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Dole ne a yi niyya da jigilar kwayoyin halitta don maganin cystic fibrosis na huhu zuwa hanyoyin iska, tun da canjin huhu na gefe ba shi da wani tasiri na warkewa.Ingancin fassarar hoto yana da alaƙa kai tsaye da lokacin zama na mai ɗaukar hoto.Koyaya, ruwan isarwa kamar masu ɗaukar kwayoyin halitta a zahiri suna yaɗuwa cikin alveoli yayin shakar numfashi, kuma ana cire ɓangarorin warkewa na kowane nau'i da sauri ta hanyar jigilar mucociliary.Tsawaita lokacin zama na masu jigilar kwayoyin halitta a cikin numfashi yana da mahimmanci amma yana da wahala a samu.Ƙwayoyin maganadisu masu haɗaɗɗiyar ɗaukar kaya waɗanda za a iya kai su zuwa saman fili na numfashi na iya inganta niyya na yanki.Saboda matsalolin da ke tattare da in vivo imaging, halayen irin waɗannan ƙananan ƙwayoyin maganadisu a saman filin iska a gaban filin maganadisu ba a fahimta sosai.Manufar wannan binciken shine a yi amfani da hoton synchrotron don gani a cikin vivo motsi na jerin abubuwan maganadisu a cikin trachea na berayen anesthetized don yin nazarin motsin rai da tsarin halayen ɓangarorin guda da girma a cikin vivo.Daga nan kuma mun tantance ko isar da ƙwayoyin maganadisu na lentiviral a gaban filin maganadisu zai ƙara haɓakar watsawa a cikin trachea na bera.Hoto na X-ray na Synchrotron yana nuna halayen ƙwayoyin maganadisu a tsaye da motsin filayen maganadisu a cikin vitro da in vivo.Ba za a iya jawo barbashi cikin sauƙi a sararin samaniyar hanyoyin iska ta hanyar amfani da maganadisu ba, amma yayin jigilar kayayyaki, ajiya suna tattarawa a fagen kallo, inda filin maganadisu ya fi ƙarfi.Hakanan an ƙara haɓaka aikin canzawa sau shida lokacin da aka isar da barbashi na maganadisu na lentiviral a gaban filin maganadisu.A hade, waɗannan sakamakon suna ba da shawarar cewa ƙwayoyin maganadisu na lentiviral da filayen maganadisu na iya zama hanyoyi masu mahimmanci don haɓaka haɓakar ƙwayoyin halitta da matakan watsawa a cikin hanyoyin iska a cikin vivo.
Cystic fibrosis (CF) yana haifar da bambance-bambance a cikin kwayar halitta guda ɗaya da ake kira CF transmembrane conductance regulator (CFTR).Sunan furotin na CFTR shine tashar ion da ke samuwa a cikin yawancin kwayoyin epithelial a ko'ina cikin jiki, ciki har da hanyoyin iska, babban wuri a cikin pathogenesis na cystic fibrosis.Rashin lahani a cikin CFTR yana haifar da jigilar ruwa mara kyau, rashin ruwa na filin jirgin sama, da rage zurfin ruwan saman sararin sama (ASL).Hakanan yana lalata ikon tsarin jigilar mucociliary (MCT) don share hanyoyin iska daga abubuwan da aka shaka da ƙwayoyin cuta.Manufarmu ita ce haɓaka ƙwayar ƙwayar ƙwayar cuta (LV) don sadar da daidaitaccen kwafin kwayar halittar CFTR da inganta ASL, MCT, da lafiyar huhu, da kuma ci gaba da haɓaka sabbin fasahohin da za su iya auna waɗannan sigogi a cikin vivo1.
LV vectors suna ɗaya daga cikin manyan ƴan takara don maganin ƙwayoyin cuta na cystic fibrosis, musamman saboda suna iya haɗa kwayar cutar ta har abada cikin ƙwayoyin basal na iska (airway stem cell).Wannan yana da mahimmanci saboda suna iya mayar da hydration na al'ada da ƙyallen ƙura ta hanyar bambancewa cikin sel masu aikin gyaran gyare-gyare na iska mai aiki tare da cystic fibrosis, yana haifar da fa'idodi na rayuwa.Dole ne a karkatar da vectors na LV akan hanyoyin iska, saboda anan ne ake fara shigar huhu a cikin CF.Isar da vector mai zurfi a cikin huhu na iya haifar da fassarar alveolar, amma wannan ba shi da tasirin warkewa a cikin cystic fibrosis.Duk da haka, ruwaye irin su masu ɗaukar kwayoyin halitta suna yin ƙaura a cikin alveoli lokacin da aka shayar da su bayan haihuwa3,4 kuma ana fitar da kwayoyin warkewa da sauri a cikin rami na baka ta hanyar MCTs.Ingancin fassarar LV yana da alaƙa kai tsaye da tsawon lokacin da vector ya kasance kusa da sel da aka yi niyya don ba da damar ɗaukar wayar salula - "lokacin zama" 5 wanda aka rage shi cikin sauƙi ta hanyar yanayin iska na yanki na yau da kullun da kuma haɗaɗɗun ƙwayar tsoka da ƙwayoyin MCT.Ga cystic fibrosis, ikon tsawaita lokacin zama na LV a cikin hanyoyin iska yana da mahimmanci don cimma manyan matakan watsawa a wannan yanki, amma ya zuwa yanzu yana da ƙalubale.
