Enkosi ngokundwendwela i-Nature.com.Inguqulelo yesikhangeli oyisebenzisayo inenkxaso enyiniweyo yeCSS.Ngowona mava angcono, sicebisa ukuba usebenzise isikhangeli esihlaziyiweyo (okanye uvale iModi yokuThelela kwi-Internet Explorer).Okwangoku, ukuqinisekisa inkxaso eqhubekayo, siya kunika isayithi ngaphandle kwezitayela kunye neJavaScript.
I-Gene vectors yonyango lwe-pulmonary cystic fibrosis kufuneka ijoliswe kwi-airways conductive, ekubeni ukuhanjiswa kwe-peripheral lung akunasiphumo sonyango.Ukusebenza kakuhle kwentsholongwane yentsholongwane kuhambelana ngokuthe ngqo nexesha lokuhlala lomthwali.Nangona kunjalo, ukuhanjiswa kwamanzi afana nabathwali be-gene ngokwemvelo basasazeka kwi-alveoli ngexesha lokuphefumla, kunye neengqungquthela zonyango zaso naziphi na iimilo zisuswa ngokukhawuleza ngokuthuthwa kwe-mucociliary.Ukwandisa ixesha lokuhlala labathwali bemfuza kwindlela yokuphefumula kubalulekile kodwa kunzima ukufezekisa.Iinqununu ze-Carrier-conjugated magnetic ezinokuthi zibhekiswe kumphezulu wendlela yokuphefumula zinokuphucula ukujoliswa kwengingqi.Ngenxa yeengxaki ze-invivo imaging, ukuziphatha kwaloo masuntswana amancinane kazibuthe kumphezulu wendlela yomoya phambi kwemagnethi esetyenzisiweyo ayiqondwa kakuhle.Injongo yolu phononongo yayikukusebenzisa i-synchrotron imaging ukubona kwi-vivo intshukumo yothotho lwamasuntswana amagnetic kwitrachea yeempuku ezine-anesthetized ukuze kufundwe amandla kunye neepatheni zokuziphatha kwamasuntswana angatshatanga kunye neenqwaba kwi-vivo.Siye saphinda savavanya ukuba ngaba ukuhanjiswa kwamasuntswana emagnethi kwi-lentiviral magnetic in the presence of the magnetic field kuyakonyusa ukusebenza kakuhle kwetransduction kwi-rat trachea.I-synchrotron imaging X-ray ibonisa ukuziphatha kwamasuntswana omagnetic kwindawo emileyo kunye neshukumayo yemagnethi kwi-vitro nakwi-vivo.Amacandelo akakwazi ukutsalwa ngokulula kumhlaba womoya ophilayo usebenzisa iimagnethi, kodwa ngexesha lothutho, iidiphozithi zigxininiswe kwindawo yokujonga, apho amandla kazibuthe anamandla.Impumelelo yoguqulo nayo yandiswe ngokuphindwe kathandathu xa amasuntswana emagnethi yelentiviral ahanjiswa kubukho bemagnethi.Zithathiwe kunye, ezi ziphumo zibonisa ukuba amasuntswana kazibuthe welentiviral kunye nemagnethi inokuba ziindlela ezixabisekileyo zokuphucula ukujoliswa kwe-gene vector kunye namanqanaba okudluliselwa kwi-conductive airways kwi-vivo.
I-Cystic fibrosis (CF) ibangelwa kukwahluka kwemfuza enye ebizwa ngokuba yi-CF transmembrane conductance regulator (CFTR).Iprotheni ye-CFTR yi-ion channel ekhoyo kwiiseli ezininzi ze-epithelial kuwo wonke umzimba, kubandakanywa neendlela zomoya, indawo enkulu kwi-pathogenesis ye-cystic fibrosis.Iziphene kwi-CFTR zikhokelela kuthutho lwamanzi olungaqhelekanga, ukuphelelwa ngamanzi emzimbeni kumphezulu wendlela yomoya, kunye nokuncipha kobunzulu bendlela yomoya yolwelo (ASL).Kwakhona kuphazamisa amandla othutho lwe-mucociliary (MCT) inkqubo yokucima i-airways ye-particle inhaled kunye ne-pathogens.Injongo yethu kukuphuhlisa i-lentiviral (LV) yonyango yofuzo ukuhambisa ikopi echanekileyo ye-CFTR gene kunye nokuphucula i-ASL, i-MCT, kunye nempilo yemiphunga, kunye nokuqhubeka nokuphuhlisa ubuchwepheshe obutsha obunokulinganisa ezi parameters kwi-vivo1.
I-LV vectors ngomnye wabaviwa abakhokelayo kunyango lwe-cystic fibrosis gene gene therapy, ikakhulu ngenxa yokuba banokudibanisa ngokusisigxina ijini yonyango kwiiseli ezisisiseko zomoya (iiseli ze-stem zomoya).Oku kubalulekile kuba banokubuyisela i-hydration eqhelekileyo kunye nokukhutshwa kwe-mucus ngokwahlula kwiiseli ezisebenzayo zomoya ezilungelelanisiweyo zomoya ezinxulumene ne-cystic fibrosis, okukhokelela kwiinzuzo zobomi bonke.Iivektha ze-LV kufuneka zijoliswe kwi-conductive airways, njengoko kulapho ubandakanyo lwemiphunga kwi-CF luqala khona.Ukuhanjiswa kwe-vector nzulu kwimiphunga kunokubangela ukuguqulwa kwe-alveolar, kodwa oku akunasiphumo sonyango kwi-cystic fibrosis.Nangona kunjalo, i-fluids efana ne-gene carriers ifudukela kwi-alveoli xa ifakwe emva kokuzalwa komntwana3,4 kunye neengqungquthela zonyango zikhutshwa ngokukhawuleza kwi-cavity yomlomo nge-MCTs.Ukusebenza kwe-LV transduction kuhambelana ngokuthe ngqo nobude bexesha i-vector ihlala isondele kwiiseli ezijoliswe kuyo ukuvumela ukunyuswa kwamaselula - "ixesha lokuhlala" i-5 efinyelelwe ngokulula yi-airflow eqhelekileyo yengingqi kunye nokulungelelaniswa kwe-mucus kunye neengqungquthela ze-MCT.Kwi-cystic fibrosis, ukukwazi ukwandisa ixesha lokuhlala kwe-LV kwiindlela zomoya kubalulekile ukufezekisa amanqanaba aphezulu okutshintshwa kule ndawo, kodwa ngoku kube ngumngeni.
