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MaGene vectors ekurapwa kwepulmonary cystic fibrosis anofanirwa kunangwa kune conductive airways, sezvo peripheral mapapu transduction isina mushonga wekurapa.Kubudirira kwehutachiona hwehutachiona hunoenderana zvakananga nenguva yekugara kwemutakuri.Nekudaro, zvinwiwa zvekutumira zvakaita semajeni anotakura zvinopararira mualveoli panguva yekufema, uye zvimedu zvekurapa zvechero chimiro zvinokurumidza kubviswa nemucociliary kutakura.Kuwedzera nguva yekugara kwevatakuri vemajini mumhepo yekufema kwakakosha asi kwakaoma kuita.Carrier-conjugated magineti particles anogona kunanga kune pamusoro pekufema turakiti anogona kuvandudza dunhu rakananga.Nekuda kwezvinetso ne in vivo imaging, hunhu hwemagineti madiki akadaro pamhepo yemhepo pamberi peiyo yakaiswa magineti haunzwisisike.Chinangwa chechidzidzo ichi chaive chekushandisa synchrotron imaging yekuona mu vivo kufamba kweakatevedzana magineti particles mutrachea yemakonzo anesthetized kuitira kudzidza dynamics uye mapatani ehunhu hweimwe uye yakawanda particles in vivo.Isu takazoongororawo kana kuendesa lentiviral magineti particles pamberi pesimba remagineti kwaizowedzera kugona kwekutapurirana mumakonzo trachea.Synchrotron X-ray imaging inoratidza maitiro ezvimedu zvemagineti munzvimbo dzakamira uye dzinofambisa magineti mu vitro uye mu vivo.Zvimedu hazvigone kudhonzerwa zviri nyore pamusoro penzvimbo yemhepo inorarama uchishandisa magineti, asi panguva yekufambisa, madhipoziti akaiswa mumunda wekuona, uko iyo magineti yakasimba.Kubudirira kwekushandura kwakawedzerawo katanhatu-katanhatu apo lentiviral magnetic particles yakaunzwa pamberi pesimba remagineti.Zvikatorwa pamwechete, mhedzisiro iyi inoratidza kuti lentiviral magineti particles uye magineti minda inogona kuve yakakosha nzira dzekuvandudza gene vector kunanga uye nhanho dzekufambisa mumhepo inofambisa muvivo.
Cystic fibrosis (CF) inokonzerwa nekusiyana kwejini rimwe chete rinonzi CF transmembrane conductance regulator (CFTR).Iyo CFTR protein inzira yeion inowanikwa mumasero akawanda epithelial mumuviri wose, kusanganisira nzira dzemhepo, nzvimbo huru mune pathogenesis ye cystic fibrosis.Kukanganisika muCFTR kunotungamirira kukutakura kwemvura kusina kujairika, kupera simba kwemvura pamusoro pemhepo, uye kuderera kwemhepo yemvura yepamusoro (ASL) kudzika.Izvo zvakare zvinokanganisa kugona kwemucociliary transport (MCT) system yekubvisa mhepo yezvimedu zvinofemerwa uye hutachiona.Chinangwa chedu ndechekugadzira lentiviral (LV) gene therapy kuendesa iyo chaiyo kopi yeCFTR gene nekuvandudza ASL, MCT, uye hutano hwemapapu, uye kuenderera mberi nekugadzira matekinoroji matsva anogona kuyera aya ma paramita mu vivo1.
MaLV vectors ndemumwe wevanotungamira cystic fibrosis gene therapy, kunyanya nekuti ivo vanogona kubatanidza zvachose jena rekurapa mumaseru emhepo ebasal (airway stem cells).Izvi zvakakosha nekuti ivo vanogona kudzoreredza yakajairika hydration uye mucus clearance nekusiyanisa mune inoshanda gene-yakagadziriswa airway pamusoro maseru ane chekuita necystic fibrosis, zvichikonzera mabhenefiti ehupenyu hwese.MaLV vectors anofanirwa kunangana nemhepo inofambisa, sezvo apa ndipo panotanga kubatanidzwa kwemapapu muCF.Kuendeswa kwevector kwakadzika mumapapu kunogona kukonzera alveolar transduction, asi izvi hazvina maitiro ekurapa mu cystic fibrosis.Zvisinei, zvinwiwa zvakadai semagene carriers zvinogara zvichienda mualveoli painofemerwa mushure mekusununguka kwemwana3,4 uye mishonga yekurapa inokurumidza kubudiswa mumuromo wemuromo neMCTs.Kubudirira kweLV transduction kwakabatana zvakananga nehurefu hwenguva iyo vector inoramba iri pedyo nemasero anotarirwa kuti abvumire kutora masero - "nguva yekugara" 5 iyo inopfupiswa nyore nyore neyakajairwa mhepo yedunhu pamwe chete nekubatanidzwa kutorwa kwemucus uye MCT particles.Kune cystic fibrosis, kugona kurebesa nguva yekugara kweLV mumhepo kwakakosha kuti uwane huwandu hwepamusoro hwekuchinjisa munzvimbo ino, asi kusvika zvino zvave zvichinetsa.
Kukunda chipingamupinyi ichi, isu tinopa zano kuti LV magineti particles (MPs) inogona kubatsira munzira mbiri dzinoenderana.Kutanga, ivo vanogona kutungamirwa nemagineti kumhepo yemhepo kuti vavandudze kunanga uye kubatsira gene inotakura zvimedu kuti zvive munzvimbo chaiyo yenzira yemhepo;uye ASL) vanopinda muchitokisi 6. Nhengo dzeParamende dzinoshandiswa zvakanyanya sedzinenge dzakanangwa mota dzekutakura zvinodhaka padzinosunga kune masoja ekudzivirira chirwere, chemotherapy mishonga, kana mamwe mamorekuru madiki anonamira kumasero membranes kana kusunga kune mamwe maseru epamusoro receptors uye anoungana panzvimbo dzebundu mukati. kuvapo kwe static magetsi.Magnetic fields for cancer therapy 7. Dzimwe nzira dze "hyperthermic" dzakanangana nekuuraya masero ebundu nekupisa maMP kana akaiswa kune oscillating magineti.Nheyo yekuchinjisa magineti, umo magineti inoshandiswa sechinhu chinotapurirana kusimudzira kufambiswa kweDNA mumaseru, inowanzoshandiswa mu vitro uchishandisa huwandu hweasiri mavhairasi uye mavhairasi gene mavectors kune yakaoma-ku-transduce cell mitsara. ..Kushanda kweLV magnetotransfection nekuunza LV MP in vitro musero mutsara webronchial epithelium pamberi peiyo static magineti yakasimbiswa, ichiwedzera kugona kwekufambisa ne186 nguva zvichienzaniswa neLV vector chete.LV MT yakashandiswawo kune in vitro modhi ye cystic fibrosis, uko magineti transfection yakawedzera LV transduction mumhepo-liquid interface tsika nechikamu che20 pamberi pe cystic fibrosis sputum10.Nekudaro, mu vivo organ magnetotransfection yakagamuchira kutariswa kushoma uye yakangoongororwa mune zvishoma zvidzidzo zvemhuka11,12,13,14,15, kunyanya mumapapu16,17.Nekudaro, mikana yekuchinjisa magineti mukurapa kwemapapu mucystic fibrosis yakajeka.Tan et al.(2020) yakataura kuti "chidzidzo chekusimbisa pane inoshanda pulmonary kuendesa magineti nanoparticles ichavhura nzira yeramangwana CFTR inhalation mazano ekuvandudza mhedzisiro yekiriniki muvarwere vane cystic fibrosis"6.