Don shawo kan wannan matsala, muna ba da shawarar cewa barbashi na maganadisu na LV (MPs) na iya taimakawa ta hanyoyi guda biyu.Na farko, ana iya jagorantar su ta hanyar maganadisu zuwa filin jirgin sama don inganta niyya da kuma taimakawa kwayoyin jigilar kwayoyin halitta su kasance a cikin yankin da ya dace na hanyar iska;da ASL) matsawa cikin cell Layer 6. Ana amfani da MPs sosai azaman motocin isar da magunguna da aka yi niyya lokacin da suke ɗaure ƙwayoyin rigakafi, magungunan chemotherapy, ko wasu ƙananan ƙwayoyin cuta waɗanda ke haɗe zuwa membranes tantanin halitta ko ɗaure ga masu karɓar saman tantanin su kuma suna taruwa a wuraren ƙari kasancewar wutar lantarki a tsaye.Filayen Magnetic don maganin ciwon daji 7. Sauran hanyoyin "hyperthermic" ana nufin kashe ƙwayoyin tumor ta hanyar dumama 'yan majalisa lokacin da aka fallasa su zuwa filayen maganadisu.Ka'idar jigilar maganadisu, wacce ake amfani da filin maganadisu azaman wakili mai canzawa don haɓaka canja wurin DNA cikin sel, ana amfani da su a cikin vitro galibi ta amfani da kewayon ƙwayoyin ƙwayoyin cuta waɗanda ba na hoto ko bidiyo mai zagaya yanar gizo ba don wahala-da-canza layin salula. ..Ingancin LV magnetotransfection tare da isar da LV MP in vitro a cikin layin tantanin halitta na epithelium na ɗan adam a gaban filin maganadisu na tsaye an kafa shi, yana haɓaka haɓakar haɓakawa ta sau 186 idan aka kwatanta da vector LV kaɗai.An kuma yi amfani da LV MT zuwa samfurin in vitro na cystic fibrosis, inda motsin maganadisu ya karu da jigilar LV a cikin al'adun mu'amalar ruwa ta hanyar 20 a gaban cystic fibrosis sputum10.Koyaya, a cikin vivo sashin jiki magnetotransfection ya sami kulawa kaɗan kuma an kimanta shi a cikin ƴan binciken dabbobi11,12,13,14,15, musamman a cikin huhu16,17.Koyaya, yuwuwar jigilar maganadisu a cikin maganin huhu a cikin cystic fibrosis a bayyane yake.Tan et al.(2020) ya bayyana cewa "binciken tabbatarwa kan ingantaccen isar da huhu na magnetic nanoparticles zai ba da hanya don dabarun inhalation na CFTR na gaba don inganta sakamakon asibiti a cikin marasa lafiya da cystic fibrosis"6.
Halin ƙananan ƙwayoyin maganadisu a saman filin numfashi a gaban filin maganadisu da aka yi amfani da shi yana da wuyar gani da nazari, don haka ba a fahimce su ba.A cikin wasu nazarin, mun haɓaka hanyar haɓakawa ta Synchrotron Based Phase Contrast X-Ray Imaging (PB-PCXI) don hoton mara amfani da ƙididdigewa na minti a cikin vivo canje-canje a cikin zurfin ASL18 da halayyar MCT19,20 don auna iskar gas kai tsaye hydration surface. kuma ana amfani dashi azaman mai nuna alamar farkon tasirin magani.Bugu da kari, hanyar mu ta maki MCT tana amfani da barbashi diamita na 10-35 µm da suka hada da alumina ko babban gilashin mai nuna alama kamar alamun MCT da ake iya gani tare da PB-PCXI21.Dukansu hanyoyin sun dace don ɗaukar nau'ikan nau'ikan barbashi, gami da 'yan majalisa.
Saboda babban ƙuduri na sararin samaniya da na ɗan lokaci, ƙididdigar mu na PB-PCXI ASL da MCT sun dace sosai don nazarin yanayin kuzari da halayen halayen guda ɗaya da girma a cikin vivo don taimaka mana fahimta da haɓaka hanyoyin isar da gene na MP.Hanyar da muke amfani da ita a nan ta dogara ne akan karatunmu ta amfani da SPring-8 BL20B2 beamline, wanda muke ganin motsin ruwa bayan isar da kashi na ƙwayar cuta a cikin hanci da na huhu na iska na beraye don taimakawa wajen bayyana yanayin bayyanar cututtuka daban-daban da aka lura. a cikin halittarmu.nazarin dabba tare da kashi mai ɗaukar nauyin 3.4 .
Manufar wannan binciken shine a yi amfani da synchrotron na PB-PCXI don gani a cikin vivo motsi na jerin 'yan majalisa a cikin trachea na berayen.Wadannan nazarin hoto na PB-PCXI an tsara su don gwada jerin MP, ƙarfin filin maganadisu, da wuri don ƙayyade tasirin su akan motsin MP.Mun ɗauka cewa filin maganadisu na waje zai taimaka wa MF ɗin da aka kawo ta tsaya ko matsawa zuwa wurin da aka yi niyya.Waɗannan karatun kuma sun ba mu damar tantance daidaitawar maganadisu waɗanda ke haɓaka adadin barbashi da suka rage a cikin bututun bayan an ajiye su.A cikin jerin karatu na biyu, mun yi nufin amfani da wannan kyakkyawan tsari don nuna tsarin canji wanda ya samo asali daga isar da vivo na LV-MPs zuwa hanyoyin iska na bera, bisa tsammanin cewa isar da LV-MPs a cikin mahallin niyya ta iska zai haifar da hakan. a cikin haɓakar haɓakawar LV..