Ukoyisa lo mqobo, sicebisa ukuba amasuntswana ombane e-LV (MPs) anokunceda ngeendlela ezimbini ezincedisanayo.Okokuqala, banokukhokelwa yimagnethi kumphezulu womoya wokuphucula ukujolisa kunye nokunceda amasuntswana othwala imfuza abe kwindawo elungileyo yomoya;kunye ne-ASL) zifudukela kumaleko weseli 6. AmaLungu ePalamente asetyenziswa ngokubanzi njengezithuthi ekujoliswe kuzo zokuhanjiswa kweziyobisi xa zibophelela kwizilwa-buhlungu, amachiza echemotherapy, okanye ezinye iimolekyuli ezincinci ezincamathela kwiinwebu zeseli okanye zibophelele kwi-cell cell receptors zazo kwaye ziqokelelane kwiindawo ezinethumba. ubukho bombane omileyo.Amasimi Magnetic unyango umhlaza 7. Ezinye iindlela "hyperthermic" zijolise ekubulaleni iiseli ithumba ngokufudumeza MPs xa evezwe oscillating amasimi magnetic.Umgaqo wokudluliselwa kwamagnetic, apho umbane wemagnethi usetyenziswa njengearhente yodluliselo ukomeleza ukudluliselwa kweDNA kwiiseli, iqhele ukusetyenziswa kwi-vitro kusetyenziswa uluhlu lwentsholongwane yentsholongwane engeyiyo intsholongwane kunye nentsholongwane yentsholongwane kwimigca yeseli ekunzima ukuyidlulisela. ..Ukusebenza kakuhle kwe-LV magnetotransfection kunye nokuhanjiswa kwe-LV MP kwi-vitro kumgca weseli ye-bronchial epithelium phambi kommandla wemagnethi omileyo kwaye yasekwa, inyusa ukusebenza kakuhle kogqithiso ngamaxesha angama-186 xa kuthelekiswa neVector yeLV kuphela.I-LV MT iye yasetyenziswa kwimodeli ye-in vitro ye-cystic fibrosis, apho ukudluliselwa kwemagneti kwandise ukudluliselwa kwe-LV kwiinkcubeko zojongano lolwelo lomoya ngomlinganiselo wama-20 xa kukho i-cystic fibrosis sputum10.Nangona kunjalo, kwi-vivo organ magnetotransfection ifumene ingqwalasela encinci kwaye ihlolwe kuphela kwizifundo zezilwanyana ezimbalwa11,12,13,14,15, ngakumbi kwimiphunga16,17.Nangona kunjalo, amathuba okudluliselwa kwemagneti kunyango lwemiphunga kwi-cystic fibrosis acacile.Tan et al.(2020) yathi "uphononongo lokuqinisekisa malunga nokuhanjiswa okusebenzayo kwemiphunga ye-nanoparticles yamagnetic iya kuvula indlela yezicwangciso ze-CFTR ze-inhalation zexesha elizayo zokuphucula iziphumo zeklinikhi kwizigulane ezine-cystic fibrosis"6.
Ukuziphatha kwamasuntswana amagnetic amancinci kumphezulu wendlela yokuphefumula phambi kombane osetyenzisiweyo kunzima ukukubona kunye nokufunda, kwaye ngenxa yoko aqondwa kakuhle.Kwezinye izifundo, siye saqulunqa indlela ye-Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) ye-imaging engahlaseli kunye nobungakanani bemizuzu kwi-vivo utshintsho kwi-ASL18 ubunzulu kunye nokuziphatha kwe-MCT19, i-20 ukulinganisa ngokuthe ngqo i-gas channel surface hydration. kwaye isetyenziswe njengesalathisi sokuqala sonyango olusebenzayo.Ukongeza, indlela yethu yokufaka amanqaku e-MCT isebenzisa i-10-35 µm i-diameter particles eyenziwe nge-alumina okanye i-high refractive index glass njengeempawu ze-MCT ezibonakalayo nge-PB-PCXI21.Zombini iindlela zifanelekile ekufanekiseni uluhlu lweentlobo zamasuntswana, kubandakanywa nama-MP.
Ngenxa yesisombululo esiphezulu sesithuba kunye nesokwexeshana, i-PB-PCXI-based ASL kunye ne-MCT assays zifaneleke kakuhle ukufunda amandla kunye neepateni zokuziphatha zamasuntswana angatshatanga kunye nobuninzi be-vivo ukusinceda siqonde kwaye sisebenzise iindlela zokuhanjiswa kofuzo lwe-MP.Indlela esiyisebenzisayo apha isekwe kwizifundo zethu sisebenzisa intsimbi ye-SPring-8 BL20B2, apho siye sabona intshukumo yolwelo emva kokunikezelwa kwedosi ye-dummy vector kwindlela yomoya yeempuku zempumlo kunye nemiphunga ukunceda ukucacisa iipateni zethu zokuchazwa kofuzo eziboniweyo. kwimfuza yethu.izifundo zezilwanyana nge-carrier dose ye-3.4.
Injongo yolu phononongo yayikukusebenzisa i-synchrotron ye-PB-PCXI ukubona ngeso lengqondo iintshukumo ze-vivo zothotho lwama-MP kuqhoqhoqho lweempuku eziphilayo.Ezi zifundo zokucinga ze-PB-PCXI zenzelwe ukuvavanya uchungechunge lwe-MP, amandla ombane wemagnethi, kunye nendawo yokumisela umphumo wabo kwintshukumo yeMP.Sasicinga ukuba indawo yangaphandle yamagnetic iya kunceda i-MF enikezelweyo ihlale okanye ihambe kwindawo ekujoliswe kuyo.Ezi zifundo zikwasivumela ukuba sigqibe ulungelelwaniso lwemagnethi olwandisa inani lamasuntswana ashiywe kuqhoqhoqho emva kokubekwa.Kuluhlu lwesibini lwezifundo, sijonge ukusebenzisa olu lungelelwaniso lulolona lufanelekileyo ukubonisa ipateni yoguqulelo oluvela kunikezelo lwe-vivo lwe-LV-MPs kwiindlela zomoya zempuku, ngentelekelelo yokuba ukuhanjiswa kwe-LV-MPs kumxholo wokujolisa kwindlela yomoya kuya kuba nesiphumo. ekwandiseni impumelelo yokuhanjiswa kwe-LV..
Zonke izifundo zezilwanyana zenziwa ngokuhambelana nemigaqo evunyiweyo yiYunivesithi yaseAdelaide (M-2019-060 kunye ne-M-2020-022) kunye neKomiti ye-SPring-8 Synchrotron Animal Ethics Committee.Imifuniselo yenziwe ngokuhambelana neziphakamiso ze-ARIVE.
Yonke imifanekiso ye-x-ray ithathwe kwi-BL20XU beamline kwi-SPring-8 synchrotron eJapan isebenzisa ukuseta okufana noko kuchazwe ngaphambili21,22.Ngokufutshane, ibhokisi yovavanyo ibekwe kwi-245 m ukusuka kwindandatho yokugcina ye-synchrotron.Isampulu ukuya kumgama we-0.6 m isetyenziselwa izifundo ze-particle imaging kunye ne-0.3 m kwi-vivo imaging studies ukudala iziphumo zesigaba sokuchasana.Kusetyenziswe umqadi we-monochromatic onamandla we-25 keV.Imifanekiso ifunyenwe kusetyenziswa i-X-ray transducer enesisombululo esiphezulu (SPring-8 BM3) idityaniswe ne-sCMOS detector.I-transducer iguqula i-X-reyi ekukhanyeni okubonakalayo isebenzisa i-10 µm thick scintillator (Gd3Al2Ga3O12), ethi ke iqondiswe kwi-sCMOS sensor isebenzisa i-× 10 (NA 0.3) injongo ye-microscope.Umtshina we-sCMOS wawuyi-Orca-Flash4.0 (iHamamatsu Photonics, eJapan) enobungakanani be-array ye-2048 × 2048 pixels kunye nobukhulu bepixel ekrwada ye-6.5 × 6.5 µm.Olu seto lunika ubungakanani bepikseli ye-isotropic esebenzayo ye-0.51 µm kunye nomhlaba wokujonga malunga ne-1.1 mm × 1.1 mm.Ixesha lokuvezwa kwe-100 ms likhethwe ukwandisa umlinganiselo wesignali ukuya kwingxolo yamasuntswana kazibuthe ngaphakathi nangaphandle kweendlela zomoya ngelixa kuncitshiswa izinto ezishukumayo ezibangelwa kukuphefumla.Kwizifundo ze-vivo, isivalo se-X-reyi esikhawulezayo safakwa kwindlela ye-X-reyi ukunciphisa idosi yemitha ngokuthintela umqadi we-X-reyi phakathi kokutyhileka.
