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Gene vectors domin lura da cystic fibrosis huhu cuta ya kamata a niyya da gudanar da iska domin na gefe huhu transduction ba ya samar da warkewa fa'ida.ProViral transduction yadda ya dace yana da alaka kai tsaye da vector zama time.Sai da haka, bayarwa ruwaye kamar gene dako ta halitta yada a cikin alveoli a lokacin wahayi, da kuma warkewa barbashi da sauri barbashi na kowane nau'i. Masu jigilar kwayoyin halitta a cikin hanyoyin iska yana da mahimmanci amma yana da wuyar cimmawa.Gene mai ɗaukar hoto mai haɗaɗɗen magnetic barbashi wanda za a iya kai tsaye zuwa saman hanyoyin iska na iya inganta haɓaka yanki.Sakamakon ƙalubalen gani a cikin vivo, halayen irin waɗannan ƙananan ƙwayoyin maganadisu a kan filin jirgin sama a gaban filin magnetic da aka yi amfani da shi ba a fahimta sosai ba. Magnetic barbashi a cikin trachea na anesthetized berayen don nazarin kuzarin kawo cikas da alamu na mutum da kuma girma barbashi hali a cikin vivo.We sa'an nan kuma tantance ko isar da lentiviral Magnetic barbashi a gaban wani Magnetic filin zai kara transduction yadda ya dace a cikin bera trachea. Synchrotron X-ray Hoto ya bayyana halin da ake ciki a cikin fili da kuma taswirar Magnetic barbashi. vivo.Barbashi ba za a iya sauƙi ja tare da surface na rayuwa iska iska tare da maganadiso, amma a lokacin sufuri, da adibas da aka mayar da hankali a cikin filin view inda Magnetic filin ne da karfi.Transduction yadda ya dace kuma ya karu sau shida a lokacin da lentiviral Magnetic barbashi da aka tsĩrar a gaban wani Magnetic filin. Tare, wadannan sakamakon bayar da shawarar cewa particles iya zama mai muhimmanci da manufa filin Magnetic filin. da haɓaka matakan watsawa a cikin tafiyar da hanyoyin iska a cikin vivo.
Cystic fibrosis (CF) yana haifar da bambanci a cikin kwayar halitta guda daya da ake kira CF transmembrane conductance regulator (CFTR) .CfTR furotin shine tashar ion tashar da ke samuwa a cikin yawancin kwayoyin epithelial a cikin jiki, ciki har da hanyoyin iska, babban shafin yanar gizon CF pathogenesis. CFTR lahani yana haifar da rashin lafiyar ruwa na ruwa, rage ruwa mai zurfi na sararin samaniya (SLA). yana ɓata ikon tsarin jigilar mucociliary (MCT) don share ƙwayoyin da aka shayar da ƙwayoyin cuta da ƙwayoyin cuta daga hanyoyin iska.Manufarmu ita ce samar da kwayoyin halitta na lentiviral (LV) don sadar da daidai kwafin kwayar halittar CFTR da inganta ASL, MCT, da lafiyar huhu, da kuma ci gaba da bunkasa sababbin fasahar da za su iya auna waɗannan sigogi a cikin vivo1.
LV vectors ne daya daga cikin manyan 'yan takara ga CF airway genetherapy, yafi saboda za su iya har abada hade therapeutic gene a cikin Airway basal Kwayoyin (airway stem cell) .Wannan yana da muhimmanci domin za su iya mayar da al'ada hydration da gamsai sharer ta hanyar bambanta a cikin aiki gene-gyara CF-haɗe da Airway surface Kwayoyin, sakamakon da ya kamata a yi amfani da iska tsawon rai. shine inda cutar huhu ta CF ta fara. Isar da vector mai zurfi a cikin huhu na iya haifar da transduction alveolar, amma wannan ba shi da fa'idar warkewa a cikin CF. Duk da haka, ruwa irin su jigilar jigilar kwayoyin halitta suna ƙaura zuwa alveoli a kan wahayi bayan bayarwa3,4 kuma ana fitar da ƙwayoyin warkewa da sauri zuwa cikin kogin baka na tsawon lokaci ta hanyar M. vector ya kasance kusa da sel da aka yi niyya don ba da damar ɗaukar salula - "lokacin zama" 5 - wanda aka rage sauƙin ragewa ta hanyar yanayin iska na yanki na al'ada da kuma daidaitawar ƙwayar ƙwayar cuta da kuma MCT. Domin CF, ikon tsawaita lokacin zama na LV a cikin hanyar iska yana da mahimmanci don cimma manyan matakan watsawa a cikin wannan yanki, amma ya zuwa yanzu ya kasance ƙalubale.
