), ndi orientation cholinga kudziwa ngati tinthu aggregation pa khoma mbali zikhoza kuwonedwa . kasinthidwe kazowongolera (Chithunzi 3a) adasankhidwa kuti azichita maphunziro onyamula ma jini a vivo.
Nyama itayesedwa mobwerezabwereza atapha mitsempha yamphamvu, kusakhala ndi minofu yosokoneza bongo kuti ithe. ". Komabe, sitingathe kuwona bwino kukhalapo ndi kuyenda kwa tinthu ta MP6.
LV-LacZ titer inali 1.8 × 108 TU / ml, ndipo pambuyo pa 1: 1 kusakaniza ndi CombiMag MP (MP6), nyama zinalandira 50 μl tracheal mlingo wa 9 × 107 TU / ml LV galimoto (ie 4.5 × 106 TU / rat). ) .Mu maphunzirowa, m'malo momasulira maginito panthawi ya ntchito, tinakonza maginito pamalo amodzi kuti tidziwe ngati kusintha kwa LV (a) kungawongoleredwe poyerekeza ndi kutumiza kwa vector popanda mphamvu ya maginito, ndipo (b) akhoza kuyang'ana kwambiri Maselo a Airway amasinthidwa kupita kumadera a maginito chandamale chapamwamba.
Kukhalapo kwa maginito ndi kugwiritsa ntchito CombiMag pamodzi ndi ma vectors a LV sikunawonekere kukhala ndi zotsatirapo zoipa pa thanzi la nyama, monga momwe tinachitira ndondomeko yathu yovomerezeka ya LV vector. kuchuluka kwa buluu LacZ kudetsa analipo mu gulu ulamuliro (mkuyu. 9b) .Quantification wa normalized X-Gal madera othimbirira anasonyeza kuti kasamalidwe LV-MP pamaso pa maginito kutulutsa pafupifupi 6-khola kusintha (mkuyu. 9c).
Chitsanzo zithunzi zophatikizika zosonyeza transduction tracheal ndi LV-MP (a) pamaso pa maginito ndi (b) pakalibe maginito.(c) Pachiwerengero kusintha kwambiri normalized LacZ transduction dera mkati trachea pamene ntchito maginito (*p = 0.029, t-mayeso, n = 3 pa gulu, zikutanthauza ± SEM).
Magawo osalowerera ndale ofiira ofiira (chitsanzo chowonetsedwa mu Supplementary Fig. 2) adawonetsa ma cell a LacZ omwe ali ndi mawonekedwe ofanana ndi malo omwe adanenedwa kale.
Vuto lalikulu la chithandizo cha jini la airway ndikukhala kolondola kwa tinthu zonyamulira kumadera osangalatsa ndikukwaniritsa magwiridwe antchito apamwamba m'mapapo osuntha pamaso pa kutuluka kwa mpweya komanso kuchotsedwa kwa ntchofu. kupititsa patsogolo transduction ali ndi ubwino poyerekeza ndi njira zina jini yobereka monga electroporation, monga angagwirizanitse kuphweka, mtengo-mwachangu, yobereka m'dera, kuchuluka dzuwa, ndi nthawi yaifupi makulitsidwe, ndipo mwina ang'onoang'ono chonyamulira dose10. mayendedwe amoyo osasunthika.
Mayesero athu a in vitro synchrotron PCXI adawonetsa kuti tinthu tating'onoting'ono tomwe tidayesa, kupatulapo polystyrene MP, tidawoneka muzithunzi zomwe tidagwiritsa ntchito.Pamaso pa maginito, a MP amapanga zingwe zomwe kutalika kwake kumakhudzana ndi mtundu wa tinthu ndi mphamvu ya maginito (ie kuyandikira ndi kuyenda kwa maginito) . kuchititsa kuti maginito ake am'deralo. Magawo osiyanawa amachititsa kuti tinthu tating'ono tating'ono tomwe tiphatikizepo ndikugwirizanitsa, ndi kayendedwe kamagulu ngati chingwe chifukwa cha mphamvu zam'deralo kuchokera ku mphamvu zokopa komanso zonyansa za tinthu tina.
Schematic anasonyeza (a, b) tinthu sitima kwaiye mkati capillaries madzi odzaza ndi (c, d) mpweya wodzazidwa trachea.Dziwani kuti ma capillaries ndi trachea si kukopeka sikelo.Panel (a) lilinso kufotokoza za MP, amene ali Fe3O4 particles anakonza mu zingwe.