Imithombo yeendaba ye-LV ayizange isetyenziswe kuyo nayiphi na i-SPring-8 PB-PCXI ye-imaging studies kuba i-BL20XU ye-imaging room ayikho i-Biosafety Level 2 eqinisekisiweyo.Endaweni yoko, sikhethe uluhlu lwamaLungu ePalamente abonakaliswe kakuhle kubathengisi ababini bezorhwebo abagubungela uluhlu lobungakanani, izixhobo, ukugxilwa kwentsimbi, kunye nokusetyenziswa, - okokuqala ukuze siqonde ukuba amandla kazibuthe akuchaphazela njani ukuhamba kwama-MP kwi-glass capillaries, kwaye emva koko iindlela zokuhlala zomoya.umphezulu.Ubungakanani beMP buyahluka ukusuka ku-0.25 ukuya kwi-18 µm kwaye yenziwe ngezinto ezahlukeneyo (jonga kwiTheyibhile 1), kodwa ukwakheka kwesampulu nganye, kubandakanywa nobungakanani bamasuntswana omagnetic kwiMP, akwaziwa.Ngokusekwe kuphononongo lwethu olubanzi lwe-MCT 19, 20, 21, 23, 24, silindele ukuba amaLungu ePalamente ukuya kutsho ku-5 µm angabonwa kumphezulu womzila womoya woqhoqhoqho, umzekelo, ngokuthabatha izakhelo ezilandelelanayo ukubona ukubonakala okuphuculweyo kwentshukumo yeMP.I-MP enye ye-0.25 µm incinci kunesisombululo sesixhobo somfanekiso, kodwa i-PB-PCXI ilindeleke ukuba ibone umahluko wevolumu kunye nokuhamba kolwelo olungaphezulu apho zifakwe khona emva kokufakwa.
Iisampulu zeMP nganye kwitheyibhile.I-1 yalungiswa kwi-20 μl yeglasi ye-capillaries (i-Drummond Microcaps, i-PA, i-USA) kunye ne-diameter yangaphakathi ye-0.63 mm.Amasuntswana e-Corpuscular afumaneka emanzini, ngelixa ii-CombiMag particles zifumaneka kulwelo lobunikazi bomenzi.Ityhubhu nganye izaliswe lulwelo (malunga ne-11 µl) kwaye ibekwe kwisibambi sesampulu (jonga uMfanekiso 1).I-glass capillaries ibekwe ngokuthe tye kwinqanaba kwigumbi lokucinga, ngokulandelanayo, kwaye ibekwe emaphethelweni olwelo.I-19 mm ububanzi (28 mm ubude) umazibuthe we-nickel-shell eyenziwe ngomhlaba onqabileyo, i-neodymium, intsimbi kunye ne-boron (NdFeB) (N35, ikati. No. LM1652, Jaycar Electronics, e-Australia) ene-remanence ye-1.17 T yancanyathiselwa kwi-a Itheyibhile yotshintshiselwano eyahlukileyo ukuphumeza ukude ukutshintsha indawo yakho ngexesha lonikezelo.I-X-reyi imaging iqala xa umazibuthe ebekwe malunga ne-30 mm ngaphezu kwesampulu kwaye imifanekiso ifunyenwe kwiifreyimu ezi-4 ngesekhondi.Ngexesha lokucinga, i-magnet yasondezwa kufuphi neglasi ye-capillary tube (kumgama omalunga ne-1 mm) kwaye emva koko yahamba kunye nombhobho ukuvavanya umphumo wamandla entsimi kunye nesikhundla.
I-in vitro imaging setup equlethe iisampulu zeMP kwiicapillaries zeglasi kwinqanaba lokuguqulelwa kwesampulu ye-xy.Indlela ye-X-ray iphawulwe ngomgca obomvu onamachaphaza.
Nje ukuba ukubonakala kwe-in vitro kwamaLungu ePalamente kusekiwe, isetyana lawo lavavanywa kwi-vivo kwiimpuku zasendle ezizii-albino zasendle (~iiveki ezili-12 ubudala, ~200 g).I-Medetomidine 0.24 mg / kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg / kg (Dormicum®, Astellas Pharma, Japan) kunye ne-butorphanol 4 mg / kg (Vetorphale®, Meiji Seika).Iimpuku zaye zafakwa i-anesthetized nge-Pharma (eJapan) umxube ngenaliti ye-intraperitoneal.Emva kwe-anesthesia, balungiselelwa ukucinga ngokususa uboya obujikelezayo kwi-trachea, ukufaka ityhubhu ye-endotracheal (ET; 16 Ga intravenous cannula, Terumo BCT), kwaye bangazinyanzeli kwindawo ye-supine kwi-plate yokucinga eyenziwe ngokwezifiso equkethe isikhwama se-thermal. ukugcina ubushushu bomzimba.22. I-imaging plate yabe ifakwe kwi-sample stage kwi-imaging box kwi-angle encinci ukulungelelanisa i-trachea ngokuthe tye kumfanekiso we-x-ray njengoko kuboniswe kuMzobo 2a.
(a) Ukuseta umfanekiso we-vivo kwiyunithi ye-imaging ye-SPring-8, indlela ye-X-reyi yomqadi ephawulwe ngomgca wamachaphaza obomvu.(b,c) Ukwenziwa kwemagnethi yoqhoqhoqho kwenziwa ukude kusetyenziswa iikhamera zeIP ezimbini ezixhonywe ngokwe-orthogonally.Kwicala lasekhohlo lomfanekiso kwisikrini, unokubona i-loop yocingo ebambe intloko kunye ne-cannula yokuhambisa efakwe ngaphakathi kwi-ET tube.
Inkqubo yepompo yesirinji elawulwa kude (UMP2, World Precision Instruments, Sarasota, FL) esebenzisa i-100 µl yeglasi yesirinji yayiqhagamshelwe kwi-PE10 tubing (0.61 mm OD, 0.28 mm ID) kusetyenziswa inaliti engama-30 Ga.Phawula ityhubhu ukuqinisekisa ukuba incam ikwindawo echanekileyo kuqhoqhoqho xa ufaka ityhubhu endotracheal.Ukusebenzisa i-micropump, i-syringe plunger yasuswa kwaye incam yombhobho yacwiliswa kwisampuli ye-MP ukuba ihanjiswe.I-tube yokuhambisa elayishiweyo emva koko ifakwe kwi-tube endotracheal, ibeka i-tip kwindawo eyomeleleyo ye-magnetic field ekulindeleke ukuba isetyenziswe.Ukufunyanwa komfanekiso kwakulawulwa kusetyenziswa umtshini wokuphefumula oqhagamshelwe kwibhokisi yethu yexesha esekwe kwi-Arduino, kwaye zonke izibonakaliso (umzekelo, ubushushu, ukuphefumla, ukuvuleka kwe-shutter / ukuvala, kunye nokufumana umfanekiso) zarekhodwa kusetyenziswa i-Powerlab kunye neLabChart (i-AD Instruments, eSydney, e-Australia) 22 Xa uFundo Xa indlu yayingafumaneki, iikhamera ezimbini ze-IP (Panasonic BB-SC382) zibekwe malunga ne-90 ° enye kwenye kwaye zisetyenziselwa ukulawula indawo yemagnethi enxulumene noqhoqho ngexesha lokucinga (Umfanekiso 2b, c).Ukunciphisa ubugcisa obushukumayo, umfanekiso omnye ngomphefumlo ufunyenwe ngexesha lendawo yokuphefumla yokuphefumla.