Don shawo kan wannan cikas, muna ba da shawarar cewa ƙwayoyin maganadisu na LV (MPs) na iya taimakawa ta hanyoyi guda biyu masu dacewa. Na farko, ana iya jagorantar su ta hanyar maganadisu zuwa saman sararin sama don inganta niyya da kuma taimakawa ƙwayoyin jigilar kwayoyin halitta su zauna a yankin da ake so; da ASL) don matsawa zuwa Layer Layer 6.MPs an yi amfani da su sosai a matsayin motocin isar da miyagun ƙwayoyi da aka yi niyya lokacin da suke ɗaure ga ƙwayoyin cuta, magungunan chemotherapeutic, ko wasu ƙananan ƙwayoyin cuta waɗanda ke haɗawa da membranes tantanin halitta ko ɗaure ga masu karɓar sararin sel masu dacewa kuma suna tarawa a wuraren kumburi a gaban wutar lantarki. Filin Magnetic don Maganin Ciwon daji 7. Sauran dabarun "hyperthermal" suna nufin zafi sama da MPs lokacin da aka fallasa su zuwa filayen maganadisu na oscillating, ta haka ne ke lalata ƙwayoyin tumor.Ka'idar jigilar maganadisu, wanda ake amfani da filin maganadisu azaman wakili mai canzawa don haɓaka canja wurin DNA zuwa sel, ana amfani da shi a cikin vitro ta amfani da kewayon ƙwayoyin ƙwayoyin cuta marasa ƙarfi da ƙwayoyin cuta. na LV magnetotransfection da aka kafa, tare da in vitro bayarwa na LV-MPs zuwa wani ɗan adam Bronchial epithelial cell line a gaban wani a tsaye Magnetic filin, ƙara transduction yadda ya dace da 186-ninka idan aka kwatanta da LV vector kadai.LV-MP an kuma yi amfani da wani in vitro CF model, inda Magnetic transfection ya karu da LV gaban transduction CF a cikin iska-folds a cikin iska - 0. sputum10.Duk da haka, a cikin vivo magnetotransfection na gabobin ya sami in mun gwada da kadan hankali da aka kawai aka kimanta a cikin 'yan dabba studies11,12,13,14,15, musamman a cikin huhu16,17. Duk da haka, da damar da Magnetic transfection a CF huhu far ne bayyananne.Tan et al. bayarwa na huhu zai ba da hanya don dabarun inhalation na CFTR na gaba don inganta sakamakon asibiti a cikin marasa lafiya na CF"6.
Halin ƙananan ƙwayoyin magnetic akan saman saman iska a gaban filin maganadisu da aka yi amfani da shi yana da wahala a iya gani da nazari, don haka ba a fahimta sosai ba.A cikin wasu nazarin, mun haɓaka hanyar haɓakawa na tushen lokaci-daidaitawar X-ray Hoto (PB-PCXI) zuwa hanyar noninvasively gani da ƙididdige minti a cikin vivo canje-canje a cikin ACT zuwa zurfin 18 da MVD0. An yi amfani da shi azaman farkon nuna alama na ingancin jiyya. Bugu da ƙari, hanyar ƙimar mu ta MCT tana amfani da 10-35 µm diamita barbashi da aka haɗa da alumina ko babban gilashin index kamar yadda alamun MCT ke bayyane ta amfani da PB-PCXI21.Dukansu dabaru sun dace da hangen nesa na nau'ikan nau'ikan nau'ikan nau'ikan, gami da MP.
Saboda ta high sarari da kuma na wucin gadi ƙuduri, mu PB-PCXI-tushen ASL da MCT bincike dabaru ne da kyau dace domin nazarin kuzarin kawo cikas da alamu na guda da girma barbashi hali a cikin vivo don taimaka mana fahimtar da inganta MP gene isar da dabaru.The m da muke aiki a nan ya samo daga karatunmu ta amfani da SPring-8 BL20B2 na gani motsi a cikin shayarwar da muke amfani da shi a cikin shayarwa. hanyoyin iska na hanci da na huhu na beraye don taimakawa wajen bayyana tsarin maganganun jinsin mu marasa daidaituwa da aka lura a cikin binciken mu na jigilar dabbobi 3,4.
Manufar wannan binciken shine don amfani da synchrotron PB-PCXI don ganin motsin cikin vivo na jerin 'yan majalisa a cikin trachea na berayen masu rai.Waɗannan nazarin hotunan PB-PCXI an tsara su ne don gwada nau'ikan MPs, ƙarfin filin maganadisu, da wurare don sanin tasirin su akan motsin MP. Mun yi tsammanin cewa filin zai taimaka wurin yin amfani da filin da aka yi niyya a waje. A cikin jerin na biyu na binciken, mun nemi yin amfani da wannan tsari mafi kyau don nuna tsarin canji wanda ya samo asali daga isar da LV-MPs zuwa hanyar iska ta bera, bisa tsammanin cewa isar da LV-MPs a cikin mahallin inganta hanyar jirgin sama zai haifar da efficiency.
Dukkan nazarin dabba an yi su ne bisa ka'idojin da Jami'ar Adelaide (M-2019-060 da M-2020-022) suka amince da su da kuma SPring-8 Synchrotron Animal Ethics Committee. An yi gwaje-gwaje bisa ga ka'idodin ARRIVE.
An yi duk hotunan X-ray a cikin BL20XU beamline a SPring-8 synchrotron a Japan, ta yin amfani da saitin kama da abin da aka bayyana a baya21,22. A takaice dai, akwatin gwaji ya kasance a 245 m daga zoben ajiya na synchrotron. An yi amfani da samfurin-to-detector nesa na 0.6 m don yin amfani da nazarin tasirin hoto na barbashi. An yi amfani da makamashin katako na monochromatic na 25 keV. An kama hotuna ta amfani da babban mai canza X-ray (SPring-8 BM3) tare da na'urar ganowa ta sCMOS.Mai canza shi yana canza hasken X zuwa haske mai gani ta amfani da 10 µm lokacin farin ciki scintillator (Gd3Al2Ga3O12), wanda aka tura abu zuwa microscope (Gd3Al2Ga3O12), wanda aka tura abu zuwa microscope. 0.3) Mai gano sCMOS shine Orca-Flash4.0 (Hamamatsu Photonics, Japan) tare da girman tsararru na 2048 × 2048 pixels da ɗanyen girman pixel na 6.5 × 6.5 µm. Wannan saitin yana samar da ingantaccen girman pixel isotropic girman 0.51 µm na tsayin tsayin mm 1.1. An zaɓi 100 ms don ƙara girman siginar siginar sauti na ƙwayoyin maganadisu a ciki da waje da hanyar iska yayin da yake rage yawan abubuwan motsi na numfashi.Domin a cikin nazarin vivo, an sanya maɓallin X-ray mai sauri a cikin hanyar X-ray don iyakance adadin radiation ta hanyar toshe igiyar X-ray tsakanin abubuwan da aka bayyana.