Dukkan nazarin dabba an gudanar da su daidai da 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 gudanar da gwaje-gwajen bisa ga shawarwarin ARRIVE.
An ɗauki duk hotunan x-ray a BL20XU beamline a SPring-8 synchrotron a Japan ta amfani da saitin kama da wanda aka bayyana a baya21,22.A taƙaice, akwatin gwajin yana da nisan mita 245 daga zoben ajiya na synchrotron.Ana amfani da nisan samfurin-zuwa-gane na 0.6 m don nazarin hotunan barbashi da 0.3 m don nazarin hoto a cikin vivo don ƙirƙirar tasirin bambancin lokaci.An yi amfani da katako na monochromatic tare da makamashi na 25 keV.Hotunan an samo su ta amfani da babban na'urar transducer X-ray (SPring-8 BM3) haɗe da na'urar gano sCMOS.Mai fassara yana jujjuya radiyon X zuwa haske mai iya gani ta amfani da scintillator mai kauri 10 µm (Gd3Al2Ga3O12), wanda sannan a tura shi zuwa firikwensin sCMOS ta amfani da maƙasudin microscope ×10 (NA 0.3).Mai ganowa na sCMOS shine Orca-Flash4.0 (Hamamatsu Photonics, Japan) mai girman jeri na 2048 × 2048 pixels da ɗanyen girman pixel 6.5 × 6.5 µm.Wannan saitin yana ba da ingantaccen girman pixel isotropic na 0.51 µm da filin kallo na kusan 1.1 mm × 1.1 mm.An zaɓi tsawon lokacin bayyanar 100 ms don ƙara girman sigina-zuwa-amo na barbashi maganadisu a ciki da wajen hanyoyin iska yayin da ake rage abubuwan motsi da numfashi ya haifar.Don a cikin nazarin vivo, an sanya murfin X-ray mai sauri a cikin hanyar X-ray don iyakance adadin radiation ta hanyar toshe katako na X-ray tsakanin abubuwan da aka bayyana.
Ba a yi amfani da kafofin watsa labarai na LV ba a cikin kowane nazarin hoto na SPring-8 PB-PCXI saboda ɗakin hoton BL20XU ba ƙwararren Matsayin Biosafety 2 ba ne.Madadin haka, mun zaɓi nau'ikan ƙwararrun ƴan majalisar wakilai daga masu siyar da kasuwanci guda biyu waɗanda ke rufe nau'ikan nau'ikan girma, kayan aiki, ƙarancin ƙarfe, da aikace-aikace, - na farko don fahimtar yadda filayen magnetic ke shafar motsi na MPs a cikin capillaries na gilashi, sannan kuma a ciki. hanyoyin iska masu rai.saman.Girman MP ya bambanta daga 0.25 zuwa 18 µm kuma an yi shi daga abubuwa daban-daban (duba Table 1), amma abun da ke tattare da kowane samfurin, gami da girman ƙwayar maganadisu a cikin MP, ba a sani ba.Dangane da faffadan karatunmu na MCT 19, 20, 21, 23, 24, muna sa ran za a iya ganin 'yan majalisar kasa har zuwa 5 µm a saman filin jirgin sama na tracheal, misali, ta hanyar cire firam a jere don ganin ingantacciyar ganin motsin MP.MP guda ɗaya na 0.25 µm ya fi ƙanƙanta da ƙudurin na'urar hoto, amma ana sa ran PB-PCXI zai gano bambancin girman su da motsin ruwan saman da aka ajiye su bayan an ajiye su.
Samfurori ga kowane MP a cikin tebur.An shirya 1 a cikin 20 μl gilashin capillaries (Drummond Microcaps, PA, USA) tare da diamita na ciki na 0.63 mm.Ana samun barbashi na jiki a cikin ruwa, yayin da abubuwan CombiMag suna samuwa a cikin ruwa na mallakar masana'anta.Kowane bututu ya cika rabin ruwa (kimanin 11 µl) kuma an sanya shi akan mariƙin samfurin (duba hoto 1).An sanya capillaries gilashin a kwance a kan mataki a cikin ɗakin hoto, bi da bi, kuma an sanya su a gefuna na ruwa.Diamita na 19 mm (tsawon mm 28) nickel-shell magnet wanda aka yi da ƙasa mai wuya, neodymium, baƙin ƙarfe da boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Australia) tare da ragowar 1.17 T an haɗa shi zuwa keɓan tebur canja wuri don cim ma Canza matsayinku Daga nisa yayin nunawa.Hoton X-ray yana farawa lokacin da maganadisu ya kasance kusan 30 mm sama da samfurin kuma ana samun hotuna a firam 4 a sakan daya.A lokacin daukar hoto, an kawo magnet kusa da bututun capillary gilashi (a nesa na kusan 1 mm) sannan kuma ya motsa tare da bututu don tantance tasirin ƙarfin filin da matsayi.
Saitin hoton in vitro mai ɗauke da samfuran MP a cikin capillaries na gilashi a matakin fassarar samfurin xy.Hanya na katakon X-ray yana da alamar ja jajayen layi.