Maitiro ediki magineti particles ari pamusoro pemhepo yekufema pamberi peiyo inoshandiswa magineti yakaoma kuona uye kudzidza, uye saka ivo havanyatso kunzwisiswa.Mune zvimwe zvidzidzo, takagadzira Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) nzira yekufungidzira isina-invasive imaging uye quantification yeminiti mu vivo shanduko muASL18 kudzika uye MCT19 maitiro, 20 kuyera zvakananga gasi chiteshi chepamusoro hydration. uye inoshandiswa sechiratidzo chekutanga kurapwa kushanda.Pamusoro pezvo, yedu MCT scoring nzira inoshandisa 10-35 µm dhayamita zvidimbu zvinoumbwa nealumina kana yakakwira refractive index girazi seMCT mamaki anooneka nePB-PCXI21.Nzira mbiri idzi dzakakodzera kufungidzira mhando dzemhando dzezvikamu, kusanganisira MP.
Nekuda kwekugadziriswa kwenzvimbo uye kwechinguvana, PB-PCXI-based ASL uye MCT assays akanyatsokodzera kudzidza dynamics uye maitiro ezvimedu zvimwechete uye zvakawanda mu vivo kutibatsira kunzwisisa nekugadzirisa MP gene nzira dzekuunza.Nzira yatinoshandisa pano yakavakirwa pazvidzidzo zvedu tichishandisa SPring-8 BL20B2 beamline, umo takaona kufamba kwemvura zvichitevera kuburitswa kwedosi redummy vector mumhino nepulmonary airways yemakonzo kubatsira kutsanangura maitiro edu egeneti ekutaura akacherechedzwa. mujene redu.zvidzidzo zvemhuka zvine mutakuri weyero ye3.4.
Chinangwa chechidzidzo ichi chaive chekushandisa iyo PB-PCXI synchrotron kuona mu vivo mafambiro ehurongwa hweMP mu trachea yemakonzo mhenyu.Izvi zvidzidzo zvePB-PCXI zvekufungidzira zvakagadzirirwa kuyedza MP yakatevedzana, simba remagineti, uye nzvimbo yekuona maitiro avo pakufambisa kweMP.Isu takafungidzira kuti yekunze magineti yaizobatsira iyo yakaunzwa MF kugara kana kuenda kunzvimbo yainotarisirwa.Zvidzidzo izvi zvakatibvumidzawo kuona magineti masisitimu ayo anowedzera huwandu hwezvikamu zvakasara mutrachea mushure mekuiswa.Mune yechipiri nhevedzano yezvidzidzo, isu takavavarira kushandisa iyi yakakwana gadziriso kuratidza maitiro ekutapurirana anobva mukuendeswa vivo kweLV-MPs kuenda kumakonzo airways, nefungidziro yekuti kuendeswa kweLV-MPs mumamiriro ekunangwa kwemhepo kwaizoguma. mukuwedzera kweLV transduction performance..
Zvidzidzo zvese zvemhuka zvakaitwa zvinoenderana nemitemo yakatenderwa neYunivhesiti yeAdelaide (M-2019-060 uye M-2020-022) uye SPring-8 Synchrotron Animal Ethics Committee.Iwo maedzo akaitwa maringe nekurudziro yeARRIVE.
Yese mifananidzo ye x-ray yakatorwa paBl20XU beamline paSPring-8 synchrotron muJapan vachishandisa setup yakafanana neyakatsanangurwa kare21,22.Muchidimbu, bhokisi rekuedza raive 245 m kubva kune synchrotron yekuchengetedza mhete.Sample-to-detector chinhambwe che 0.6 m chinoshandiswa kuita particle imaging studies uye 0.3 m for in vivo imaging zvidzidzo kugadzira chikamu chakasiyana mhedzisiro.Danda re monochromatic rine simba re25 keV rakashandiswa.Mifananidzo yacho yakatorwa pachishandiswa high resolution X-ray transducer (SPring-8 BM3) yakabatanidzwa kune sCMOS detector.Transducer inoshandura X-rays kuita chiedza chinooneka uchishandisa 10 µm gobvu scintillator (Gd3Al2Ga3O12), iyo inobva yanangidzirwa kune sCMOS sensor uchishandisa × 10 (NA 0.3) maikorosikopu chinangwa.Iyo sCMOS detector yaive Orca-Flash4.0 (Hamamatsu Photonics, Japan) ine array saizi ye2048 × 2048 pixels uye mbishi pixel saizi ye6.5 × 6.5 µm.Kuseta uku kunopa inoshanda isotropic pixel saizi ye0.51 µm uye ndima yekuona inosvika 1.1 mm × 1.1 mm.Nguva yekuratidzwa ye100 ms yakasarudzwa kuti iwedzere chiratidzo-ku-ruzha reshiyo yezvikamu zvemagineti mukati nekunze kwenzira dzemhepo uku ichideredza mafambiro anokonzerwa nekufema.Kune muzvidzidzo zve vivo, chivharo cheX-ray chekukurumidza chakaiswa munzira yeX-ray kudzikamisa dhamu remwaranzi nekuvharira X-ray danda pakati pekufumurwa.
LV midhiya haina kushandiswa mune chero SPring-8 PB-PCXI imaging zvidzidzo nekuti iyo BL20XU imaging chamber haisi Biosafety Level 2 yakasimbiswa.Pane kudaro, takasarudza huwandu hwevamiriri veMP kubva kune vatengesi vaviri vekutengesa vanofukidza huwandu hwehukuru, zvigadzirwa, iron concentrations, uye mashandisirwo, - kutanga kuitira kuti tinzwisise kuti magineti anoita sei kufamba kweMP mugirazi capillaries, uyezve mukati. kurarama airways.pamusoro.Saizi yeMP inosiyana kubva pa0.25 kusvika 18 µm uye inogadzirwa kubva kune zvakasiyana siyana (ona Tafura 1), asi maumbirwo emuenzaniso wega wega, kusanganisira saizi yemagineti muMP, hazvizivikanwe.Zvichienderana nezvidzidzo zvedu zvakakura zveMCT 19, 20, 21, 23, 24, isu tinotarisira kuti MPs kusvika ku5 µm inogona kuoneka patracheal airway pamusoro, semuenzaniso, nekubvisa akateedzana mafuremu kuti uone kuoneka kwakavandudzwa kweMP kufamba.Imwe MP ye0.25 µm idiki pane kugadziriswa kwechigadzirwa chekufungidzira, asi PB-PCXI inotarisirwa kuona kusiyana kwavo kwevhoriyamu uye kufamba kwemvura yepamusoro painoiswa mushure mekuiswa.