Ba a yi amfani da mai ɗaukar LV ba a cikin kowane karatun hoto na SPring-8 PB-PCXI saboda ɗakin hoto na BL20XU ba ƙimar Biosafety Level 2 ba ce. Maimakon haka, mun zaɓi kewayon ƙwararrun ƴan majalisar wakilai daga masu samar da kasuwanci guda biyu-wanda ke rufe nau'ikan girma, kayan, ƙarfin ƙarfe, da aikace-aikacen motsi a cikin filin rayuwa sannan kuma a cikin filin MPilla don fahimtar tasirin gilashin gilashin da farko. hanyoyin iska. a kan surface.MPs kewayo a cikin girman daga 0.25 zuwa 18 μm kuma an yi su daga wani iri-iri na kayan (duba Table 1), amma abun da ke ciki na kowane samfurin, ciki har da girman da Magnetic barbashi a cikin MP, shi ne unknown.Based a kan mu m MCT karatu 19, 20, 21, 23, 24, muna sa ran cewa MPs a matsayin kananan kamar yadda za a iya gani a cikin iska ta hanyar 5. Rage firam ɗin jere don ganin ingantaccen gani na motsin MP.Mai girman girman μm 0.25 μm ya fi ƙanƙanta da ƙudurin na'urar hoto, amma ana sa ran PB-PCXI zai gano bambancin ƙarar su da motsin ruwan saman da aka ajiye su bayan an ajiye su.
An shirya samfurori ga kowane MP a cikin Table 1 a cikin 20 μl gilashin capillaries (Drummond Microcaps, PA, Amurka) tare da diamita na ciki na 0.63 mm. Ƙwararren ƙwanƙwasa suna samuwa a cikin ruwa, yayin da CombiMag barbashi suna samuwa a cikin ma'auni na masu sana'a. Kowane tube yana da rabi cike da ruwa (kimanin samfurin) da gilashin 11). capillaries da aka sanya horizontally a kan samfurin mataki a cikin hoton akwatin, bi da bi, da kuma positioned gefuna na ruwa. A 19 mm diamita (28 mm tsawo) nickel harsashi rare duniya neodymium iron boron (NdFeB) maganadisu (N35, cat. no. LM1652, Jaycar Electronics, Australia) tare da saura cimma magnetization na 1. a lokacin daukar hoto. Hoton hoton X-ray yana farawa lokacin da magnet ya kasance kimanin 30 mm sama da samfurin, kuma ana samun hotuna a cikin nau'i na 4 Frames a kowace dakika. A lokacin hoton, an kawo magnet kusa da gilashin capillary tube (kimanin 1 mm nesa) sa'an nan kuma an fassara shi tare da bututu don tantance tasirin ƙarfin filin da matsayi.
Saitin hoto na in vitro mai ɗauke da samfuran MP a cikin capillaries na gilashi akan matakin fassarar samfurin xy. Hanya na katako na X-ray yana da alama tare da layin ja mai ja.
Da zarar an tabbatar da ganin in vitro na MPs, an gwada wani yanki na su a cikin vivo a cikin nau'in ratsan mata na albino Wistar (~ 12 makonni da haihuwa, ~ 200 g) .0.24 mg / kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg / kg midazolam (Dormicum® , mg / Japan) amma P. (Vetorphale®, Meiji Seika) An kwantar da berayen tare da cakuda Pharma), Japan) ta hanyar allurar intraperitoneal. Bayan maganin sa barci, an shirya su don yin hoto ta hanyar cire Jawo a kusa da trachea, shigar da bututun endotracheal (ET; 16 Ga iv cannula, Terumo BCT) da kuma canza yanayin zafin jiki wanda ke ƙunshe da yanayin zafin jiki. 22 .Sa'an nan kuma an haɗa farantin hoto zuwa matakin fassarar samfurin a cikin akwatin hoto a wani ɗan kusurwa don daidaita trachea a kwance a cikin hoton X-ray, kamar yadda aka nuna a cikin Hoto 2a.
(a) A cikin saitin hoto na vivo a cikin akwatin hoto na SPring-8, hanyar X-ray beam alama ce ta layin ja mai ja.
Tsarin famfo na sirinji mai sarrafawa mai nisa (UMP2, Kayan Kayan Kayan Duniya, Sarasota, FL) ta amfani da sirinji na gilashin 100 μl an haɗa shi da tubing PE10 (OD 0.61 mm, ID 0.28 mm) ta hanyar allurar 30 Ga. Alama bututu don tabbatar da cewa tip yana cikin madaidaicin bututun ET.U. syringe plunger aka janye yayin da tip na tube da aka nutsar a cikin MP samfurin da za a bayarwa.The lodi bayarwa tube aka sa'an nan saka a cikin endotracheal tube, ajiye da tip a cikin mafi karfi na mu sa ran shafi Magnetic filin.Image saye da aka sarrafa ta amfani da wani numfashi ganowa da alaka da mu Arduino tushen lokaci akwatin, da kuma duk alamun da aka rubuta ta amfani da yanayin zafin jiki, misali, zazzabi reclosing (misali. Powerlab da LabChart (AD Instruments, Sydney, Ostiraliya) 22. Lokacin da hoto Lokacin da shinge bai isa ba, kyamarorin IP guda biyu (Panasonic BB-SC382) an sanya su a kusan 90 ° ga juna kuma an yi amfani da su don saka idanu da matsayi na maganadisu dangane da trachea a lokacin hoto (Fig. 2b,c) don rage girman motsin motsin motsi, don rage girman motsin motsi. plateau.