Da zarar an kafa ganin in vitro na 'yan majalisar, an gwada wani bangare na su a cikin vivo akan berayen Wistar albino na mace na daji (~ makonni 12, ~ 200 g).Medetomidine 0.24 mg/kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg/kg (Dormicum®, Astellas Pharma, Japan) da butorphanol 4 mg/kg (Vetorphale®, Meiji Seika).An kwantar da berayen tare da cakuda Pharma (Japan) ta allurar intraperitoneal.Bayan maganin sa barci, an shirya su don yin hoto ta hanyar cire gashin gashi a kusa da trachea, shigar da bututun endotracheal (ET; 16 Ga intravenous cannula, Terumo BCT), da kuma hana su a cikin matsayi na baya a kan farantin hoto na al'ada wanda ya ƙunshi jakar zafi. don kula da zafin jiki.22. Sa'an nan kuma an haɗa farantin hoto zuwa matakin samfurin a cikin akwatin hoto a wani ɗan kusurwa don daidaita trachea a kwance akan hoton x-ray kamar yadda aka nuna a cikin Hoto 2a.
(a) A cikin saitin hoto na vivo a cikin sashin hoto na SPring-8, hanyar katako na X-ray mai alama da layin ja mai digo.(b,c) An gudanar da gano yanayin maganan tracheal daga nesa ta amfani da kyamarorin IP guda biyu da aka ɗora su.A gefen hagu na hoton akan allon, zaku iya ganin madauki na waya yana riƙe da kai da cannula isar da aka shigar a cikin bututun ET.
Tsarin famfo mai sarrafa sirinji mai nisa (UMP2, Instruments Precision World, Sarasota, FL) ta amfani da sirinji 100 µl gilashin an haɗa shi da bututun PE10 (0.61 mm OD, ID na 0.28 mm) ta amfani da allura 30 Ga.Alama bututu don tabbatar da cewa tip ɗin yana cikin matsayi daidai a cikin trachea lokacin shigar da bututun endotracheal.Yin amfani da micropump, an cire plunger ɗin sirinji kuma an nutsar da titin bututun a cikin samfurin MP don isar da shi.An shigar da bututun isar da kaya a cikin bututun endotracheal, sanya tip a mafi ƙarfi na filin maganadisu da ake sa ran mu.An sarrafa sayan hoto ta amfani da na'urar gano numfashi da aka haɗa zuwa akwatin lokaci na tushen Arduino, kuma duk sigina (misali, zafin jiki, numfashi, buɗewa / rufewa, da siyan hoto) an yi rikodin su ta amfani da Powerlab da LabChart (AD Instruments, Sydney, Australia) 22 Lokacin da Hoto Lokacin da babu mahalli, kyamarorin IP guda biyu (Panasonic BB-SC382) an sanya su a kusan 90 ° zuwa juna kuma ana amfani da su don sarrafa matsayi na maganadisu dangane da trachea yayin hoto (Hoto 2b, c).Don rage kayan tarihi na motsi, an sami hoto ɗaya a kowane numfashi yayin tudun numfashi na ƙarshe.
Magnet ɗin yana haɗe zuwa mataki na biyu, wanda zai iya kasancewa a nesa daga wajen jikin hoton.An gwada wurare daban-daban da kuma daidaitawar maganadisu, ciki har da: an sanya shi a kusurwar kusan 30 ° sama da trachea (an nuna saiti a cikin Figures 2a da 3a);daya maganadisu a saman dabba da sauran a kasa, tare da sanduna da aka saita don jan hankali (Hoto 3b)., magnet guda ɗaya a sama da dabba da ɗaya a ƙasa, tare da sandunan da aka saita don tunkudewa (Hoto 3c), da magnet guda ɗaya a sama kuma daidai da trachea (Figure 3d).Bayan saita dabba da maganadisu da loda MP a ƙarƙashin gwaji a cikin famfon sirinji, isar da kashi 50 µl a ƙimar 4 µl/sec akan samun hotuna.Ana matsar da maganadisu baya da baya tare ko ƙetare trachea yayin da ake ci gaba da samun hotuna.
Tsarin Magnet don in vivo Hoto (a) Magnet ɗaya sama da trachea a kusurwar kusan 30 °, (b) Magnet guda biyu da aka saita don jan hankali, (c) Magnet guda biyu da aka saita don tunkuɗewa, (d) magnet guda ɗaya a sama kuma daidai gwargwado ga trachea.Mai kallo ya kalli kasa daga baki zuwa huhu ta hanyar bututun iska sannan hasken X-ray ya bi ta bangaren hagu na bera ya fita bangaren dama.Ana matsar da maganadisu tare da tsawon hanyar iska ko hagu da dama sama da trachea zuwa hanyar katakon X-ray.
Mun kuma nemi sanin ganuwa da halayen barbashi a cikin hanyoyin iska idan babu gaurayawan numfashi da bugun zuciya.Sabili da haka, a ƙarshen lokacin hoton, dabbobi sun kasance masu lalata da mutuntaka saboda yawan adadin pentobarbital (Somnopentyl, Pitman-Moore, Washington Crossing, Amurka; ~ 65 mg/kg ip).An bar wasu dabbobi a kan dandalin hotunan, kuma bayan dakatarwar numfashi da bugun zuciya, an sake maimaita tsarin hoton, yana ƙara ƙarin adadin MP idan babu MP a kan filin iska.
Hotunan da aka gyara an gyara su don filin lebur da duhu 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).