Mienzaniso yeMP yega yega iri patafura.1 yakagadzirwa mu20 μl girazi capillaries (Drummond Microcaps, PA, USA) ine dhayamita yemukati ye 0.63 mm.Zvimedu zveCorpuscular zvinowanikwa mumvura, nepo CombiMag zvimedu zvinowanikwa mumugadziri wemvura.Imwe neimwe chubhu inozadzwa nehafu nemvura (inenge 11 µl) uye inoiswa pamubati wemuenzaniso (ona Mufananidzo 1).Iwo magirazi capillaries akaiswa akatwasuka pachikuva mukamuri yekufungidzira, zvichiteerana, uye akaiswa kumicheto yemvura.A 19 mm dhayamita (28 mm kureba) nickel-shell magineti akagadzirwa nepasi risingawanzo, neodymium, iron uye boron (NdFeB) (N35, katsi no. LM1652, Jaycar Electronics, Australia) ine remanence ye1.17 T yaive yakanamatira kune a patsanura tafura yekuchinjisa kuti uwane Kure shandura chinzvimbo chako panguva yekupa.X-ray imaging inotanga kana magineti yakamira ingangoita 30 mm pamusoro pesample uye mifananidzo inotorwa pamafuremu mana pasekondi.Panguva yekufungidzira, magineti akaunzwa pedyo negirazi capillary chubhu (pachinhambwe chingangoita 1 mm) uye yobva yafamba ichitevedza chubhu kuti iongorore mhedzisiro yesimba remunda uye chinzvimbo.
Iyo in vitro imaging setup ine MP samples mune girazi capillaries padanho rekududzirwa kweiyo xy sample.Nzira ye X-ray danda inotaridzwa nemutsetse mutsvuku.
Pakangotangwa kuonekwa kwemuvitro kweMPs, chikamu chidiki chavo chakaedzwa mu vivo pamhando dzesango dzechikadzi Wistar albino rats (~ mavhiki gumi nemaviri, ~ 200 g).Medetomidine 0.24 mg/kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg/kg (Dormicum®, Astellas Pharma, Japan) uye butorphanol 4 mg/kg (Vetorphale®, Meiji Seika).Makonzo akaiswa anesthetized nePharma (Japan) musanganiswa nejekiseni remukati memuviri.Mushure meanesthesia, vakagadzirirwa kufungidzira kuburikidza nekubvisa furo yakakomberedza trachea, kuisa endotracheal tube (ET; 16 Ga intravenous cannula, Terumo BCT), uye kuvamisa munzvimbo yepamusoro pane imwe tsika yakagadzirwa yekufungidzira ndiro ine bhegi rinopisa. kuchengetedza tembiricha yemuviri.22. Iplate yekufungidzira yakabva yanamatirwa kumuenzaniso nhanho mubhokisi rekufungidzira pane imwe kona kuti ienzanise trachea yakatwasuka pamufananidzo we x-ray sezvakaratidzwa mumufananidzo 2a.
(a) In vivo imaging setup muSPring-8 imaging unit, X-ray beam nzira yakanyorwa nemutsara une doti dzvuku.(b,c) Tracheal magineti localization yakaitwa kure uchishandisa maviri orthogonally akaiswa IP kamera.Kurutivi rworuboshwe rwechifananidzo pachiratidziro, unogona kuona wire loop yakabata musoro uye yekusvitsa cannula yakaiswa mukati meET chubhu.
Sirinji inodzorwa kure kure (UMP2, World Precision Instruments, Sarasota, FL) ichishandisa sirinji yegirazi ye100 µl yakanga yakabatana nePE10 tubing (0.61 mm OD, 0.28 mm ID) uchishandisa tsono ye30 Ga.Maka chubhu kuti uone kuti muromo uri panzvimbo chaiyo mutrachea paunenge uchiisa endotracheal chubhu.Uchishandisa micropump, sirinji plunger yakabviswa uye muromo wechubhu yakanyudzwa muMP sampu kuti iunzwe.Iyo yakatakurwa yekutakura chubhu yakazopinzwa mu endotracheal chubhu, ichiisa iyo tip pane yakasimba chikamu chedu chinotarisirwa chinoshandiswa magineti.Kutorwa kwemifananidzo kwaidzorwa pachishandiswa dhizaini yekufema yakabatana nebhokisi redu reArduino-based timing, uye zviratidzo zvese (semuenzaniso, tembiricha, kufema, kuvhurika / kuvhara, uye kutora mifananidzo) zvakanyorwa pachishandiswa Powerlab neLabChart (AD Instruments, Sydney, Australia) 22 Pakufananidzira Apo imba yacho yakanga isipo, makamera maviri eIP (Panasonic BB-SC382) akaiswa paanenge 90 ° kune mumwe nemumwe uye akashandiswa kudzora nzvimbo yemagineti maererano netrachea panguva yekufungidzira (Mufananidzo 2b, c).Kudzikisira zvinhu zvinofamba, mufananidzo mumwechete pakufema wakawanikwa panguva yekupera kwekufema kuyerera.
Iyo magineti inonamirwa kune yechipiri nhanho, iyo inogona kunge iri kure nechekunze kwemuviri wekufungidzira.Nzvimbo dzakasiyana-siyana uye magadzirirwo emagineti akaedzwa, kusanganisira: akaiswa paakona inenge 30 ° pamusoro petrachea (magadzirirwo anoratidzwa muMifananidzo 2a uye 3a);imwe magineti pamusoro pemhuka uye imwe iri pazasi, ine mapango akagadzirirwa kukwezva (Mufananidzo 3b)., imwe magineti pamusoro pemhuka uye imwe pazasi, ine mapango akaiswa kuti adzoke (Mufananidzo 3c), uye imwe magineti kumusoro uye perpendicular kune trachea (Mufananidzo 3d).Mushure mekumisa mhuka nemagineti uye kuisa MP pasi pekuedzwa mupombi yesirinji, endesa muyero we50 µl pamwero we4 µl/sec pakutorwa kwemifananidzo.Iyo magineti inodzoserwa kumashure nekudzoka pamwe kana kuyambuka trachea ichienderera mberi nekutora mifananidzo.