Ana haɗe magnet zuwa mataki na biyu wanda za'a iya kasancewa a nesa daga waje da gidaje masu hoto. An gwada wurare daban-daban da kuma daidaitawa, ciki har da: An ɗora a kusurwar kusan 30 ° sama da trachea (tsarin da aka nuna a cikin Figures 2a da 3a); daya maganadisu a sama da dabba da sauran a kasa, tare da sanduna da aka saita don jawo hankali (Hoto 3b); daya maganadisu a sama da dabba da sauran a kasa, tare da sanduna da aka saita don tunkude (Hoto 3c); kuma daya maganadisu a sama da perpendicular zuwa trachea (Figure 3d) .Da zarar an daidaita dabba da maganadisu kuma an ɗora MP ɗin da za a gwada a cikin famfon sirinji, sadar da kashi 50 μl a cikin adadin 4 μl / sec yayin da ake samun hotuna. Ana tura magnet zuwa gaba da gaba tare ko a baya a kan hanyar samun hotuna yayin ci gaba da samun hotuna.
Tsarin Magnet don in vivo imaging (a) maganadisu guda ɗaya a saman trachea a kusurwar kusan 30 °, (b) magneto biyu saita don jawo hankali, (c) magneti guda biyu saita don tunkude, (d) magnet guda ɗaya a sama da daidaitaccen a cikin trachea. ya motsa tare da tsawon hanyar iska ko hagu da dama a sama da trachea zuwa hanyar X-ray katako.
Mun kuma nemi sanin ganuwa da halayyar barbashi a cikin iska ba tare da rikicewar numfashi da motsin zuciya ba.Saboda haka, a ƙarshen lokacin daukar hoto, an kashe dabbobi da mutuntawa don yawan adadin pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, Amurka; ~ 65 mg / kg ip dandali, da zarar an dakatar da dabbar da ke cikin zuciya). An sake maimaita tsarin hoto, ƙara ƙarin kashi na MP idan ba a ga MP a saman iska ba.
Hotunan da aka samo an gyara su a fili da duhu-filin sannan aka haɗa su cikin fim (firam 20 a sakan daya; 15-25 × na al'ada gudun dangane da ƙimar numfashi) ta amfani da rubutun al'ada da aka rubuta a cikin MATLAB (R2020a, The Mathworks).
Duk LV gene vector isar da karatun da aka gudanar a Laboratory Animal Research Facility a Jami'ar Adelaide da nufin yin amfani da sakamakon SPring-8 gwaji don tantance ko LV-MP bayarwa a gaban wani Magnetic filin zai iya bunkasa gene canja wurin a vivo.To tantance sakamakon MP da Magnetic filin, biyu kungiyoyin dabbobi da aka bi: daya rukuni aka bai LV-MP tare da wani rukuni na Magnetic sanya LV, da kuma wani rukuni na LV.
LV gene vectors an samar da su ta hanyar amfani da hanyoyin da aka bayyana a baya 25, 26 .LacZ vector yana bayyana kwayoyin beta-galactosidase na nukiliya-localized ta hanyar mai gabatarwa MPSV (LV-LacZ), wanda ke samar da samfurin amsawa mai launin shuɗi a cikin sel da aka canza, bayyane a cikin huhu nama gaba da kuma sassan nama da aka yi da lambar da aka yi ta hanyar al'ada ta LacZ a cikin al'ada ta hanyar Lacz. hemocytometer don lissafta titer a cikin TU / ml. Ana adana masu ɗaukar kaya a -80 ° C, narke kafin amfani da su, kuma an ɗaure su zuwa CombiMag ta hanyar haɗawa a cikin rabo na 1: 1 da incubating akan kankara don akalla minti 30 kafin bayarwa.
Al'ada Sprague Dawley berayen (n = 3 / rukuni, ~ 2-3 an anesthetized intraperitoneally tare da cakuda 0.4 mg / kg medetomidine (Domitor, Ilium, Australia) da kuma 60 mg / kg ketamine (Ilium, Ostiraliya) wata-wata) ip) allura da wadanda ba tiyata na baka cannulation iya tabbatar da iska ta hanyar iska 16. nama yana karɓar LV transduction, an sharadi ta amfani da ka'idodin mu na injiniya da aka bayyana a baya, wanda a cikin abin da aka lalatar da filin jirgin sama na tracheal axially tare da kwandon waya (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Tracheal aminci da aka yi a minti na L. hargitsa.
An daidaita filin magnetic da aka yi amfani da shi a cikin wannan gwaji a cikin irin wannan hanyar zuwa nazarin hoton X-ray a cikin vivo, tare da nau'i-nau'i iri-iri da aka gudanar a sama da trachea ta amfani da shirye-shiryen stent distillation (Figure 4) .A 50 μl (2 × 25 μl aliquots) na LV-MP an ba da shi a cikin trachea (n = 3 da dabbobi) da aka bayyana a baya ta hanyar amfani da bututun tip. 3 dabbobi) sun sami LV-MPs iri ɗaya ba tare da yin amfani da magnet ba.Bayan jiko ya cika, an cire cannula daga bututun ET kuma an cire dabbar.Magnet ɗin ya kasance a wurin na minti 10, sa'an nan kuma an cire shi. Berayen sun karbi kashi na subcutaneous na meloxicam (1 ml / kg) (Ilium, Ostiraliya) tare da reversal na 1 mg / chloride azol. (Antisedan, Zoetis, Ostiraliya) .An kiyaye berayen dumi kuma ana kula da su har sai an dawo da su daga maganin sa barci.