An gudanar da duk nazarin akan isar da vector na LV a Cibiyar Nazarin Dabbobi ta Jami'ar Adelaide Laboratory Animal Research Center kuma da nufin yin amfani da sakamakon gwajin SPring-8 don tantance ko isar da LV-MP a gaban filin maganadisu na iya haɓaka jigilar kwayoyin halitta a cikin vivo. .Don kimanta tasirin MF da filin maganadisu, an bi da ƙungiyoyi biyu na dabbobi: an yi wa rukuni ɗaya allura tare da LV MF tare da sanya magnet, kuma ɗayan ƙungiyar an allura tare da ƙungiyar kulawa tare da LV MF ba tare da maganadisu ba.
An samar da kwayoyin halittar LV ta hanyar amfani da hanyoyin da aka bayyana a baya 25, 26.LacZ vector yana bayyana kwayar halittar beta-galactosidase ta nukiliya ta hanyar MPSV mai haɓakawa (LV-LacZ), wanda ke samar da samfurin amsa shuɗi a cikin ƙwayoyin da aka canza, bayyane a gaba da sassan nama na huhu.An yi titration a cikin al'adun tantanin halitta ta hanyar kirga adadin sel masu kyau na LacZ da hannu ta amfani da hemocytometer don ƙididdige titer a TU/ml.Ana adana masu ɗaukar kaya a -80 ° C, narke kafin amfani, kuma an ɗaure su zuwa CombiMag ta hanyar haɗawa 1: 1 da cuba akan kankara na akalla mintuna 30 kafin bayarwa.
Al'ada Sprague Dawley berayen (n = 3 / rukuni, ~ 2-3 anesthetized ip tare da cakuda 0.4mg/kg medetomidine (Domitor, Ilium, Ostiraliya) da 60mg/kg ketamine (Ilium, Australia) a 1 watan shekaru) ip ) allura da ƙwanƙolin baka wanda ba na tiyata ba tare da 16 Ga cannula na ciki.Don tabbatar da cewa nama na iska na tracheal ya karbi LV transduction, an yi amfani da shi ta hanyar amfani da ka'idar rikice-rikice na inji wanda aka kwatanta a baya wanda aka shafe filin jirgin sama na tracheal axially tare da kwandon waya (N-Circle, nitinol dutse mai cirewa ba tare da tip NTSE-022115) -UDH , Cook Medical, Amurka) 30 p28.Sa'an nan, kamar minti 10 bayan tashin hankali a cikin majalisar kula da lafiyar halittu, an gudanar da aikin tracheal na LV-MP.
Filin maganadisu da aka yi amfani da shi a cikin wannan gwaji an daidaita shi daidai da binciken in vivo x-ray, tare da maganadisu iri ɗaya da aka riƙe a saman trachea tare da ƙuƙumman stent (Hoto 4).An isar da ƙarar 50 µl (2 x 25 µl aliquots) na LV-MP zuwa trachea (n = 3 dabbobi) ta amfani da pipette mai gel-tipped kamar yadda aka bayyana a baya.Ƙungiyar sarrafawa (n = 3 dabbobi) sun sami LV-MP iri ɗaya ba tare da amfani da maganadisu ba.Bayan kammala jiko, an cire cannula daga bututun endotracheal kuma an fitar da dabba.Magnet ɗin yana nan a wurin na tsawon mintuna 10 kafin a cire shi.An yi amfani da berayen a cikin ƙasa tare da meloxicam (1 ml/kg) (Ilium, Ostiraliya) sannan an cire maganin sa barci ta hanyar allurar intraperitoneal na 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia).An kiyaye berayen da dumi kuma ana lura da su har sai an dawo da su gaba ɗaya daga maganin sa barci.
LV-MP na'urar isarwa a cikin ma'ajin aminci na halitta.Kuna iya ganin cewa hannun rigar Luer-lock mai haske na bututun ET yana fitowa daga baki, kuma tip ɗin gel pipette da aka nuna a cikin adadi an saka shi ta cikin bututun ET zuwa zurfin da ake so a cikin trachea.