Umazibuthe uncanyathiselwe kwinqanaba lesibini, elinokuthi libekwe ukude ngaphandle komzimba womfanekiso.Izikhundla ezahlukeneyo kunye nokulungelelaniswa kwemagneti zavavanywa, kubandakanywa: ibekwe kwi-angle emalunga ne-30 ° ngaphezu kwe-trachea (uqwalaselo luboniswa kwiMifanekiso 2a kunye ne-3a);omnye umazibuthe ngaphezu kwesilwanyana kunye nomnye ongezantsi, kunye neepali ezimiselwe ukutsala (Figure 3b)., enye imagnethi ngaphezu kwesilwanyana kunye enye ingezantsi, kunye nezibonda ezimiselwe ukuchithwa (Umfanekiso 3c), kunye nemagnethi enye ngaphezulu kunye ne-perpendicular to trachea (Umfanekiso 3d).Emva kokuseta isilwanyana kunye nemagnethi kunye nokulayisha iMP phantsi kovavanyo kwimpompo yesirinji, hambisa idosi ye-50 µl ngesantya se-4 µl/sec ekufumaneni imifanekiso.Umazibuthe uhanjiswa emva naphambili ecaleni okanye ngapha kwetrachea ngelixa uqhubeka nokufumana imifanekiso.
Ulungelelwaniso lwemagnethi kumfanekiso we-vivo (a) umazibuthe omnye ngentla koqhoqhoqho kwi-engile emalunga nama-30°, (b) iimagnethi ezimbini ezimiselwe ukutsala umtsalane, (c) iimagnethi ezimbini ezimiselwe ukurhoxa, (d) umazibuthe omnye ngasentla kunye nependicular uqhoqhoqho.Umbukeli wajonga phantsi ukusuka emlonyeni ukuya emiphungeni ngokusebenzisa uqhoqhoqho kwaye umqadi we-X-ray wadlula kwicala lasekhohlo lempuku kwaye waphuma kwicala lasekunene.Umazibuthe ushukunyiswa kubude bendlela yomoya okanye ekhohlo kwaye ekunene phezu koqhoqhoqho kwicala le-X-reyi.
Siphinde safuna ukufumanisa ukubonakala kunye nokuziphatha kweengqungquthela kwiindlela zomoya ngokungabikho kokuxuba ukuphefumula kunye nentliziyo.Ngoko ke, ekupheleni kwexesha lokucinga, izilwanyana zaye zabulawa ngenxa ye-pentobarbital overdose (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg ip).Ezinye izilwanyana zashiywa kwiqonga lokucinga, kwaye emva kokuyeka ukuphefumla kunye nentliziyo, inkqubo yokucinga iphindwe, yongeza i-dose eyongezelelweyo ye-MP ukuba akukho MP ibonakala kwi-airway surface.
Imifanekiso efunyenweyo yalungiswa kwindawo ethe tyaba nemnyama emva koko yadityaniswa kwimuvi (iifreyimu ezingama-20 ngesekhondi; 15–25 × isantya esiqhelekileyo kuxhomekeke kwisantya sokuphefumla) kusetyenziswa iskripthi esibhalwe kwiMATLAB (R2020a, iMathworks).
Lonke uphononongo kunikezelo lweVector ye-LV lwenziwe kwiYunivesithi yaseAdelaide Laboratory yeZiko loPhando lweZilwanyana kwaye lujolise ekusebenziseni iziphumo zovavanyo lwe-SPring-8 ukuvavanya ukuba unikezelo lwe-LV-MP kubukho bomhlaba wemagnethi lunokuphucula ukudluliselwa kwemfuza kwi-vivo. .Ukuvavanya imiphumo ye-MF kunye nentsimi yamagnetic, amaqela amabini ezilwanyana aphathwa: elinye iqela lifakwe i-LV MF ngokubeka imagnethi, kwaye elinye iqela lajongwa kunye neqela lokulawula kunye ne-LV MF ngaphandle kwemagnethi.
I-LV gene vectors yenziwe ngokusebenzisa iindlela ezichazwe ngaphambili ze-25, i-26.IVector ye-LacZ ivakalisa ujini we-beta-galactosidase wenyukliya wasekhaya oqhutywa yiMPSV constitutive promoter (LV-LacZ), evelisa imveliso yeblue reaction kwiiseli eziguqulweyo, ezibonakala kumphambili kunye nakumacandelo ezithishu zemiphunga.I-Titration yenziwa kwiinkcubeko zeeseli ngokubala ngesandla inani le-LacZ-positive cells usebenzisa i-hemocytometer ukubala i-titer kwi-TU / ml.Abathwali be-cryopreserved kwi--80 ° C, bancibilika ngaphambi kokusetyenziswa, kwaye baboshwe kwi-CombiMag ngokuxuba i-1: i-1 kunye ne-incubating kwi-ice ubuncinane imizuzu engama-30 ngaphambi kokunikezelwa.
Iigundane eziqhelekileyo ze-Sprague Dawley (n = 3 / iqela, ~ 2-3 i-anesthetized ip kunye nomxube we-0.4mg / kg medetomidine (iDomitor, Ilium, Australia) kunye ne-60mg / kg ketamine (Ilium, e-Australia) kwi-1 inyanga yeminyaka) ip ) isitofu kunye ne-cannula yomlomo engeyiyo yotyando kunye ne-16 Ga ye-cannula ye-intravenous.Ukuqinisekisa ukuba izicubu ze-tracheal airway zifumana ukuguqulwa kwe-LV, i-conditioned usebenzisa i-protocol yethu ye-mechanical perturbation echazwe ngaphambili apho i-tracheal airway surface yaxutywa nge-axially ngebhasikithi yocingo (i-N-Circle, i-nitinol i-extractor yamatye ngaphandle kwe-tip NTSE-022115 ) -UDH , Cook Medical, USA) 30 p28.Emva koko, malunga nemizuzu eyi-10 emva kokuphazamiseka kwikhabhinethi ye-biosafety, ukulawulwa kwe-tracheal ye-LV-MP kwenziwa.
Indawo kazibuthe esetyenzisiweyo kolu lingelo yacwangciswa ngokufanayo kuphononongo lwe-x-reyi ye-vivo, kunye nemagnethi efanayo ebanjwe phezu koqhoqhoqho kunye ne-distillation stent clamps (Figure 4).Umthamo we-50 µl (2 x 25 µl aliquots) we-LV-MP uhanjiswe kuqhoqhoqho (n = izilwanyana ezi-3) kusetyenziswa ipipetti ene-gel-tipped njengoko kuchaziwe ngaphambili.Iqela lokulawula (n = izilwanyana ezi-3) lifumene i-LV-MP efanayo ngaphandle kokusetyenziswa kwemagnethi.Emva kokugqitywa kwe-infusion, i-cannula iyasuswa kwi-tube endotracheal kwaye isilwanyana sikhutshwe.Umazibuthe uhlala kwindawo kangangemizuzu eli-10 phambi kokuba ususwe.Iigundane zahlanjululwa ngaphantsi kwe-meloxicam (1 ml / kg) (Ilium, e-Australia) ilandelwa ukuhoxiswa kwe-anesthesia nge-injection ye-intraperitoneal ye-1 mg / kg i-atipamazole hydrochloride (Antisedan, Zoetis, Australia).Iigundane zigcinwe zifudumele kwaye zigcinwe de zibuyiswe ngokupheleleyo kwi-anesthesia.
Isixhobo sokuhanjiswa kwe-LV-MP kwikhabhinethi yokhuseleko lwebhayoloji.Uyabona ukuba ukukhanya okungwevu kwe-Luer-lock sleeve yetyhubhu ye-ET iphuma emlonyeni, kunye nencam ye-gel pipette eboniswe kumzobo ifakwe kwi-ET tube ukuya kubunzulu obufunwayo kwi-trachea.