LV-MP na'urar isar da saƙo a cikin ma'ajin aminci na halitta. Ana iya ganin haske mai launin toka Luer hub na bututun ET yana fitowa daga baki kuma an saka tip gel na pipette da aka nuna a cikin hoton ta cikin bututun ET zuwa zurfin da ake so a cikin trachea.
Mako daya bayan tsarin maganin LV-MP, an kashe dabbobi da mutuntawa ta hanyar 100% CO2 inhalation da kuma LacZ magana da aka kimanta ta yin amfani da mu misali X-gal magani.The uku caudal mafi yawan cartilaginous zobe da aka cire don tabbatar da cewa duk wani inji lalacewa ko ruwa riƙewa daga endotracheal tube jeri ba a hada a cikin analysis.Kowane trachea da aka yanke a cikin tantanin halitta, kuma an halicce su a cikin dogon lokaci. silicone rubber (Sylgard, Dow Inc) ta yin amfani da allurar Minutien (Kyakkyawan Kimiyyar Kimiyya) don ganin hangen nesa mai haske. An tabbatar da rarrabawa da kuma tsarin kwayoyin halitta ta hanyar daukar hoto ta gaba ta hanyar amfani da microscope na Nikon (SMZ1500) tare da kyamarar DigiLite da software na TCapture (Tucsen Photonics, China) Hotuna20 sun sami girman girman girman girman girman girman girman girman girman girman girman girman girman girman girman girman girman girman China. trachea), tare da dukan tsayin trachea wanda aka kwatanta mataki-mataki, yana tabbatar da isasshen daidaituwa tsakanin kowane hoto don ba da damar yin amfani da hoton "stitching". Rubutun MATLAB (R2020a, MathWorks) kamar yadda aka bayyana a baya, ta amfani da saitunan 0.35
Kowane trachea an saka shi a cikin paraffin kuma an yanke sassan μm 5. An ƙirƙira sassan tare da ja mai tsaka tsaki mai sauri don 5 min kuma an samo hotuna ta amfani da microscope Nikon Eclipse E400, kyamarar DS-Fi3 da software na daukar nauyin NIS (version 5.20.00).
An yi amfani da duk ƙididdigar ƙididdiga a cikin GraphPad Prism v9 (GraphPad Software, Inc.) . An saita mahimmancin ƙididdiga a p ≤ 0.05. An tabbatar da al'ada ta amfani da gwajin Shapiro-Wilk, kuma an kiyasta bambance-bambance a cikin LacZ tabo ta amfani da t-gwajin da ba a haɗa ba.
An yi nazarin MPs shida da aka kwatanta a cikin Table 1 ta amfani da PCXI, kuma an kwatanta hangen nesa a cikin Table 2. Biyu polystyrene MPs (MP1 da MP2; 18 μm da 0.25 μm, bi da bi) ba a bayyane a karkashin PCXI ba, amma sauran samfurori sun kasance masu ganewa (misali an nuna su a cikin Hoto 5% 5 (MP3.5-5) μm da 0.9 μm, bi da bi) suna suma bayyane.Ko da yake dauke da wasu daga cikin kananan barbashi gwada, MP5 (98% Fe3O4; 0.25 μm) shi ne mafi pronounced.The CombiMag samfurin MP6 yana da wuya a tabo. A duk lokuta, mu ikon gane MP aka muhimmanci inganta da fassara da maganadisu da koma baya. capillary, barbashi mika cikin dogon kirtani, amma kamar yadda maganadiso samu kusa da Magnetic filin ƙarfi ƙara, barbashi kirtani taqaitaccen kamar yadda barbashi yi hijira zuwa saman surface na capillary (duba Ƙarin Video S1: MP4), ƙara The barbashi yawa na surface.Conversely, lokacin da maganadiso da aka cire daga cikin capillary mai tsawo restrings da tsawo restringing MP4. saman saman capillary (duba Ƙarin Bidiyo S2: MP4) .Bayan maganadisu ya daina motsi, barbashi suna ci gaba da motsawa na ɗan gajeren lokaci bayan sun kai matsayin ma'auni. Yayin da MP ke motsawa zuwa sama da nisa daga saman saman capillary, ƙwayoyin maganadisu yawanci suna jan tarkace ta cikin ruwa.
Ganuwa na MP a karkashin PCXI ya bambanta sosai tsakanin samfurori.(a) MP3, (b) MP4, (c) MP5 da (d) MP6. Duk hotunan da aka nuna a nan an ɗauke su tare da magnet wanda yake kusan 10 mm kai tsaye a sama da capillary. Babban da'irori na fili shine kumfa mai iska a cikin capillaries, wanda ke nuna alamar baƙar fata da fari mai banƙyama wanda ke nuna bambanci na launi na lokaci-lokaci. Diamita na ƙirar maganadisu a cikin dukkan lambobi ba za su yi girma ba kuma sun fi girma kusan sau 100 fiye da yadda aka nuna.