Mako guda bayan tsarin gudanarwa na LV-MP, an yi hadaya da dabbobi cikin mutuntaka ta hanyar shakar 100% CO2 kuma an tantance furcin LacZ ta amfani da daidaitaccen jiyyarmu ta X-gal.An cire mafi yawan zoben guringuntsi guda uku don tabbatar da cewa duk wani lahani na inji ko riƙewar ruwa saboda sanya bututun endotracheal ba za a haɗa shi cikin bincike ba.An yanke kowace trachea tsawon tsayi don samun rabi biyu don bincike kuma an sanya shi a cikin kofi mai dauke da siliki (Sylgard, Dow Inc) ta amfani da allurar Minutien (Kayan Aikin Kimiyya mai Kyau) don ganin fuskar haske.An tabbatar da rarrabawa da halayen sel da aka canza ta hanyar daukar hoto ta gaba ta amfani da microscope na Nikon (SMZ1500) tare da kyamarar DigiLite da software na TCapture (Tucsen Photonics, China).An samo hotuna a girman girman 20x (ciki har da madaidaicin saiti don cikakken nisa na trachea), tare da tsawon tsayin trachea wanda aka nuna mataki-mataki, yana ba da isasshen haɗuwa tsakanin kowane hoto don ba da damar hotuna su zama "dike".Hotunan daga kowace trachea an haɗa su zuwa hoto guda ɗaya ta amfani da Sigar Editan Hoto Mai Haɗin 2.0.3 (Binciken Microsoft) ta amfani da algorithm motsi na planar. An ƙididdige yanki na bayanin LacZ a cikin hotunan da aka haɗar tracheal daga kowace dabba ta amfani da rubutun MATLAB mai sarrafa kansa (R2020a, MathWorks) kamar yadda aka bayyana a baya28, ta amfani da saitunan 0.35 <Hue <0.58, Saturation> 0.15, da ƙimar <0.7. An ƙididdige yanki na bayanin LacZ a cikin hotuna masu haɗakarwa daga kowace dabba ta amfani da rubutun MATLAB mai sarrafa kansa (R2020a, MathWorks) kamar yadda aka bayyana a baya28, ta amfani da saitunan 0.35 <Hue <0.58, Saturation> 0.15, da ƙimar <0.7. Площадь экспрессии LacZ в составных изображениях трахеи от каждого животного была количественно определена с использованием автоматизированного сценария MATLAB (R2020a, MathWorks), как описано ранее28, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0 ,7. An ƙididdige yankin bayanin LacZ a cikin tarin hotunan tracheal daga kowace dabba ta amfani da rubutun MATLAB mai sarrafa kansa (R2020a, MathWorks) kamar yadda aka bayyana a baya28 ta amfani da saitunan 0.350.15 da darajar <0.7.如 前所, 自动 Malaab 脚本 (R2020a, Matworks) 对 来自 图像 中 中 中 中 中 中 的 的 中> 0.15 和值> 0.15 和值 <和值. 0.15 和值 <的.如 自动, 自动 Mattab 脚本 ((R2020a, Matworks) 来自来自 色调每 的 色调每 的 气管复合 的 气管每 的 气管复合 的 气管复合 的 气管复合 的 气管每表达 的 气管复合 的 气管复合表达 的 气管复合 的 气管复合 的使用………………………………………… Области экспрессии LacZ на составных изображениях трахеи каждого животного количественно определяли с использованием автоматизированного сценария MATLAB (R2020a, MathWorks), как описано ранее, с использованием настроек 0,35 <оттенок <0,58, насыщенность> 0,15 и значение <0,7 . An ƙididdige wuraren maganganun LacZ akan abubuwan da aka haɗa na trachea na kowace dabba ta amfani da rubutun MATLAB mai sarrafa kansa (R2020a, MathWorks) kamar yadda aka bayyana a baya ta amfani da saitunan 0.35 <hue <0.58, jikewa> 0.15 da ƙimar <0.7.Ta hanyar bin diddigin nama a cikin GIMP v2.10.24, an ƙirƙiri abin rufe fuska da hannu don kowane hoto mai haɗaka don gano yankin nama da hana duk wani gano ƙarya a waje da nama na tracheal.Wuraren da aka tabo daga dukkan hotuna masu haɗaka daga kowace dabba an tattara su don ba da jimillar yanki na dabbar.Sannan an raba yankin fentin ta wurin jimlar abin rufe fuska don samun yanki mai daidaitacce.
An saka kowace bututun ruwa a cikin paraffin kuma an raba shi cikin kauri 5 µm.An ƙirƙira sassan da ja mai sauri na mintuna 5 kuma an samo hotuna ta amfani da microscope Nikon Eclipse E400, kyamarar DS-Fi3 da software na kama abubuwan NIS (sigar 5.20.00).
An yi duk ƙididdigar ƙididdiga a cikin GraphPad Prism v9 (GraphPad Software, Inc.).An saita mahimmancin ƙididdiga a p ≤ 0.05.An gwada al'ada ta amfani da gwajin Shapiro-Wilk da bambance-bambance a cikin launi na LacZ an kimanta ta amfani da t-gwajin da ba a haɗa ba.
'Yan majalisar shida da aka kwatanta a cikin Table 1 PCXI sun bincikar su, kuma an kwatanta hangen nesa a cikin Table 2. MPs polystyrene guda biyu (MP1 da MP2; 18 µm da 0.25 µm, bi da bi) ba su ganuwa ta PCXI, amma sauran samfurori za a iya gano su. (misali ana nuna su a hoto na 5).MP3 da MP4 suna da rauni a bayyane (10-15% Fe3O4; 0.25 µm da 0.9 µm, bi da bi).Kodayake MP5 (98% Fe3O4; 0.25 µm) ya ƙunshi wasu ƙananan ƙwayoyin da aka gwada, ita ce mafi yawan furci.Samfurin CombiMag MP6 yana da wuyar ganewa.A kowane hali, ikonmu na gano MFs ya inganta sosai ta hanyar matsar da maganadisu baya da gaba daidai da capillary.Yayin da maganadisu ke nisa daga capillary, an fitar da barbashi cikin dogayen sarkoki, amma yayin da maganadisu ke gabatowa kuma ƙarfin filin maganadisu ya ƙaru, sassan sassan jikin sun ragu yayin da barbashi ke ƙaura zuwa saman saman capillary (duba Ƙarin Bidiyo S1). : MP4), ƙara yawan ƙwayar barbashi a saman.Akasin haka, lokacin da aka cire maganadisu daga capillary, ƙarfin filin yana raguwa kuma ’yan majalisar suna sake tsarawa zuwa dogayen sarƙoƙi waɗanda ke shimfiɗa daga saman saman capillary (duba Ƙarin Bidiyo S2: MP4).Bayan maganadisu ya daina motsi, ɓangarorin suna ci gaba da motsawa na ɗan lokaci bayan sun kai matsayin ma'auni.Yayin da MP ɗin ke motsawa zuwa da nesa daga saman saman capillary, ɓangarorin maganadisu sukan jawo tarkace ta cikin ruwa.