Kwiveki enye emva kwenkqubo yolawulo lwe-LV-MP, izilwanyana zenziwa idini ngobuntu ngokuphefumlelwa i-100% ye-CO2 kunye nokubonakaliswa kwe-LacZ kwavavanywa kusetyenziswa unyango lwethu oluqhelekileyo lwe-X-gal.Izindandatho ezintathu ze-caudal cartilage zisuswe ukuze kuqinisekiswe ukuba nawuphi na umonakalo womatshini okanye ukugcinwa kwamanzi ngenxa yokubekwa kwe-tube endotracheal akuyi kufakwa kuhlalutyo.Uqhoqhoqho ngalunye lwalusikwe ngobude ukuze kufumaneke iziqingatha ezibini zokuhlalutya kwaye zibekwe kwikomityi enerabha ye-silicone (i-Sylgard, i-Dow Inc) kusetyenziswa inaliti ye-Minutien (iZixhobo zeSayensi eziFine) ukujonga umphezulu wokukhanya.Ukuhanjiswa kunye nomlingiswa weeseli ezitshintshiweyo zaqinisekiswa ngokufota okungaphambili kusetyenziswa i-Nikon microscope (SMZ1500) enekhamera yeDigiLite kunye nesoftware ye-TCapture (Tucsen Photonics, China).Imifanekiso ifunyenwe kwi-20x yokukhulisa (kubandakanya ukusetwa okuphezulu kobubanzi obupheleleyo be-trachea), kunye nobude bonke be-trachea ebonakaliswe ngesinyathelo ngesinyathelo, ukubonelela ngokugqithiswa okwaneleyo phakathi komfanekiso ngamnye ukuvumela imifanekiso ukuba "ithungwe".Imifanekiso esuka kuqhoqhoqho ngamnye yadityaniswa ibe ngumfanekiso omnye odibeneyo usebenzisa i-Composite Image Editor version 2.0.3 (uPhando lweMicrosoft) kusetyenziswa i-algorithm ye-planar motion. Ummandla wenkcazo ye-LacZ ngaphakathi kwemifanekiso edibeneyo ye-tracheal evela kwisilwanyana ngasinye yalinganiswa ngokusebenzisa i-script ye-MATLAB ezenzekelayo (i-R2020a, i-MathWorks) njengoko ichazwe ngaphambili28, isebenzisa izicwangciso ze-0.35 <Hue <0.58, i-Saturation> 0.15, kunye ne-Value <0.7. Ummandla wenkcazo yeLacZ ngaphakathi kwemifanekiso edibeneyo yoqhoqhoqho kwisilwanyana ngasinye yalinganiswa ngokusebenzisa i-script ye-MATLAB ezenzekelayo (R2020a, i-MathWorks) njengoko ichazwe ngaphambili28, isebenzisa izicwangciso ze-0.35 <Hue <0.58, Saturation> 0.15, kunye nexabiso <0.7. Площадь экспрессии LacZ в составных изображениях трахеи от каждого животного была количественно определена с использованием MAZAR20,60 thWorks), как описано ранее28, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0 ,7. Indawo yokubonakaliswa kweLacZ kwimifanekiso edibeneyo yoqhoqhoqho kwisilwanyana ngasinye yalinganiswa kusetyenziswa iskripthi esizihambelayo seMATLAB (R2020a, MathWorks) njengoko kuchaziwe ngaphambili28 kusetyenziswa useto lwe-0.350.15 kunye nexabiso <0 .7.如前所述，使用自动MATLAB 脚本（R2020a，MathWorks) 0.58, i-饱和度> 0.15 kunye ne-< 0.7 的设置。如 前所述 自动 自动 Matlab 脚本 （r2020a ， Mathworks） 來自 每 只 的 气管 合 图像 的 的 只 . . 5 <色调 <0.58,> 0.15 kunye ne- <0.7 的。。。 .................. Inkcazelo ngokuthe gabalala I-LacZ на составных изображениях трахеи каждого количественно определяли с использованием автоматизер, 2, 2014 к описано ранее, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0,7 . Iindawo zokubonakaliswa kweLacZ kwimifanekiso edibeneyo ye-trachea yesilwanyana ngasinye zilinganiswe ngokusebenzisa i-script ye-MATLAB ezenzekelayo (i-R2020a, i-MathWorks) njengoko ichazwe ngaphambili usebenzisa izicwangciso ze-0.35 <hue <0.58, saturation> 0.15 kunye nexabiso <0.7.Ngokulandelela i-tissue contours kwi-GIMP v2.10.24, imaski yenziwa ngesandla kumfanekiso ngamnye odibeneyo ukuchonga indawo yethishu kunye nokuthintela nakuphi na ukufunyanwa kobuxoki ngaphandle kwethishu yoqhoqhoqho.Imimandla enamabala avela kuyo yonke imifanekiso eyintlanganisela evela kwisilwanyana ngasinye yaqikelelwa ukunika indawo enebala elipheleleyo yeso silwanyana.Indawo epeyintiweyo ke yahlulwa yindawo yonke yemaski ukufumana indawo eqhelekileyo.
Uqhoqhoqho ngalunye lwaluzinziswe kwiparafini kwaye yahlulwe nge-5 µm ubukhulu.Amacandelo aye athintelwa ngokungathathi hlangothi obomvu ngokukhawuleza kwimizuzu emi-5 kwaye imifanekiso yafunyanwa kusetyenziswa i-microscope ye-Nikon Eclipse E400, ikhamera ye-DS-Fi3 kunye ne-NIS element yokubamba isoftware (uhlobo 5.20.00).
Lonke uhlalutyo lwamanani lwenziwa kwiGraphPad Prism v9 (iGraphPad Software, Inc.).Ukubaluleka kwamanani kubekwe kwi-p ≤ 0.05.Isiqhelo savavanywa kusetyenziswa uvavanyo lwe-Shapiro-Wilk kunye nokwahluka kwi-LacZ staining kwavavanywa kusetyenziswa i-t-test engasebenziyo.
AmaLungu ePalamente amathandathu achazwe kwiThebhile yoku-1 avavanywa yi-PCXI, kwaye ukubonakala kuchazwe kwiThebhile 2. Ii-MP1 ze-polystyrene ezimbini (MP1 kunye ne-MP2; 18 µm kunye ne-0.25 µm, ngokulandelanayo) zazingabonakali yi-PCXI, kodwa iisampulu eziseleyo zazinokuchongwa. (imizekelo iboniswe kuMfanekiso 5).IMP3 kunye neMP4 zibonakala buthathaka (10-15% Fe3O4; 0.25 µm kunye ne-0.9 µm, ngokulandelelanayo).Nangona iMP5 (98% Fe3O4; 0.25 µm) iqulathe amanye amasuntswana amancinane avavanyiweyo, yayiyeyona ivakala kakhulu.Imveliso yeCombiMag MP6 kunzima ukuyohlula.Kuzo zonke iimeko, ukukwazi kwethu ukubona ii-MFs kwaphuculwa kakhulu ngokuhambisa umazibuthe emva naphambili ngokuhambelana ne-capillary.Njengoko imagnethi isuka kude ne-capillary, amasuntswana atsalwa ngaphandle kumatyathanga amade, kodwa njengoko imagnethi isondela kwaye amandla emagnethi anyuka, amakhonkco amasuntswana afinyezwa njengoko amasuntswana afudukela kumphezulu we-capillary (jonga iVidiyo eyoNgezelelweyo S1 : MP4), ukwandisa ukuxinana kwamasuntswana kumphezulu.Ngokwahlukileyo, xa umazibuthe esuswa kwi-capillary, amandla entsimi ayancipha kwaye i-MPs iphinda ilungelelanise amatyathanga amade asuka kumphezulu we-capillary (jonga i-Supplementary Video S2: MP4).Emva kokuba umazibuthe eyeka ukuhamba, amasuntswana aqhubeka ehamba ixesha elithile emva kokufikelela kwindawo yokulinganisa.Njengoko i-MP isiya kwaye isuka kumphezulu we-capillary, amasuntswana omagnetic athanda ukutsala inkunkuma kulwelo.