Magnet kumisikidza ye in vivo imaging (a) imwe magineti pamusoro petrachea pakona inoita 30°, (b) magineti maviri akagadzirirwa kukwezva, (c) magineti maviri akagadzirirwa kudzoreredza, (d) magineti imwe kumusoro uye perpendicular kune trachea.Mucherechedzi akatarisa pasi kubva pamuromo kuenda kumapapu kuburikidza netrachea uye X-ray danda rakapfuura nekuruboshwe rwegonzo ndokubuda nekurudyi.Iyo magineti inogona kufambiswa kureba kwenzira yemhepo kana kuruboshwe uye kurudyi pamusoro petrachea munzira yeX-ray danda.
Isu takatsvagawo kuona kuoneka uye maitiro ezvimedu mumhepo pasina kusanganiswa kwekufema uye kurova kwemoyo.Nokudaro, pakupera kwenguva yekufungidzira, mhuka dzakasunungurwa nenzira yevanhu nekuda kwepentobarbital overdose (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg ip).Dzimwe mhuka dzakasiiwa pachikuva chekufungidzira, uye mushure mekupera kwekufema uye kurova kwemoyo, maitiro ekufungidzira akadzokororwa, achiwedzera imwe dhigirii yeMP kana pasina MP yaionekwa pamhepo.
Mifananidzo yakazobuda yakagadziridzwa yemunda wakadzikama uye wakasviba wobva waunganidzwa kuita bhaisikopo (makumi maviri mafuremu pasekondi; 15–25 × yakajairika kumhanya zvichienderana nekufema) uchishandisa tsika script yakanyorwa muMATLAB (R2020a, Iyo Mathworks).
Zvidzidzo zvese zveLV gene vector delivery zvakaitwa paUniversity yeAdelaide Laboratory Animal Research Center uye zvine chinangwa chekushandisa zvakabuda muyedzo yeSPring-8 kuona kana LV-MP kuendesa pamberi pemagineti inogona kusimudzira kutamiswa kwemajini mu vivo. .Kuti uongorore migumisiro yeMF uye magnetic field, mapoka maviri emhuka akabatwa: boka rimwe rakaiswa jekiseni neLV MF rine magineti kuiswa, uye rimwe boka rakaiswa jekiseni neboka rekutonga neLV MF risina magineti.
LV gene vectors yakagadzirwa uchishandisa nzira dzakambotsanangurwa 25, 26.Iyo LacZ vector inoratidzira nyukireya yenzvimbo yebeta-galactosidase gene inofambiswa neMPSV constitutive promotioner (LV-LacZ), iyo inogadzira chigadzirwa cheblue reaction mumaseru akachinjwa, anoonekwa kumberi uye nezvikamu zvetishu yemapapu.Titration yakaitwa mumasero tsika nekuverenga nemaoko nhamba yeLacZ-positive masero uchishandisa hemocytometer kuverenga titer muTU / ml.Vanotakura vari cryopreserved pa -80 ° C, yakanyungudutswa isati yashandiswa, uye inosungwa kuCombiMag nekusanganisa 1: 1 uye incubating pachando kwemaminitsi makumi matatu asati asvika.
Normal Sprague Dawley rats (n = 3 / boka, ~ 2-3 anesthetized ip ine musanganiswa we 0.4mg / kg medetomidine (Domitor, Ilium, Australia) uye 60mg / kg ketamine (Ilium, Australia) pamwedzi 1 wezera) ip ) jekiseni uye kwete-kuvhiya nemuromo cannulation ine 16 Ga intravenous cannula.Kuti ive nechokwadi chekuti tracheal airway tissue inogamuchira LV transduction, yakagadziriswa kushandisa yedu yakatsanangurwa kare mechanical perturbation protocol umo tracheal airway surface yakakwizwa axially newaya basket (N-Circle, nitinol stone extractor pasina tip NTSE-022115 ) -UDH , Cook Medical, USA) 30 p28.Zvino, anenge maminetsi gumi mushure mekuvhiringidzika mubhayosafety cabinet, tracheal administration yeLV-MP yakaitwa.
Iyo magineti yakashandiswa muchiyedzo ichi yakagadziridzwa zvakafanana neye in vivo x-ray kudzidza, iine magineti akafanana akabatwa pamusoro petrachea ane distillation stent clamps (Mufananidzo 4).A 50 µl vhoriyamu (2 x 25 µl aliquots) yeLV-MP yakaunzwa kune trachea (n = 3 mhuka) uchishandisa gel-tipped pipette sezvakatsanangurwa kare.Boka rinodzora (n = mhuka nhatu) rakagamuchira yakafanana LV-MP pasina kushandisa magineti.Mushure mokupedzwa kwekuputira, cannula inobviswa kubva mu endotracheal tube uye mhuka inobudiswa.Iyo magineti inoramba iripo kwemaminetsi gumi isati yabviswa.Makonzo akaiswa pasi pecutaneously ne meloxicam (1 ml / kg) (Ilium, Australia) inoteverwa neanesthesia kubvisa ne intraperitoneal jekiseni ye 1 mg / kg atipamazole hydrochloride (Antisedan, Zoetis, Australia).Makonzo aichengetwa achidziya uye akacherechedzwa kusvikira kupora kwakazara kubva kune anesthesia.
LV-MP yekuendesa mudziyo mune biological kuchengetedza kabati.Iwe unogona kuona kuti chiedza chegrey Luer-lock sleeve yeET chubhu inobuda kubva mumuromo, uye gel pipette tip inoratidzwa mumufananidzo inoiswa kuburikidza neET chubhu kusvika pakadzika hunodiwa mu trachea.