Ganin MP a ƙarƙashin PCXI ya bambanta sosai tsakanin samfuran.(a) MP3, (b) MP4, (c) MP5 da (d) MP6.Dukkanin hotuna da aka nuna anan an ɗauke su tare da maganadisu da aka sanya kusan mm 10 kai tsaye sama da capillary.Manyan da'irar da aka bayyana su ne kumfa na iska da aka makale a cikin capillaries, suna nuna a fili baƙar fata da fari na sifofin yanayin yanayin yanayin.Akwatin ja yana nuna haɓakawa wanda ke haɓaka bambanci.Lura cewa diamita na da'irorin 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 maganadisu ke motsawa hagu da dama tare da saman capillary, kusurwar kirtan MP tana canzawa don daidaitawa da maganadisu (duba Hoto 6), don haka yana zayyana layin filin maganadisu.Don MP3-5, bayan maƙarƙashiyar ta isa kusurwar ƙofar, barbashi suna ja tare da saman saman capillary.Wannan sau da yawa yana haifar da ƴan majalisa suna taruwa zuwa manyan ƙungiyoyi kusa da inda filin maganadisu ya fi ƙarfi (duba Ƙarin Bidiyo S3: MP5).Wannan kuma yana bayyana musamman lokacin da ake yin hoto kusa da ƙarshen capillary, wanda ke sa MP ɗin ya tattara kuma ya mai da hankali a wurin haɗin ruwa-iska.Barbashi a cikin MP6, waɗanda ke da wahalar bambancewa fiye da waɗanda ke cikin MP3-5, ba su ja lokacin da magnet ɗin ke motsawa tare da capillary ba, amma igiyoyin MP sun rabu, suna barin barbashi a gani (duba Ƙarin Bidiyo S4: MP6).A wasu lokuta, lokacin da filin maganadisu da aka yi amfani da shi ya ragu ta hanyar matsar da maganadisu mai nisa mai nisa daga wurin hoton, duk wasu 'yan majalisar da suka rage a hankali suna gangarowa zuwa ƙasan bututu da nauyi, suna cikin kirtani (duba Ƙarin Bidiyo S5: MP3) .
Matsakaicin kirtan MP yana canzawa yayin da maganadisu ke motsawa zuwa dama sama da capillary.(a) MP3, (b) MP4, (c) MP5 da (d) MP6.Akwatin ja yana nuna haɓakawa wanda ke haɓaka bambanci.Lura cewa ƙarin bidiyon don dalilai ne na bayanai yayin da suke bayyana mahimman tsari na ɓangarorin da bayanai masu ƙarfi waɗanda ba za a iya gani a cikin waɗannan hotuna masu tsayi ba.
Gwaje-gwajenmu sun nuna cewa motsi da maganadisu baya da gaba a hankali tare da trachea yana sauƙaƙe hangen nesa na MF a cikin mahallin hadaddun motsi a cikin vivo.Ba a yi gwajin in vivo ba saboda ba a iya ganin beads na polystyrene (MP1 da MP2) a cikin capillary.Kowane ɗayan MF guda huɗu da suka rage an gwada su a cikin vivo tare da doguwar axis na maganadisu da aka sanya a kan trachea a kusurwar kusan 30 ° zuwa tsaye (duba Figures 2b da 3a), saboda wannan ya haifar da sarƙoƙin MF masu tsayi kuma ya fi tasiri. fiye da maganadisu..daidaitawa ya ƙare.MP3, MP4 da MP6 ba a sami su a cikin bututun kowace dabba mai rai ba.A lokacin da ake hango hanyoyin numfashi na berayen bayan kashe dabbobi ta hanyar mutuntaka, barbashi sun kasance ba a ganuwa ko da an ƙara ƙarin ƙara ta amfani da famfon sirinji.MP5 yana da mafi girman abun ciki na baƙin ƙarfe oxide kuma shine ɓangarorin da ake iya gani kawai, don haka an yi amfani da shi don kimantawa da siffanta halayen MP a vivo.
Sanya magnet akan trachea yayin shigar da MF ya haifar da da yawa, amma ba duka ba, MFs sun mai da hankali a fagen kallo.An fi lura da shigar barbashi na tracheal a cikin dabbobin da ba su da lafiya.Hoto na 7 da Ƙarin Bidiyo S6: MP5 yana nuna saurin kama maganadisu da daidaita ɓangarorin a saman bututun iska, yana nuni da cewa za a iya niyya 'yan majalisar zuwa wuraren da ake so na trachea.Lokacin da ake bincika mafi nisa tare da trachea bayan isar da MF, an sami wasu MFs kusa da carina, wanda ke nuna ƙarancin filin maganadisu don tattarawa da riƙe duk MFs, tunda an isar da su ta yankin mafi girman ƙarfin filin maganadisu yayin gudanar da ruwa.tsari.Koyaya, yawan adadin MP na bayan haihuwa ya kasance mafi girma a kusa da wurin hoton, yana nuna cewa 'yan majalisar da yawa sun kasance a yankunan hanyar iska inda ƙarfin maganadisu ya fi girma.