Ukubonakala kweMP phantsi kwePCXI kuyahluka kakhulu phakathi kweesampuli.(a) MP3, (b) MP4, (c) MP5 kunye (d) MP6.Yonke imifanekiso eboniswe apha ithathwe ngemagnethi ebekwe malunga ne-10 mm ngokuthe ngqo ngaphezulu kwe-capillary.Izangqa ezinkulu ezibonakalayo ziyi-bubbles zomoya ezibanjwe kwii-capillaries, ezibonisa ngokucacileyo iimpawu ezimnyama kunye ezimhlophe zecala lomfanekiso wesigaba sokungafani.Ibhokisi ebomvu ibonisa ulwandiso olwandisa umahluko.Qaphela ukuba iidayamitha zeesekethe zemagnethi kuwo onke amanani azinakulinganiswa kwaye zimalunga namaxesha angama-100 amakhulu kunoko kubonisiwe.
Njengoko umazibuthe uhamba ekhohlo nasekunene ecaleni komphezulu wecapillary, i-engile yomtya weMP iyatshintsha ukuze ihambelane nemagnethi (jonga uMzobo 6), ngaloo ndlela uchaza imigca yemagnethi.Kwi-MP3-5, emva kokuba i-chord ifikelele kwi-angle yombundu, iinqununu zidonsa kwindawo ephezulu ye-capillary.Oku kudla ngokukhokelela ekubeni amalungu ePalamente adibane abe ngamaqela amakhulu kufutshane nalapho imagnethi yomelele khona (jonga iVidiyo eyoNgezelelweyo S3: MP5).Oku kubonakala ngakumbi xa umfanekiso usondele ekupheleni kwe-capillary, obangela ukuba i-MP idibanise kwaye igxininise kwi-interface yomoya we-liquid.Amasuntswana akwiMP6, ebekunzima ukwahlula kunalawo akwiMP3-5, awazange atsale xa umazibuthe ehamba ecaleni kwecapillary, kodwa iintambo zeMP zahlukana, zishiya amasuntswana embonakalo (bona iSupplementary Video S4: MP6).Kwezinye iimeko, xa imagnethi esetyenzisiweyo yancitshiswa ngokuhambisa umazibuthe umgama omde ukusuka kwindawo yokucinga, nawaphi na amaLungu ePalamente aseleyo aye ehla ngokuthe chu ukuya kumphezulu wetyhubhu ngomxhuzulane, ashiyeke kumtya (jonga iVidiyo eyoNgezelelweyo S5: MP3) .
I-engile yomtya weMP iyatshintsha njengoko umazibuthe esiya ngasekunene ngaphezu kwecapillary.(a) MP3, (b) MP4, (c) MP5 kunye (d) MP6.Ibhokisi ebomvu ibonisa ulwandiso olwandisa umahluko.Nceda uqaphele ukuba iividiyo ezongezelelweyo zenzelwe iinjongo zolwazi njengoko zityhila isakhiwo se-particle ebalulekileyo kunye nolwazi oluguquguqukayo olungenakubonwa kule mifanekiso emileyo.
Iimvavanyo zethu zibonise ukuba ukuhambisa umazibuthe ngasemva naphambili kancinci ecaleni kwetrachea kuququzelela ukubonwa kwe-MF kumxholo wentshukumo entsonkothileyo kwi-vivo.Akukho vavanyo lwe-vivo lwenziwa ngenxa yokuba amaso e-polystyrene (MP1 kunye ne-MP2) ayengabonakali kwi-capillary.Nganye kwezine eziseleyo ze-MF zavavanywa kwi-vivo kunye ne-axis ende yemagnethi ebekwe phezu koqhoqhoqho kwi-engile emalunga ne-30 ° ukuya ngokuthe nkqo (jonga iMifanekiso 2b kunye ne-3a), njengoko oku kubangele amatyathanga amade e-MF kwaye yayisebenza ngakumbi. kunemagnethi..uqwalaselo luphelisiwe.I-MP3, iMP4 kunye ne-MP6 ayifumanekanga kwi-trachea yazo naziphi na izilwanyana eziphilayo.Xa kubonwa indlela yokuphefumula yeegundane emva kokubulala izilwanyana ngobuntu, iinqununu zahlala zingabonakali nangona ivolumu eyongezelelweyo yongezwa usebenzisa ipompo yesirinji.I-MP5 yayinomxholo ophezulu we-iron oxide kwaye yayiyeyona nto ibonakalayo kuphela, ngoko yayisetyenziselwa ukuvavanya nokubonakalisa ukuziphatha kwe-MP kwi-vivo.
Ukubekwa kwemagnethi phezu kwetrachea ngexesha lokufakwa kwe-MF kubangele ezininzi, kodwa kungekhona zonke, i-MFs igxininiswe kwintsimi yokujonga.Ukungena kwetracheal of particles kubonwa kakuhle kwizilwanyana ezibulawa ngabantu.Umzobo we-7 kunye neVidiyo eyoNgezelelweyo ye-S6: I-MP5 ibonisa ukubamba ngokukhawuleza kwamagnetic kunye nokulungelelaniswa kwamasuntswana ebusweni be-trachea ye-ventral, ebonisa ukuba ii-MPs zinokujoliswa kwiindawo ezifunwayo ze-trachea.Xa kukhangelwa kude kakhulu kuqhoqhoqho emva kokuhanjiswa kweMF, ezinye iiMFs zafunyanwa kufutshane necarina, nto leyo ebonisa ukungonelanga kwamandla emagnethi okuqokelela nokubamba zonke ii-MFs, ekubeni bezisiwa kummandla wamandla amakhulu emagnethi ngexesha lolawulo lolwelo.inkqubo.Nangona kunjalo, ugxininiso lwe-MP emva kokubeleka lwaluphezulu malunga nommandla wemifanekiso, ebonisa ukuba amaLungu ePalamente amaninzi ahlala kwimimandla yeendlela zomoya apho amandla okusebenza kazibuthe ayephezulu.
Imifanekiso (a) phambi kunye (b) emva kokuziswa kweMP5 kuqhoqhoqho lwempuku esanda kubulawa ngemagnethi ebekwe ngasentla kwendawo yokucinga.Indawo ebonisiweyo iphakathi kwamakhonkco amabini e-cartilaginous.Kukho ulwelo kwimizila yomoya phambi kokuba iMP iziswe.Ibhokisi ebomvu ibonisa ulwandiso olwandisa umahluko.Le mifanekiso ithathwe kwividiyo eboniswe kwi-S6: Ividiyo yeMP5 eyoNgezelelweyo.
Ukuhambisa umazibuthe ecaleni koqhoqhoqho kwi-vivo kubangele utshintsho kwi-engile yekhonkco le-MP kwindawo yomoya, efana naleyo ibonwa kwii-capillaries (jonga i-Figure 8 kunye neVidiyo eyoNgezelelweyo ye-S7: MP5).Nangona kunjalo, kuphononongo lwethu, amaLungu ePalamente awakwazanga ukutsalwa kumphezulu wamaphecana aphilayo wokuphefumla, njengoko ii-capillaries zinokwenza.Kwezinye iimeko, ikhonkco leMP liyakhula njengoko umazibuthe ushukuma ekhohlo nasekunene.Okubangela umdla kukuba, siye safumanisa ukuba ikhonkco lesuntswana litshintsha ubunzulu bomaleko womphezulu wolwelo xa umazibuthe ushukunyiswa ixesha elide ecaleni koqhoqhoqho, kwaye uyanda xa umazibuthe ushukunyiswa phezu kwentloko ngqo kwaye ikhonkco lesuntswana lijikeleziswa kwindawo ethe nkqo (bona. Ividiyo eyongezelelweyo S7).: MP5 ngo-0:09, ezantsi ekunene).Ipatheni yentshukumo yeempawu yatshintsha xa umazibuthe washenxiswa ecaleni kumphezulu woqhoqhoqho (oko kukuthi, ukuya ekhohlo okanye ngasekunene kwesilwanyana, kunokuba kubude boqhoqhoqho).Iingqungquthela zazisabonakala ngokucacileyo ngexesha lokuhamba kwazo, kodwa xa i-magnet isusiwe kwi-trachea, iingcebiso zeentambo zeengqungquthela zabonakala (jonga i-Supplementary Video S8: MP5, eqala ngo-0:08).Oku kuvumelana nokuziphatha okubonwayo kwemagnethi phantsi kwesenzo semagnethi esetyenzisiweyo kwi-capillary yeglasi.