Vhiki imwe mushure memaitiro ekutonga kweLV-MP, mhuka dzakabayirwa nehunhu nekufemerwa kwe100% CO2 uye kutaura kweLacZ kwakaongororwa pachishandiswa kurapa kwedu kweX-gal.Zvindori zvitatu zvakanyanya caudal cartilage zvakabviswa kuti zvive nechokwadi chokuti chero kukanganisa kwemagetsi kana kuchengetwa kwemvura nekuda kwe endotracheal tube kuiswa kwaisazobatanidzwa mukuongorora.Imwe neimwe trachea yakachekwa kureba kuti iwane mahafu maviri ekuongorora uye yakaiswa mukapu ine silicone rabha (Sylgard, Dow Inc) uchishandisa Minutien tsono (Yakanaka Sainzi Zvishandiso) kuti utarise nzvimbo yechiedza.Kugovewa uye hunhu hwemaseru akachinjirwa akasimbiswa nekutorwa mafoto uchishandisa Nikon microscope (SMZ1500) ine DigiLite kamera uye TCapture software (Tucsen Photonics, China).Mifananidzo yakawanikwa pa 20x magnification (kusanganisira kukwirira kwepamusoro kwehupamhi hwakazara hwetrachea), nehurefu hwose hwetrachea hunoratidzwa nhanho nhanho, zvichipa kukwana kwakakwana pakati pechifananidzo chimwe nechimwe kubvumira mifananidzo "kubatanidzwa".Mifananidzo kubva mutrachea yega yega yakabva yasanganiswa kuita mufananidzo mumwe chete uchishandisa Composite Image Editor shanduro 2.0.3 (Microsoft Research) uchishandisa planar motion algorithm. Nzvimbo yeLacZ yekutaura mukati me tracheal composite mifananidzo kubva kumhuka yega yega yakaverengerwa pachishandiswa otomatiki MATLAB script (R2020a, MathWorks) sezvakambotsanangurwa28, vachishandisa marongero e0.35 <Hue <0.58, Saturation> 0.15, uye Kukosha <0.7. Nzvimbo yeLacZ kutaura mukati me tracheal composite mifananidzo kubva kumhuka yega yega yakaverengerwa pachishandiswa otomatiki MATLAB script (R2020a, MathWorks) sezvakambotsanangurwa28, vachishandisa marongero e0.35 <Hue <0.58, Saturation> 0.15, uye Kukosha <0.7. Площадь экспрессии LacZ в составных изображениях трахеи от каждого животного была количественно определена с использованием зивотново2, MATLAKO 2, 2014 thWorks), как описано ранее28, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0 ,7. Nzvimbo yekutaura kweLacZ mumifananidzo inosanganiswa yetracheal kubva kumhuka yega yega yakaverengerwa pachishandiswa otomatiki MATLAB script (R2020a, MathWorks) sezvakambotsanangurwa28 uchishandisa marongero e0.350.15 uye kukosha <0 .7.如前所述，使用自动MATLAB 脚本（R2020a，MathWorks) 0.58, 饱和度> 0.15 ne值< 0.7 的设置。如 前所述 自动 自动 Matlab 脚本 (使（r2020a ， Mathworks） 來自 每 只 的 气管 合 图像 的 的 只5 <色调 <0.58 、> 0.15 和值 <0.7 的。。. ............ Области экспрессии LacZ на составных изображениях трахеи каждого количественно определяли с использованием автоматистование, 2014 автоматизер, 2 к описано ранее, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0,7 . Nzvimbo dzekutaura kweLacZ pamifananidzo inoumbwa yetrachea yemhuka yega yega dzakaverengerwa pachishandiswa otomatiki MATLAB script (R2020a, MathWorks) sezvakatsanangurwa kare uchishandisa marongero e0.35 <hue <0.58, saturation> 0.15 uye kukosha <0.7.Nekutevera maturusi contours muGIMP v2.10.24, mask yakagadzirwa nemaoko kumufananidzo wega wega unoumbwa kuti uone nzvimbo yetishu uye kudzivirira chero kuwanikwa kwenhema kunze kwetracheal tishu.Nzvimbo dzakasvibiswa kubva kumifananidzo yose yakaumbwa kubva kumhuka yega yega dzakapfupikiswa kuti dzipe nzvimbo yakazara ine mavara emhuka iyoyo.Nzvimbo yependi yakazopatsanurwa nenzvimbo yese yemask kuti iwane nzvimbo yakajairwa.
Imwe neimwe trachea yakaiswa muparafini uye yakakamurwa 5 µm gobvu.Zvikamu zvakadzorwa nekusarerekera nekukasira tsvuku kwemaminetsi mashanu uye mifananidzo yakatorwa pachishandiswa Nikon Eclipse E400 maikorosikopu, DS-Fi3 kamera uye NIS element yekutora software (vhezheni 5.20.00).
Yese ongororo yenhamba yakaitwa muGraphPad Prism v9 (GraphPad Software, Inc.).Nhamba yakakosha yakaiswa pa p ≤ 0.05.Zvakajairwa zvakaedzwa uchishandisa iyo Shapiro-Wilk bvunzo uye misiyano muLacZ staining yakaongororwa uchishandisa isina t-bvunzo.
MaParamende matanhatu anotsanangurwa muTable 1 akaongororwa nePCXI, uye kuonekwa kunotsanangurwa muTebhu 2. Nhengo mbiri dze polystyrene MP (MP1 uye MP2; 18 µm uye 0.25 µm, maererano) dzakanga dzisingaonekwi nePCXI, asi sampuli dzakasara dzinogona kuonekwa. (mienzaniso inoratidzwa mumufananidzo 5).MP3 neMP4 zvinoonekwa zvisina simba (10-15% Fe3O4; 0.25 µm uye 0.9 µm, zvichiteerana).Kunyangwe MP5 (98% Fe3O4; 0.25 µm) yaive nezvimwe zvidiki-diki zvakaedzwa, ndiyo yaive yakanyanya kutaurwa.Iyo CombiMag MP6 chigadzirwa chakaoma kusiyanisa.Muzviitiko zvese, kugona kwedu kuona maMF kwakavandudzwa zvakanyanya nekufambisa magineti kumashure nekudzoka kunoenderana necapillary.Sezvo magineti aisimuka kubva pacapillary, zvimedu zvakadhonzwa kunze mumaketani marefu, asi sezvo magineti aiswedera uye simba remagineti richiwedzera, cheni dzakapfupika sezvo zvidimbu zvaifamba zvichienda kumusoro kwecapillary (ona Supplemental Vhidhiyo S1 : MP4), kuwedzera chidimbu density pamusoro.Sezvineiwo, kana magineti yabviswa kubva pacapillary, simba remunda rinodzikira uye MP vanorongazve mumaketani akareba anotambanuka kubva kumusoro kwecapillary (ona Supplementary Vhidhiyo S2: MP4).Mushure mekunge magineti yamira kufamba, zvimedu zvinoramba zvichifamba kwenguva yakati mushure mekunge zvasvika pakuenzanisa.Sezvo iyo MP inofamba ichienda uye kure nepamusoro peiyo capillary, magineti particles anowanzo dhonza marara kuburikidza nemvura.
Kuonekwa kweMP pasi pePCXI kunosiyana zvakanyanya pakati pemasampuli.(a) MP3, (b) MP4, (c) MP5 uye (d) MP6.Mifananidzo yese inoratidzwa pano yakatorwa negineti yakamira ingangoita 10 mm pamusoro pecapillary.Iwo anooneka akakura madenderedzwa mabhuru emhepo akavharirwa mumacapillaries, achiratidza zvakajeka matema uye machena epamucheto maficha echikamu chekusiyanisa mufananidzo.Bhokisi dzvuku rinoratidza kukwidziridzwa kunowedzera musiyano.Ziva kuti madhayamita emasekete emagineti munhamba dzese haafanire kuyera uye akakura zvakapetwa ka100 pane akaratidzwa.