Yayin da aka fassara maganadisu hagu da dama tare da saman capillary, kusurwar kirtan MP yana canzawa don daidaitawa tare da magnet (duba Hoto 6), don haka yana nuna layin filin maganadisu.Domin MP3-5, bayan da maƙarƙashiya ya kai kusurwar bakin kofa, ana jan barbashi tare da saman saman capillary. Wannan sau da yawa yana haifar da mafi girma a cikin MPs apples a kusa da gungu na bidiyo zuwa ga mafi girma filin filin bidiyo. S3: MP5) Wannan kuma yana bayyana musamman lokacin da hoton kusa da ƙarshen capillary, wanda ke haifar da 'yan majalisa su tara da kuma mai da hankali a cikin yanayin iska mai iska. Barbashi a cikin MP6, waɗanda suka fi wahalar ganewa fiye da MP3-5, ba a jawo su yayin da magnet ke motsawa tare da capillary, amma igiyoyin MP sun rabu, suna barin filin wasan bidiyo. lokuta, lokacin da filin maganadisu da aka yi amfani da shi ya ragu ta hanyar matsar da maganadisu mai nisa mai nisa daga wurin hoto, duk wasu 'yan majalisar da suka rage a hankali suna gangarowa zuwa ƙasan bututu ta nauyi yayin da suke cikin kirtani (duba Ƙarin Bidiyo S5: MP3).
Ƙaƙwalwar igiyar MP tana canzawa yayin da aka fassara maganadisu zuwa dama sama da capillary.(a) MP3, (b) MP4, (c) MP5 da (d) MP6. Akwatin ja yana ƙunshe da haɓakar haɓakawa.
Gwaje-gwajenmu sun nuna cewa motsi da maganadisu sannu a hankali baya da gaba tare da trachea yana sauƙaƙe hangen nesa na MP a cikin mahallin hadaddun motsi a cikin vivo.In vivo gwajin ba a yi ba kamar yadda polystyrene beads (MP1 da MP2) ba a bayyane a cikin capillary.Kowane daga cikin sauran MPs hudu da aka gwada a cikin vivo tare da magnet dogon axis saita sama da trachea na kusa da 0 Figures a kan wani 0 Figures. 3a), saboda wannan ya haifar da dogon sarƙoƙi na MP kuma ya fi tasiri fiye da ƙayyadaddun ƙayyadaddun maganadisu.MP3, MP4 da MP6 ba a gano su ba a cikin trachea na kowane dabbobi masu rai. Lokacin da aka kwatanta hanyoyin iska na bera bayan an kashe dabbobin da mutuntaka, barbashi ya kasance marar ganuwa ko da lokacin da aka ƙara ƙarar ƙarar ta amfani da famfon sirinji.MP5e yana da mafi girman abun ciki kuma an yi amfani da oxidize don haka kawai an yi amfani da oxidize don haka an yi amfani da oxidize mafi girma kuma an yi amfani da sinadarin baƙin ƙarfe don yin famfo. a vivo hali na MP.
Sanya maganadisu akan trachea yayin isar da MP5 ya haifar da da yawa, amma ba duka ba, 'yan majalisar suna mai da hankali a fagen hangen nesa. Barbasa da ke shiga cikin bututun an fi lura da su a cikin dabbobin da aka sadaukar da su. Hoto na 7 da Ƙarin Bidiyo S6: MP5 yana nuna saurin kamawar maganadisu da daidaitawar barbashi a saman fage na ventral trachea, lokacin da MP5 ke nuna yanayin da ake so. distally tare da trachea bayan MP bayarwa, an sami wasu 'yan majalisar kusa da carina, suna nuna cewa ƙarfin filin maganadisu bai isa ba don tattarawa da riƙe duk 'yan majalisar, yayin da aka isar da su ta yankin mafi girman ƙarfin filin maganadisu yayin aiwatar da ruwa.
Hotuna daga (a) kafin da (b) bayan isar da MP5 a cikin trachea na bera da aka kashe kwanan nan tare da magnet da aka sanya kai tsaye a sama da wurin hoto. Wurin da aka kwatanta yana tsakanin zoben guringuntsi guda biyu. Kafin isar da MP, akwai wani ruwa a cikin iska. Akwatin ja yana ƙunshe da haɓakar haɓakawa.
Fassara maganadisu tare da trachea a cikin vivo ya sa sarkar MP ta canza kusurwa a cikin filin jirgin sama kamar yadda aka gani a cikin capillaries (duba Hoto na 8 da Ƙarin Bidiyo S7: MP5) .Duk da haka, a cikin bincikenmu, 'yan majalisa ba za a iya jawo su tare da saman filin jirgin sama ba kamar yadda za su iya tare da capillaries. yana bayyana yana canza zurfin saman Layer ruwa lokacin da maganadisu ke motsawa a tsaye tare da trachea, kuma yana faɗaɗa lokacin da magnet ɗin ke motsa kai tsaye kuma aka juya zaren barbashi zuwa matsayi a tsaye (duba Ƙarin Bidiyo S7). : MP5 a 0:09, kasa dama).Halayen halayen motsi sun canza lokacin da aka fassara magnet a saman saman trachea a kaikaice (wato, hagu ko dama na dabba maimakon tare da tsayin trachea) . Barbashi sun kasance a bayyane a fili yayin da suke motsawa, amma lokacin da aka cire magnet daga trachea, tukwici na barbashi na trachea ya zama bayyane a fili a Spplement. 0: 08) Wannan ya yi daidai da halin MP da muka lura a ƙarƙashin filin maganadisu da aka yi amfani da shi a cikin gilashin gilashi.