Isampulu yemifanekiso ebonisa iMP5 kuqhoqhoqho lwempuku ephilayo ene-anesthetized.(a) Umazibuthe usetyenziselwa ukufumana imifanekiso ngaphezulu nasekhohlo kuqhoqhoqho, emva koko (b) emva kokususa umazibuthe ekunene.Ibhokisi ebomvu ibonisa ulwandiso olwandisa umahluko.Le mifanekiso ivela kwividiyo eboniswe kwividiyo eyoNgezelelweyo ye-S7: MP5.
Xa iipali ezimbini zilungelelaniswe kwicala elingasentla ukuya kumzantsi ngaphezulu nangaphantsi koqhoqhoqho (oko kukuthi, ukutsala; Umzobo 3b), iichords zeMP zabonakala zinde kwaye zazibekwe eludongeni olusecaleni loqhoqho kunokuba kumphezulu womqolo wetrachea. uqhoqhoqho (jonga kwiSihlomelo).Ividiyo S9:MP5).Nangona kunjalo, ugxininiso oluphezulu lwamasuntswana kwindawo enye (oko kukuthi, umphezulu womqolo woqhoqhoqho) awufunyaniswanga emva kokulawulwa kolwelo kusetyenziswa isixhobo esinemagnethi ezimbini, esikholisa ukwenzeka ngesixhobo esinye semagnethi.Emva koko, xa enye imagnethi ilungiselelwe ukugxotha izibonda ezichaseneyo (Umfanekiso 3c), inani leengqungquthela ezibonakalayo kwintsimi yembono ayizange inyuke emva kokunikezelwa.Ukuseta zombini ulungelelwaniso lwemagnethi lucelomngeni ngenxa yamandla emagnethi aphezulu athi etsala okanye atyhale iimagnethi ngokulandelelanayo.Ukuseta emva koko kwatshintshwa kwimagnethi enye ehambelana neendlela zomoya kodwa idlula kwi-airways kwi-angle ye-90 degree ukuze imigca yamandla iwele udonga lwe-tracheal orthogonally (Figure 3d), uqhelaniso olunenjongo yokugqiba ukuba nokwenzeka kokuhlanganiswa kwamasuntswana udonga olusecaleni.ukuqwalaselwa.Nangona kunjalo, kolu lungelelwaniso, bekungekho ntshukumo ibonakalayo yokuqokelela kweMF okanye intshukumo yemagnethi.Ngokusekelwe kuzo zonke ezi ziphumo, ukucwangciswa kunye nemagnethi enye kunye ne-30-degree orientation yakhethwa kwizifundo ze-vivo ze-gene carriers (Fig. 3a).
Xa isilwanyana sasifanekiselwa izihlandlo ezininzi ngoko nangoko emva kokubingelelwa ngobuntu, ukungabikho kokuphazamiseka kwentshukumo yethishu kwakuthetha ukuba imigca emifutshane, emifutshane yamasuntswana inokuqondwa kwindawo ecacileyo ye-intercartilaginous, 'ukushukuma' ngokuhambelana nentshukumo yokuguqulela umazibuthe.bona ngokucacileyo ubukho kunye nokuhamba kwamasuntswana eMP6.
I-titer ye-LV-LacZ yayiyi-1.8 x 108 IU/mL, kwaye emva kokuxuba i-1:1 ne-CombiMag MP (MP6), izilwanyana zatofwa nge-50 µl yedosi yoqhoqhoqho ye-9 x 107 IU/ml yesithuthi se-LV (okt 4.5) x 106 TU/impuku).)).Kwezi zifundo, endaweni yokuhambisa umazibuthe ngexesha lomsebenzi, simise umazibuthe kwindawo enye ukugqiba ukuba ngaba ukuhanjiswa kwe-LV (a) kungaphuculwa xa kuthelekiswa nokuhanjiswa kwe-vector ngokungabikho kwendawo yamagnetic, kwaye (b) ukuba indlela yomoya inokuthi isebenze. gxila.Iiseli zitshintshelwa kwiindawo ekujoliswe kuzo zimagnetic kwindawo ephezulu yokuphefumla.
Ubukho bemagnethi kunye nokusetyenziswa kwe-CombiMag ngokudityaniswa ne-LV vectors akuzange kubonakale kuchaphazela kakubi impilo yezilwanyana, njengoko senza iprotocol yethu yokuhambisa iVector yeLV.Imifanekiso yangaphambili yommandla we-tracheal ephantsi kokuphazamiseka komatshini (i-Supplementary Fig. 1) ibonise ukuba iqela eliphathwayo le-LV-MP lalinamanqanaba aphezulu kakhulu okutshintshwa phambi kombane (Fig. 9a).Kuphela inani elincinci le-blue LacZ staining lalikhona kwiqela lolawulo (Umfanekiso 9b).Ubungakanani bemimandla eqhelekileyo ene-X-Gal-stained ibonise ukuba ulawulo lwe-LV-MP kubukho bemagnethi ibangele uphuculo oluphindwe ka-6 (Fig. 9c).
Umzekelo wemifanekiso ehlanganisiweyo ebonisa ukuhanjiswa koqhoqhoqho nge-LV-MP (a) kubukho bemagnethi kunye (b) nokungabikho kwemagnethi.(c) Uphuculo olubalulekileyo ngokwezibalo kwindawo eqhelekileyo yeLacZ transduction kwitrachea kusetyenziswa umazibuthe (*p = 0.029, t-test, n = 3 ngeqela ngalinye, ithetha ± impazamo eqhelekileyo yentsingiselo).
Amacandelo abomvu angathathi hlangothi ngokukhawuleza (umzekelo oboniswe kwi-Supplementary Fig. 2) ibonise ukuba iiseli ze-LacZ-stained zikhoyo kwisampuli efanayo nakwindawo efanayo njengoko kuchazwe ngaphambili.
Umceli mngeni ophambili kunyango lwejene yomoya uhlala uyindawo echanekileyo yeengqungquthela zomthwali kwiindawo ezinomdla kunye nokufezekiswa kwezinga eliphezulu lokugqithiswa kakuhle kwimiphunga ehambayo phambi kokuhamba komoya kunye nokukhutshwa kwe-mucus esebenzayo.Kubathwali be-LV ejoliswe kunyango lwezifo zokuphefumla kwi-cystic fibrosis, ukunyusa ixesha lokuhlala kwamasuntswana othwala kwiindlela zomoya eziqhubayo ukuza kuthi ga ngoku kube yinjongo engenakufikelelwa.Njengoko kubonisiwe nguCastellani et al., Ukusetyenziswa kwemimandla yemagnethi ukonyusa ukudluliselwa kunenzuzo ngaphezu kwezinye iindlela zokuhanjiswa kofuzo ezifana ne-electroporation kuba inokudibanisa ukulula, uqoqosho, ukuhanjiswa kwendawo, ukonyuka kobuchule, kunye nexesha elifutshane lokufukamela.kwaye mhlawumbi nedosi esezantsi yesithuthi10.Nangona kunjalo, kwi-vivo ukubekwa kunye nokuziphatha kwamasuntswana kazibuthe kwimizila yomoya phantsi kwempembelelo yamandla kazibuthe angaphandle akuzange kuchazwe, kwaye eneneni ukukwazi kwale ndlela ukunyusa amanqanaba embonakalo yemfuza kwindlela ephilayo yomoya ayikabonakaliswa kwi-vivo.