Sezvo magineti achifamba kuruboshwe nekurudyi kumusoro kwecapillary, kona yetambo yeMP inoshanduka kuti ienderane nemagineti (ona Mufananidzo 6), nokudaro ichitsanangura mitsetse yemagineti.YeMP3-5, mushure mekunge chord yasvika pachikumbaridzo kona, zvimedu zvinokwevera pamwe nepamusoro pepamusoro pecapillary.Izvi zvinowanzokonzera kuti vamiriri veParamende vaungane kuita mapoka makuru pedyo nepaine simba remagineti (ona Supplementary Video S3: MP5).Izvi zvinonyanya kuoneka kana kufungidzira padhuze nekupera kwecapillary, izvo zvinoita kuti MP iunganidze uye kutarisisa pane yemvura-yemhepo interface.Zvimedu zviri muMP6, izvo zvaive zvakaoma kusiyanisa pane izvo zviri muMP3-5, hazvina kukweva apo magineti akafamba pacapillary, asi tambo dzeMP dzakaparadzaniswa, dzichisiya zvidimbu zvichionekwa (ona Supplementary Video S4: MP6).Mune zvimwe zviitiko, apo simba remagineti rakaiswa rakaderedzwa nekufambisa magineti kureba kubva panzvimbo yekufungidzira, chero maMP akasara akadzika zvishoma nezvishoma kusvika pasi pechubhu negiravhiti, akasara mutambo (ona Supplementary Video S5: MP3) .
Kona yetambo yeMP inoshanduka sezvo magineti inofamba ichienda kurudyi pamusoro pecapillary.(a) MP3, (b) MP4, (c) MP5 uye (d) MP6.Bhokisi dzvuku rinoratidza kukwidziridzwa kunowedzera musiyano.Ndokumbira utarise kuti mavhidhiyo ekuwedzera ndeezvinangwa zveruzivo sezvo achiburitsa akakosha chidimbu chimiro uye ruzivo rune simba rusingagone kuoneswa mumifananidzo iyi.
Miedzo yedu yakaratidza kuti kufambisa magineti kumashure uye mberi zvishoma nezvishoma pamwe netrachea kunofambisa kuratidzwa kweMF mumamiriro ekufamba kwakaoma mu vivo.Kwete in vivo bvunzo dzakaitwa nekuti polystyrene bead (MP1 uye MP2) aisaonekwa mucapillary.Imwe neimwe yeasara mana maMF akaedzwa muvivo neakisi refu yemagneti yakamira pamusoro petrachea pakona ye 30 ° kuenda kumberi (ona Figure 2b ne3a), sezvo izvi zvakakonzera kureba kweMF cheni uye yakashanda zvakanyanya. kupfuura magineti..configuration yapera.MP3, MP4 uye MP6 haina kuwanikwa mutrachea yechero mhuka mhenyu.Paunenge uchiona nzira yekufema yemakonzo mushure mekuuraya mhuka dzevanhu, zvimedu zvakaramba zvisingaonekwe kunyangwe pakawedzerwa vhoriyamu pachishandiswa sirinji pombi.MP5 yaive nepamusoro pe iron oxide yemukati uye ndiyo yega yaioneka chidimbu, saka yaishandiswa kuongorora uye kuratidza MP maitiro muvivo.
Kuiswa kwemagineti pamusoro petrachea panguva yekuiswa kweMF kwakakonzera akawanda, asi kwete ese, maMF akatarisana mumunda wekuona.Tracheal kupinda kwezvimedu kunonyanya kucherechedzwa mumhuka dzakaburitswa nevanhu.Mufananidzo 7 uye Supplementary Vhidhiyo S6: MP5 inoratidza kukurumidza kubatwa kwemagineti uye kurongeka kwezvikamu zviri pamusoro pe ventral trachea, zvichiratidza kuti MPs inogona kunangwa kunzvimbo dzinodiwa dzetrachea.Pakutsvaga zvakanyanya kure netrachea mushure mekutumira MF, mamwe maMF akawanikwa ari padyo necarina, izvo zvinoratidza kusakwana kwemagineti simba rekuunganidza uye kubata maMF ese, sezvo akaunzwa kuburikidza nenharaunda yehukuru hwemagineti simba panguva yekutonga kwemvura.process.Nekudaro, maMP ekumashure kwekuzvarwa aive akakwira kutenderedza nzvimbo yemufananidzo, zvichiratidza kuti maMP mazhinji airamba ari munzvimbo dzemhepo uko simba remagineti rakashandiswa raive rakakwirira.
Mifananidzo ye (a) isati yasvika uye (b) mushure mekuburitswa kweMP5 mutrachea yegonzo richangoburwa rine magineti yakaiswa pamusoro penzvimbo yekufungidzira.Iyo nzvimbo inoratidzwa iri pakati pezvindori zviviri zvecartilaginous.Pane imwe mvura mumigwagwa yemhepo MP isati yaunzwa.Bhokisi dzvuku rinoratidza kukwidziridzwa kunowedzera musiyano.Iyi mifananidzo inotorwa kubva muvhidhiyo inoratidzwa muS6: MP5 Supplementary Video.
Kufambisa magineti parutivi rwetrachea mu vivo kwakakonzera shanduko yekona yeMP cheni pamhepo, yakafanana neyakaonekwa mumacapillaries (ona Mufananidzo 8 uye Supplementary Vhidhiyo S7: MP5).Nekudaro, muchidzidzo chedu, MPs yaisakwanisa kudhonzwa pamusoro pematurakiti ekufema mapenyu, semacapillaries aigona kuita.Mune zvimwe zviitiko, MP cheni inorebesa sezvo magineti inofamba kuruboshwe nekurudyi.Sezvineiwo, isu takaona zvakare kuti cheni cheni inoshandura kudzika kwepamusoro pemvura yemvura kana magineti ichifambiswa kureba parutivi rwetrachea, uye inowedzera kana magineti ichifambiswa zvakananga kumusoro uye cheni inotenderedzwa ichienda panzvimbo yakatwasuka (ona. Yekuwedzera Vhidhiyo S7).: MP5 na0:09, pasi kurudyi).Mafambiro ehunhu akachinja apo magineti aifambiswa nechemberi nepamusoro petrachea (kureva, kuruboshwe kana kurudyi kwemhuka, pane kureba kwetrachea).Zvimedu zvakanga zvichiri kuoneka zvakajeka panguva yekufamba kwavo, asi apo magineti yakabviswa kubva mutrachea, mazano ezvinyorwa zvechidimbu zvakaonekwa (ona Supplementary Video S8: MP5, kutanga pa0: 08).Izvi zvinobvumirana nemaitiro akacherechedzwa eiyo magineti pasi pekuita kweiyo yakaiswa magineti mugirazi capillary.