Hotunan misalin da ke nuna MP5 a cikin trachea na bera mai raye-raye.(a) Ana amfani da maganadisu don samun hotuna a sama da hagu na trachea, sannan (b) bayan magnet ɗin an motsa shi zuwa dama. Akwatin ja yana ƙunshe da haɓakar haɓakawa. Waɗannan hotuna suna daga bidiyon da aka nuna a Ƙarin Bidiyo S7: MP5.
Lokacin da aka saita sandunan biyu a cikin fuskantar arewa-kudu a sama da ƙasa da trachea (watau jan hankali; Hoto 3b), ƙwararrun MP sun bayyana tsawon lokaci kuma suna kan gefen bangon trachea maimakon a kan dorsal tracheal surface (duba Ƙarin Bidiyo S9: MP5) .Duk da haka, babban taro na barbashi a wuri guda (watau ruwa ba a gano shi ba a wuri guda). An yi amfani da na'ura mai dual-magnet, wanda yawanci yakan faru ne lokacin da ake amfani da na'ura mai mahimmanci guda ɗaya. Sannan lokacin da aka saita magnet guda ɗaya don korar sandunan ya juya (Fig. 3c), adadin ƙwayoyin da ake gani a cikin filin kallo bai bayyana ya karu ba bayan bayarwa. a layi daya zuwa hanyar iska amma wucewa ta hanyar iska a digiri 90 don haka layin filin sun ƙetare bangon tracheal orthogonally (Fig. 3d), wani tsari wanda aka tsara don ƙayyade idan za'a iya lura da haɗuwar ƙwayar cuta a kan bangon gefe. 3a) an zaba don nazarin jigilar jigilar kwayoyin halitta a cikin vivo.
Lokacin da aka maimaita hoton dabbar nan da nan bayan kisan ɗan adam, rashin motsin nama mai ruɗani yana nufin cewa za a iya gane layukan barbashi mafi ƙanƙanta da guntu a cikin fili na tsaka-tsakin tsaka-tsakin, “mai raɗaɗi” daidai da motsin fassarar maganadisu. Duk da haka , har yanzu ba zai iya gani a fili gaban da motsi na MP6 barbashi.
LV-LacZ titer ya kasance 1.8 × 108 TU / ml, kuma bayan 1: 1 haɗuwa tare da CombiMag MP (MP6), dabbobi sun sami nauyin 50 μl na tracheal na 9 × 107 TU / ml LV (watau 4.5 × 106 TU / bera). .A cikin waɗannan karatun, maimakon fassarar maganadisu a lokacin aiki, mun gyara magnet a wuri ɗaya don sanin ko za a iya inganta aikin LV (a) idan aka kwatanta da isar da vector a cikin rashin filin maganadisu, kuma (b) za a iya mayar da hankali ga ƙwayoyin Airway ana canza su zuwa yankunan da aka yi amfani da su na magnetic na babban jirgin sama.
Kasancewar maganadisu da kuma yin amfani da CombiMag hade tare da LV vectors bai bayyana a yi mummunan tasiri a kan lafiyar dabba ba, kamar yadda tsarin mu na LV vector bayarwa ya yi. Hotunan gaba na yankin tracheal da aka yi wa lalacewa na inji (Ƙarin Fig. 1) ya nuna cewa akwai matakan da suka fi girma a cikin rukuni na dabbobin da aka bi da su tare da ƙananan adadin LV-MP).n. LacZ tabo ya kasance a cikin ƙungiyar kulawa (Fig. 9b) Ƙididdigar wuraren da aka tsara na X-Gal na al'ada ya nuna cewa gudanarwa na LV-MP a gaban filin magnetic ya samar da haɓakar kusan 6-ninka (Fig. 9c).
Misalin hotuna masu haɗaka da ke nuna fassarar tracheal ta LV-MP (a) a gaban filin maganadisu da (b) a cikin rashin magnet.
Sassan ja-ja-jaja masu saurin tsaka tsaki (misali da aka nuna a Ƙarin Fig. 2) ya nuna ƙwayoyin LacZ da ke cikin irin wannan tsari da wuri kamar yadda aka ruwaito a baya.
Mahimmin ƙalubalen don maganin ƙwayar cutar ta iska ya kasance daidaitaccen yanki na sassan masu ɗaukar kaya zuwa yankuna masu sha'awa da kuma samun babban matakan haɓakawa a cikin huhu mai motsi a gaban iska mai iska da kuma cirewa mai aiki. Ga masu ɗaukar kaya na LV da aka tsara don magance cutar ta CF, ƙara yawan lokacin zama na kwayoyin halitta a cikin hanyoyin jiragen sama sun kasance manufa ta hanyar amfani da iska. Magnetic filayen don inganta transduction yana da abũbuwan amfãni idan aka kwatanta da sauran gene isar da hanyoyin kamar electroporation, kamar yadda zai iya hada sauki, kudin-tasiri, bayarwa localization, ƙãra yadda ya dace, da kuma guntu shiryawa sau , kuma yiwu a karami m dose10.Duk da haka, da a cikin vivo deposition da hali na Magnetic barbashi a cikin iska, kuma ba a zahiri ya bayyana irin ƙarfin lantarki a cikin hanyar iska ba, kamar yadda sojojin da aka bayyana a zahiri. nunawa a cikin vivo don haɓaka matakan maganganun kwayoyin halitta a cikin ingantattun hanyoyin iska.
Gwajin mu na in vitro synchrotron PCXI ya nuna cewa duk barbashi da muka gwada, ban da polystyrene MP, suna bayyane a cikin saitin hoton da muka yi amfani da su. Filin tserewa na gida.Thesesesesan gaba suna haifar da wasu barbashi iri ɗaya don tara da haɗuwa, tare da kusurwoyi na rukuni-rukuni saboda mahaɗan wasu barbashi na gida.