Pamene maginito anasuntha pamwamba capillary, ngodya ya tinthu chingwe anafika pachimake yovuta kwa MP3-5 munali Fe3O4, kenako tinthu chingwe sanalinso anakhala mu udindo wapachiyambi, koma anasamukira kumtunda ku malo atsopano.magnet. Tinthu tating'onoting'ono tating'onoting'ono, tokhala ndi zokutira kapena ma charger osiyanasiyana, kapena madzimadzi onyamula katundu adasokoneza kuthekera kwawo kusuntha. Kusiyanitsa kwa tinthu tating'ono ta CombiMag ndikocheperako, kutanthauza kuti madzimadzi ndi tinthu tating'onoting'ono titha kukhala ndi kachulukidwe kofananira kotero kuti sizingasunthike mosavuta. Tinthu tating'onoting'ono tithanso kukakamira ngati maginito akuyenda mwachangu kwambiri, kuwonetsa kuti mphamvu ya maginito imatha kugonja nthawi zonse. N'zosadabwitsa kuti maginito mphamvu ndi mtunda pakati pa maginito ndi chandamale dera Chofunika kwambiri.Kutengera pamodzi, zotsatirazi zikusonyezanso kuti, pamene maginito angathe kugwira MPs ambiri amene amayenda m'dera chandamale, n'zokayikitsa kuti maginito akhoza kudaliridwa kusuntha CombiMag particles pamodzi pamwamba pa trachea.Choncho, ife timaganiza kuti mu vivo static chandamale chandamale maginito LV-MP m'munda wa maginito maphunziro ayenera kukhala ndi mphamvu ya maginito. mtengo.
Pamene particles amaperekedwa mu thupi, n'zovuta kuzindikira mu nkhani ya zovuta kusuntha minofu minofu, koma luso kuzindikira iwo anali kulimbikitsidwa ndi kumasulira maginito horizontally pamwamba pa trachea kuti "wiggle" MP zingwe. Mosiyana ndi maphunziro a in vitro, tinthu tating'onoting'ono sitingakokedwe m'mphepete mwa trachea pomasulira maginito. Izi zimagwirizana ndi momwe ntchofu yomwe imaphimba pamwamba pa trachea imayendetsa tinthu tating'onoting'ono tomwe timapumira, ndikuyiyika mu ntchofu ndipo kenako imatsukidwa ndi mucociliary chilolezo.
Tinkaganiza kuti kugwiritsa ntchito maginito kukopa pamwamba ndi pansi pa trachea (mkuyu 3b) kungapangitse kuti pakhale maginito ofanana kwambiri, osati mphamvu yamagetsi yomwe imakhala yokhazikika kwambiri panthawi imodzi, zomwe zingayambitse kugawidwa kofanana kwa particles. zambiri tinthu mafunsidwe mu fano area.Awa anapeza awiri zikusonyeza kuti wapawiri-maginito khwekhwe si kwambiri kusintha m'dera ulamuliro wa MP chandamale, ndi kuti chifukwa amphamvu maginito mphamvu n'zovuta sintha, kupanga njira imeneyi zochepa practical.Similarly, orienting maginito pamwamba ndi kudzera trachea (mkuyu. 3d) Komanso sanali kuonjezera chiwerengero cha particles njira zina kusungidwa chifukwa m'dera lachifaniziro sangasungidwe bwino. Chifukwa chake, kasinthidwe ka maginito a digirii 30 (Chithunzi 3a) imatengedwa kuti ndiyo njira yosavuta komanso yothandiza kwambiri yoyesera mu vivo.
Kafukufuku wa LV-MP adawonetsa kuti ma vector a LV ataphatikizidwa ndi CombiMag ndikuperekedwa pambuyo pa kusokonezeka kwakuthupi pamaso pa maginito, milingo ya transduction idawonjezeka kwambiri mu trachea poyerekeza ndi zowongolera. zingapangitse kuti zikhale zogwira mtima kwambiri pamene zimachepetsa ma titers operekedwa, kupititsa patsogolo, kutupa ndi chitetezo cha mthupi, komanso ndalama zonyamula ma jini.Chofunika kwambiri, malinga ndi wopanga, CombiMag ingagwiritsidwe ntchito limodzi ndi njira zina zotumizira ma gene, kuphatikizapo ma vectors ena (monga AAV) ndi nucleic acids.