Mienzaniso yemifananidzo inoratidza MP5 mu trachea ye mhenyu anesthetized rat.(a) Iyo magineti inoshandiswa kutora mifananidzo kumusoro uye kuruboshwe kwetrachea, ipapo (b) mushure mekufambisa magineti kurudyi.Bhokisi dzvuku rinoratidza kukwidziridzwa kunowedzera musiyano.Iyi mifananidzo inobva muvhidhiyo inoratidzwa muS7′s Supplementary Video: MP5.
Apo matanda maviri akanga akataridzwa kuchamhembe-kumaodzanyemba pamusoro uye pasi pe trachea (kureva, kukwezva; mufananidzo 3b), maMP chords akaonekwa kwenguva refu uye akanga ari pamadziro emadziro e trachea kwete panzvimbo yedorsal ye. trachea (ona Appendikisi).Vhidhiyo S9:MP5).Zvisinei, kuwanda kwezvimedu panzvimbo imwe chete (kureva, dorsal surface yetrachea) hazvina kuonekwa mushure mekutonga kwemvura uchishandisa maviri magineti mudziyo, unowanzoitika nemudziyo mumwe chete wemagineti.Zvadaro, apo imwe magineti yakagadziriswa kuti idzore matanda akatarisana (Mufananidzo 3c), nhamba yezvikamu zvinoonekwa mumunda wekuona haina kuwedzera mushure mekusununguka.Kumisikidza ese maviri magineti masisitimu kunonetsa nekuda kweiyo yakakwira magineti simba iyo inokwezva kana kusundira magineti zvichiteerana.Iyo setup yakabva yachinjwa kuita magineti imwe chete inofambirana nemhepo asi ichipfuura nemunzira dzemhepo pa90 degree angle zvekuti mitsetse yesimba yakayambuka tracheal wall orthogonally (Figure 3d), nhangemutange yaida kuona mukana wekubatanidzwa kwechikamu pa. the lateral wall.kucherechedzwa.Nekudaro, mukumisikidzwa uku, pakanga pasina inozivikanwa MF kuunganidza kufamba kana kufamba kwemagineti.Zvichienderana nemhedzisiro iyi yese, gadziriso ine magineti imwe chete uye 30-degree orientation yakasarudzwa kune in vivo zvidzidzo zvevatakuri vemajini (Fig. 3a).
Mhuka payaifananidzwa kakawanda mushure mekubayirwa sevanhu, kusavapo kwekukanganisa kufamba kwematishu kwaireva kuti mitsetse yakapfava, mipfupi yaigona kuonekwa mumunda wakajeka weintercartilaginous, 'kuzununguka' zvinoenderana neshanduko yekufamba kwemagineti.ona zvakajeka kuvapo uye kufamba kweMP6 zvidimbu.
Iyo titer yeLV-LacZ yaive 1.8 x 108 IU/mL, uye mushure mekusanganisa 1: 1 neCombiMag MP (MP6), mhuka dzakabayiwa ne50 µl yetracheal dose ye9 x 107 IU/ml yeLV mota (kureva 4.5 x 106 TU/gonzo).)).Muzvidzidzo izvi, pachinzvimbo chekufambisa magineti panguva yebasa, takaisa magineti panzvimbo imwe chete kuti tione kana LV transduction inogona (a) kuvandudzwa kana ichienzaniswa nevector kuburitsa pasina simba remagineti, uye (b) kana nzira yemhepo yaigona. isa pfungwa dzako.Masero ari kufambiswa munzvimbo dzemagineti dzepamusoro pekufema turakiti.
Kuvapo kwemagineti uye kushandiswa kweCombiMag pamwe chete neLV vectors hakuna kuita sekukanganisa hutano hwemhuka, sezvakaita yedu standard LV vector delivery protocol.Mifananidzo yepamberi yenharaunda yetracheal yakatarisana ne mechanical perturbation (Supplementary Fig. 1) yakaratidza kuti LV-MP yakarapwa boka yakanga ine mazinga akakwirira zvikuru ekushandurwa pamberi pemagineti (Fig. 9a).Ingori shoma shoma yebhuruu LacZ tsvina yaivepo muboka rekutonga (Mufananidzo 9b).Quantification yenzvimbo dzeX-Gal-stained normalized yakaratidza kuti kutonga kweLV-MP muhupo hwemagineti kwakaguma nekuvandudzika kwe6-fold (Fig. 9c).
Muenzaniso wemifananidzo inoumbwa inoratidza tracheal transduction neLV-MP (a) pamberi pemagineti uye (b) pasina magineti.(c) Statistically yakakosha kuvandudzwa munzvimbo yakajairwa yeLacZ transduction mutrachea nekushandisa magineti (* p = 0.029, t-test, n = 3 paboka, zvinoreva ± chiyero chikanganiso chezvinoreva).
Neutral fast-red-stained sections (muenzaniso unoratidzwa muSupplementary Fig. 2) yakaratidza kuti masero eLacZ-akasvibiswa akanga aripo mumuenzaniso wakafanana uye munzvimbo imwechete sezvakambotaurwa.
Chinetso chikuru mumhepo yejene therapy inoramba iri iyo chaiyo yenzvimbo yezvimedu zvekutakura munzvimbo dzinofarira uye kubudirira kwepamusoro-soro yekufambisa kwepamusoro mumapapu ekufambisa pamberi pemhepo uye kushanda kwemucus clearance.Kune vatakuri veLV vakagadzirirwa kurapwa kwezvirwere zvekufema mu cystic fibrosis, kuwedzera nguva yekugara yezvimedu zvinotakura mumigwagwa inofambisa mhepo kusvika zvino chave chinangwa chisingawanikwe.Sezvakataurwa naCastellani et al., kushandiswa kwemagineti kusimudzira transduction kune zvakanakira pane dzimwe nzira dzekuburitsa majini senge electroporation nekuti inogona kusanganisa kureruka, hupfumi, kuendesa kwenzvimbo, kuwedzera kugona, uye ipfupi incubation nguva.uye pamwe mwero wakaderera wemotokari10.Nekudaro, mu vivo deposition uye hunhu hwemagineti particles mumhepo pasi pesimba rekunze remagineti masimba haasati ambotsanangurwa, uye kutaura zvazviri kugona kweiyi nzira yekuwedzera magene ekutaura mazinga mumararamiro emhepo asina kuratidzwa muvivo.