Ƙwayoyin da ke nuna (a,b) jiragen ƙasa da aka samar a cikin ruwa mai cike da ruwa da (c,d) mai cike da iska. Lura cewa ba a zana capillaries da trachea zuwa sikelin.Panel (a) yana ƙunshe da bayanin MP, wanda ya ƙunshi nau'in Fe3O4 da aka shirya a cikin igiyoyi.
Lokacin da maganadisu ya motsa sama da capillary, kwana na barbashi kirtani ya kai wani m bakin kofa ga MP3-5 dauke da Fe3O4, bayan da barbashi kirtani daina zama a cikin asali matsayi, amma koma tare da surface zuwa wani sabon matsayi.magnet.Wannan sakamako ne mai yiwuwa ya faru saboda gilashin capillary surface ne santsi isa ya ba da damar wannan motsi ya faru. da barbashi sun kasance karami, yana da daban-daban coatings ko surface zargin, ko mallaki m ruwa ya shafi su ikon motsi.The image bambanci na CombiMag barbashi ne kuma mai rauni, bayar da shawarar cewa ruwa da barbashi na iya samun irin wannan densities sabili da haka ba sauƙi matsawa zuwa juna. Barbashi kuma iya samun makale idan da wani m m ruwa ya shafi su ikon motsi.The image bambanci na CombiMag barbashi ne kuma mai rauni, bayar da shawarar cewa ruwa da barbashi na iya samun irin wannan yawa kuma sabili da haka ba sauki matsawa zuwa juna. Ba abin mamaki ba cewa Magnetic filin ƙarfi da nisa tsakanin maganadisu da manufa yankin Very muhimmanci.Take tare, wadannan sakamakon kuma bayar da shawarar cewa, yayin da maganadiso iya kama da yawa MPs cewa gudãna ta cikin manufa yankin, shi ne mai wuya cewa maganadiso za a iya dogara a kan matsar CombiMag barbashi tare da surface na trachea.Saboda haka, mun kammala cewa a cikin vivo LV-MP binciken ya kamata a yi amfani da takamaiman igiyoyin iska zuwa filin jirgin sama na sararin samaniya.
Lokacin da barbashi da aka tsĩrar a cikin jiki, suna da wuya a gane a cikin mahallin na hadaddun motsi jiki nama, amma da ikon gano su da aka inganta ta hanyar fassara da maganadisu a kwance sama da trachea zuwa "juyawa" da MP kirtani. Ko da yake live Hoto yana yiwuwa, yana da sauki don gane barbashi motsi da zarar dabba da aka mutunta mutunta kashe.MP taro yawanci ya fi girma a wannan wuri, ko da yake da particle aka samu mafi girma a wannan wuri a lokacin da wani bangare na magana da aka samu a sama da wani wuri a sama da wani bangare na hoton. trachea.Ya bambanta da nazarin in vitro, ba za a iya jan barbashi tare da trachea ta hanyar fassarar magnet. Wannan binciken ya yi daidai da yadda ƙwayar da ke rufe saman trachea yakan tafiyar da kwayoyin halitta, yana kama su a cikin ƙumburi kuma daga bisani ya share su ta hanyar tsarin cirewa na mucociliary.
Mun yi hasashe cewa yin amfani da maganadisu don jan hankali a sama da ƙasa da trachea (Fig. 3b) na iya haifar da filin maganadisu iri ɗaya, maimakon filin maganadisu wanda ke da hankali sosai a lokaci ɗaya, wanda zai iya haifar da ƙarin rarraba nau'ikan ƙwayoyin cuta. Duk da haka, binciken mu na farko bai sami tabbataccen shaida don tallafawa wannan hasashe ba. Barbashi shaida a cikin imaged area.These biyu binciken nuna cewa dual-magnet saitin ba ya muhimmanci inganta gida iko na MP niyya, da kuma cewa sakamakon karfi Magnetic sojojin da wuya a daidaita, yin wannan hanya m m. Hakazalika, orienting da maganadiso a sama da kuma ta hanyar trachea (Fig. 3d) Har ila yau, bai ƙara yawan barbashi na iya zama mai nasara a cikin yankin da aka ci gaba da yin amfani da su. Ƙarfin filin maganadisu a cikin wurin ajiya.Saboda haka, saitin maganadisu na kusurwa 30-digiri guda ɗaya (Figure 3a) ana ɗaukar hanya mafi sauƙi kuma mafi inganci don gwajin vivo.
Nazarin LV-MP ya nuna cewa lokacin da aka haɗu da vectors na LV tare da CombiMag kuma an ba da su bayan ɓarna ta jiki a gaban filin magnetic, matakan transduction sun karu sosai a cikin trachea idan aka kwatanta da sarrafawa.Based a kan nazarin hotunan synchrotron da sakamakon LacZ, filin magnetic ya bayyana a fili yana iya adana LV a cikin trachea kuma ya rage adadin abubuwan da ke cikin ɓarna nan da nan. na iya haifar da inganci mafi girma yayin da rage ƙwanƙwasa titers, fassarar kashe-manufa, cututtuka masu kumburi da rigakafi, da kuma farashin jigilar kwayoyin halitta.Mahimmanci, bisa ga masana'anta, CombiMag za a iya amfani da shi tare da sauran hanyoyin canja wurin kwayoyin halitta, ciki har da sauran ƙwayoyin cuta (irin su AAV) da kuma nucleic acid.
Lokacin aikawa: Yuli-16-2022