Uvavanyo lwethu lwe-in vitro kwi-synchrotron ye-PCXI lubonise ukuba zonke iinqununu esizivavanyileyo, ngaphandle kwe-polystyrene ye-MP, zibonakala kwi-imaging setup esiyisebenzisayo.Phambi kommandla wamagnetic, imimandla yamagnetic yenza iintambo, ubude bayo buhambelana nohlobo lwamasuntswana kunye namandla ombane wemagnethi (oko kukuthi, ukusondela kunye nokuhamba kwemagnethi).Njengoko kubonisiwe kuMzobo we-10, imitya esiyijongileyo yenziwa njengoko isuntswana ngalinye liba nguzibuthe kwaye livelise indawo yalo yasekhaya.Ezi nkalo ezihlukeneyo zibangela ukuba ezinye iinqununu ezifanayo ziqokelele kwaye zidibanise kunye neentshukumo zentambo yeqela ngenxa yemikhosi yendawo evela kwimikhosi yendawo yokukhanga kunye nokunyanzeliswa kwamanye amaqhekeza.
Umzobo obonisa (a, b) amatyathanga amasuntswana enza ngaphakathi kwee-capillaries ezizaliswe lulwelo kunye (c, d) ne-trachea ezaliswe ngumoya.Qaphela ukuba ii-capillaries kunye ne-trachea azitsalwanga kwisikali.Iphaneli (a) nayo iqulethe inkcazo ye-MF equkethe amaqhekeza e-Fe3O4 ahlelwe ngamatyathanga.
Xa imagnethi ishukuma phezu kwe-capillary, i-angle ye-particle string yafikelela kwinqanaba elibalulekileyo le-MP3-5 equlethe i-Fe3O4, emva koko umtya we-particle awuzange uhlale kwindawo yawo yangaphambili, kodwa uqhubele phambili kwindawo entsha.umazibuthe.Esi siphumo sinokwenzeka ngenxa yokuba umphezulu we-capillary yeglasi ugudile ngokwaneleyo ukuvumela ukuba le ntshukumo yenzeke.Okuthakazelisayo kukuba, i-MP6 (i-CombiMag) ayizange iziphathe ngale ndlela, mhlawumbi ngenxa yokuba iincinci zazincinci, zine-coating eyahlukileyo okanye intlawulo ephezulu, okanye i-carrier carrier fluid yachaphazela amandla abo okuhamba.Umahluko kumfanekiso wamasuntswana weCombiMag nawo ubuthathaka, ucebisa ukuba ulwelo kunye namasuntswana anokuba noxinaniso olufanayo kwaye ngenxa yoko akanakuhamba ngokulula ukuya kwenye.Amalungu angaxinga ukuba umazibuthe uhamba ngokukhawuleza kakhulu, nto leyo ebonisa ukuba amandla emagnethi akakwazi ukusoloko esoyisa ukukhuhlana phakathi kwamasuntswana kulwelo, nto leyo ebonisa ukuba amandla emagnethi kunye nomgama phakathi kwemagnethi kunye nendawo ekujoliswe kuyo akufuneki ize njenge. ukothuka.kubalulekile.Ezi ziphumo zikwabonisa ukuba nangona iimagnethi zinokubamba i-microparticles ezininzi ezihamba kwindawo ekujoliswe kuyo, akunakwenzeka ukuba imagnethi inokuthenjwa ukuhambisa amasuntswana e-CombiMag kumphezulu wetrachea.Ke ngoko, sigqibe kwelokuba izifundo ze-LV MF ze-vivo kufuneka zisebenzise amandla kazibuthe ukujolisa ngokwasemzimbeni iindawo ezithile zomthi womoya.
Nje ukuba amasuntswana ahanjiswe emzimbeni, kunzima ukuchonga kumxholo wezicubu ezihambayo zomzimba, kodwa amandla abo okufumanisa aphuculwe ngokuhambisa umazibuthe ngokuthe tye phezu koqhoqhoqho ukuze "ujike" iintambo zeMP.Ngelixa umfanekiso wexesha lokwenyani unokwenzeka, kulula ukubona intshukumo yamasuntswana emva kokuba isilwanyana sibulewe ngobuntu.Ugxininiso lweMP lwaludla ngokuba phezulu kule ndawo xa umazibuthe wayebekwe phezu kwendawo yokucinga, nangona amanye amasuntswana ayedla ngokufunyanwa ezantsi kuqhoqhoqho.Ngokungafaniyo nezifundo ze-in vitro, amasuntswana awakwazi ukutsalwa phantsi koqhoqhoqho ngokushukuma kwemagnethi.Oku kufunyaniswayo kuhambelana nendlela i-mucus egubungela ngayo umphezulu we-trachea ngokuqhelekileyo iqhuba amaqhekeza afakwe ngaphakathi, ukuwafaka kwi-mucus kwaye emva koko ihlambulule ngokusebenzisa i-muco-ciliary clearance mechanism.
Sacinga ukuba ukusebenzisa iimagnethi ngaphezulu nangaphantsi kwetrachea yokutsala (umzobo 3b) kunokubangela ukuba kubekho umbane olinganayo, kunokuba ube nemagnethi egxininiswe kakhulu kwinqanaba elinye, okunokubangela ukuhanjiswa okufanayo kwamasuntswana..Nangona kunjalo, uphando lwethu lokuqala aluzange lufumane ubungqina obucacileyo bokuxhasa le ngcamango.Ngokufanayo, ukuseta iperi yamagnethi ukugxotha (Umfanekiso 3c) akuzange kubangele ukuba i-particle eninzi ihlale kwindawo yomfanekiso.Ezi zinto zimbini zifunyanisiweyo zibonisa ukuba ukuseta i-double-magnetic setup ayiphuculi kakhulu ulawulo lwasekhaya lwe-MP ekhombayo, kwaye isiphumo samandla anamandla omagnetic kunzima ukuwahambisa, okwenza le ndlela ingasebenzi.Ngokufanayo, ukuqhelanisa imagnethi ngaphezulu nangaphaya kwetrachea (Umzobo 3d) nawo awuzange ukhulise inani lamasuntswana asele kwindawo efanekileyo.Olunye kolu lungelelwaniso lunolunye lusenokungaphumeleli njengoko lukhokelela ekucuthekeni kwamandla emagnethi kwindawo yokubeka.Ngaloo ndlela, ukucwangciswa kwemagnethi enye kwii-degrees ze-30 (umzobo 3a) ithathwa njengeyona ndlela ilula kwaye isebenzayo kwindlela yokuvavanya i-vivo.
Uphononongo lwe-LV-MP lubonise ukuba xa iivektha ze-LV zidityanisiwe kunye ne-CombiMag kwaye zihanjiswa emva kokuphazamiseka ngokwasemzimbeni kubukho bemagnethi, amanqanaba okuhanjiswa anyuke kakhulu kuqhoqhoqho xa kuthelekiswa nolawulo.Ngokusekwe kwizifundo ze-synchrotron imaging kunye neziphumo ze-LacZ, i-magnetic field ibonakala ikwazi ukugcina i-LV kwi-trachea kunye nokunciphisa inani lamasuntswana e-vector angena ngokukhawuleza kwimiphunga.Ukuphuculwa kokujolisa okunjalo kunokukhokelela ekusebenzeni okuphezulu ngelixa kunciphisa i-titers ezinikezelweyo, ukuhanjiswa okungajoliswanga, ukuvutha kunye nemiphumo emibi ye-immune, kunye neendleko zokudluliselwa kwemfuza.Okubalulekileyo, ngokutsho komenzi, i-CombiMag ingasetyenziselwa kunye nezinye iindlela zokudlulisa i-gene, kubandakanywa nezinye ii-viral vectors (ezifana ne-AAV) kunye ne-nucleic acids.