Yedu in vitro miyedzo paPCXI synchrotron yakaratidza kuti zvese zvimedu zvatakaedza, kunze kweMP polystyrene, zvaionekwa mukumisikidza kwekufungidzira kwatakashandisa.Muhupo hwemagineti, magineti anoumba tambo, kureba kwacho kunoenderana nemhando yezvimedu uye simba remagineti (kureva, kuswedera uye kufamba kwemagineti).Sezvinoratidzwa muMufananidzo 10, tambo dzatinoona dzinoumbwa sezvo chidimbu chega chega chichiita magineti uye chinokwevera magineti enzvimbo.Iyi minda yakaparadzana inokonzeresa zvimwe zvimedu zvakafanana kuti zviunganidze uye zvibatane nekufamba kwetambo dzeboka nekuda kwemasimba enzvimbo kubva kumasimba emunharaunda ekukwezva uye kudzorwa kwezvimwe zvimedu.
Dhiagiramu inoratidza (a, b) cheni dzezvimedu zvinoumba mukati mecapillaries akazadzwa nemvura uye (c, d) trachea izere nemhepo.Cherechedza kuti capillaries uye trachea hazvina kukweverwa pachiyero.Panel (a) inewo tsananguro yeMF ine Fe3O4 zvidimbu zvakarongwa mumaketani.
Apo magineti akafamba pamusoro pe capillary, kona yetambo yechidimbu yakasvika pachikumbaridzo chakakosha cheMP3-5 ine Fe3O4, mushure mezvo tambo yechidimbu haina kuramba iri panzvimbo yayo yepakutanga, asi yakatama pamusoro pepamusoro kune imwe nzvimbo.magineti.Izvi zvingangoitika nekuti pamusoro pegirazi capillary inotsvedzerera zvakakwana kubvumira kufamba uku kuti kuitike.Sezvineiwo, MP6 (CombiMag) haina kuzvibata nenzira iyi, pamwe nekuti zvidimbu zvacho zvaive zvidiki, zvaive nerayiti yakasiyana kana yekuchajiswa kwepamusoro, kana iyo inotakura inotakura mvura yakakanganisa kugona kwavo kufamba.Musiyano uri muCombiMag particle mufananidzo zvakare hauna kusimba, zvichiratidza kuti mvura uye zvimedu zvinogona kunge zvine density yakafanana uye nekudaro haigone kufamba yakanangana.Zvicherwa zvinokwanisawo kunamira kana magineti akamhanya zvakanyanya, zvichiratidza kuti simba remagineti harigone kugara richikunda kupokana kuri pakati pezvikamu zviri mumvura, zvichiratidza kuti simba remagineti uye chinhambwe chiri pakati pemagineti nenzvimbo yakananga haifanire kuuya se kushamisika.zvakakosha.Mhedzisiro iyi inoratidzawo kuti kunyangwe magineti achigona kubata akawanda mamicroparticles anoyerera nepakati penzvimbo yakanangwa, hazvigoneke kuti magineti anogona kuvimbwa nawo kuti afambise maCombiMag zvimedu pamusoro petrachea.Nekudaro, isu takagumisa kuti mu vivo LV MF zvidzidzo zvinofanirwa kushandisa static magineti kutarisisa chaiko nzvimbo dzemuti wemhepo.
Kana zvimedu zvaunzwa mumuviri, zvinonetsa kuziva mukati meiyo yakaoma inofamba tishu yemuviri, asi kugona kwavo kwekuona kwakagadziridzwa nekufambisa magineti yakatwasuka pamusoro petrachea kuti "wiggle" MP tambo.Nepo kufungidzira chaiko-nguva kuchigoneka, zviri nyore kuona kufamba kwechikamu mushure mekunge mhuka yaurayiwa.Kumisikidzwa kweMP kwaiwanzo kwirisa panzvimbo iyi kana magineti yakamira pamusoro penzvimbo yekufungidzira, kunyangwe zvimwe zvimedu zvaiwanzo kuwanikwa zvichidzika pasi petrachea.Kusiyana nezvidzidzo zve in vitro, zvimedu hazvigone kudhonzwa pasi trachea nekufamba kwemagineti.Kuwanikwa uku kunopindirana nemabatiro anoita mucus anovhara pamusoro petrachea anowanzo gadzira zvimedu zvinofemerwa, zvichivateya mumhata uye zvozozvibvisa kuburikidza nemuco-ciliary clearance mechanism.
Isu takafungidzira kuti kushandisa magineti kumusoro uye pasi petrachea yekukwezva (Fig. 3b) inogona kuguma kune imwe yunifomu yemagineti, pane iyo magineti yakanyanyisa kunyura pane imwe nguva, zvichigona kuguma nekumwe yunifomu kugoverwa kwezvikamu..Zvisinei, chidzidzo chedu chekutanga hachina kuwana humbowo hwakajeka hwekutsigira iyi fungidziro.Saizvozvowo, kuisa magineti maviri kuti adzore (Fig. 3c) hazvina kukonzera kuti chidimbu chigadzikane munzvimbo yemufananidzo.Izvi zviviri zvakawanikwa zvinoratidza kuti maviri-magineti setup haanyanyi kuvandudza kudzora kwenzvimbo kweMP kunongedza, uye kuti mhedzisiro yakasimba magineti masimba inonetsa kuita, zvichiita kuti nzira iyi isashande.Saizvozvo, kutungamira magineti kumusoro uye kuyambuka trachea (Mufananidzo 3d) zvakare haina kuwedzera huwandu hwezvikamu zvakasara munzvimbo yemufananidzo.Zvimwe zvezvigadziriso izvi zvinogona kusabudirira sezvo zvichikonzera kudzikiswa kwesimba remagineti munzvimbo yekuisa.Saka, iyo imwe chete magineti configuration pa 30 madhigirii (Fig. 3a) inoonekwa seyakareruka uye inoshanda zvakanyanya mu vivo yekuyedza nzira.
Chidzidzo cheLV-MP chakaratidza kuti kana maVector eLV akabatanidzwa neCombiMag uye akaunzwa mushure mekukanganiswa mumuviri pamberi pemagineti, mazinga ekutapurirana akawedzera zvakanyanya mutrachea zvichienzaniswa nekudzora.Zvichienderana nezvidzidzo zvekufungidzira zvesynchrotron uye mhedzisiro yeLacZ, iyo magineti yaitaridza kunge ichikwanisa kuchengeta LV mutrachea uye kuderedza huwandu hwemavector particles akabva apinda mukati memapapu.Kuvandudzwa kwekutarisa kwakadai kunogona kutungamira mukubudirira kwepamusoro uku uchideredza kuendeswa titers, kusiri-kwakananga transduction, kuzvimba uye immune side mhedzisiro, uye gene kutamisa mari.Zvakakosha, maererano nemugadziri, CombiMag inogona kushandiswa pamwe chete nedzimwe nzira dzekutumira majini, kusanganisira mamwe mavhairasi (akadai seAAV) uye nucleic acids.