Hotunan (a) kafin da (b) bayan isar da MP5 a cikin bututun bera da aka kashe kwanan nan tare da maganadisu da aka sanya a saman wurin hoto.Wurin da aka kwatanta yana tsakanin zoben cartilaginous guda biyu.Akwai wani ruwa a cikin hanyoyin iska kafin a kai MP.Akwatin ja yana nuna haɓakawa wanda ke haɓaka bambanci.Ana ɗaukar waɗannan hotuna daga bidiyon da aka nuna a cikin S6: MP5 Ƙarin Bidiyo.
Matsar da maganadisu tare da trachea a cikin vivo ya haifar da canji a kusurwar sarkar MP a saman filin jirgin sama, kamar wanda aka gani a cikin capillaries (duba Hoto 8 da Ƙarin Bidiyo S7: MP5).Koyaya, a cikin bincikenmu, 'yan majalisar ba za a iya jan su a saman hanyoyin numfashi masu rai ba, kamar yadda capillaries ke iya yi.A wasu lokuta, sarkar MP tana tsayi yayin da magnet ke motsawa hagu da dama.Abin sha'awa shine, mun kuma gano cewa sarkar barbashi tana canza zurfin saman saman ruwan lokacin da magnet ɗin ke motsawa a tsayi tare da trachea, kuma yana faɗaɗa lokacin da magnet ɗin ya motsa kai tsaye sama da sarkar barbashi yana jujjuya zuwa matsayi a tsaye (duba. Ƙarin Bidiyo S7).MP5 a 0:09, kasa dama).Halin yanayin motsi ya canza lokacin da magnet ɗin ya motsa a gefe a saman saman trachea (watau zuwa hagu ko dama na dabba, maimakon tare da tsawon trachea).Barbashin sun kasance a bayyane a fili yayin motsinsu, amma lokacin da aka cire maganadisu daga trachea, tukwici na zaren barbashi ya zama bayyane (duba Ƙarin Bidiyo S8: MP5, farawa daga 0:08).Wannan ya yarda da halayen da aka lura na filin maganadisu ƙarƙashin aikin filin maganadisu da aka yi amfani da shi a cikin capillary gilashi.
Hotunan samfurin da ke nuna MP5 a cikin trachea na bera mai rai.(a) Ana amfani da magnet don samun hotuna a sama da hagu na trachea, sannan (b) bayan motsa magnet zuwa dama.Akwatin ja yana nuna haɓakawa wanda ke haɓaka bambanci.Waɗannan hotunan suna daga bidiyon da aka nuna a cikin Ƙarin Bidiyo na S7: MP5.
Lokacin da aka daidaita sandunan biyu a cikin fuskantar arewa-kudu a sama da ƙasa da trachea (watau jan hankali; Hoto 3b), ƙwanƙolin MP ɗin ya bayyana ya fi tsayi kuma suna kan bangon gefen trachea maimakon a saman dorsal na ƙofofin. trachea (duba Karin Bayani).Bidiyo S9: MP5).Duk da haka, ba a gano yawan adadin barbashi a wuri ɗaya (watau saman bayan bututun ruwa) ba bayan gudanar da ruwa ta amfani da na'urar maganadisu biyu, wanda yawanci yana faruwa da na'urar maganadisu ɗaya.Sa'an nan, lokacin da aka saita magnet guda ɗaya don tunkuɗe igiyoyi masu adawa (Hoto na 3c), adadin barbashi da ake gani a fagen kallo bai ƙaru ba bayan bayarwa.Ƙirƙirar saiti biyu na maganadisu yana da ƙalubale saboda babban ƙarfin filin maganadisu wanda ke jan hankali ko tura maganadisu bi da bi.Daga nan sai aka canza saitin zuwa magnet guda ɗaya daidai da hanyoyin iska amma yana wucewa ta hanyoyin iska a kusurwar digiri 90 ta yadda layin ƙarfin ya ketare bangon tracheal bisa ka'ida (Hoto 3d), fuskantarwa wanda aka yi niyya don tantance yiwuwar tara abubuwan da ke kan gaba. bangon gefe.a kiyaye.Koyaya, a cikin wannan tsarin, babu wani motsi na tarawa na MF ko motsin maganadisu.Dangane da duk waɗannan sakamakon, an zaɓi daidaitawa tare da maganadisu ɗaya da madaidaicin digiri na 30 don nazarin vivo na masu jigilar kwayoyin halitta (Fig. 3a).
Lokacin da aka yi hoton dabbar sau da yawa nan da nan bayan an yi hadaya ta mutumtaka, rashin tsoma baki motsi na nama yana nufin cewa mafi ƙarancin, gajeriyar layukan barbashi za a iya gane su a cikin fili tsakanin cartilaginous fili, 'swaying' daidai da motsin fassarar maganadisu.gani a fili gaban da motsi na MP6 barbashi.
Matsakaicin adadin LV-LacZ shine 1.8 x 108 IU/mL, kuma bayan haɗe 1:1 tare da CombiMag MP (MP6), an yiwa dabbobi allura da 50 µl na ƙwayar tracheal na 9 x 107 IU/ml na motar LV (watau 4.5). x 106 TU/rat).)).A cikin waɗannan karatun, maimakon motsa maganadisu yayin aiki, mun gyara maganadisu a wuri ɗaya don sanin ko za a iya haɓakawa na LV (a) idan aka kwatanta da isar da vector idan babu filin maganadisu, da (b) idan hanyar iska zata iya. a mai da hankali.Kwayoyin da ake canza su a cikin wuraren da ake nufi da maganadisu na sashin numfashi na sama.
Kasancewar maganadiso da kuma amfani da CombiMag a haɗe tare da ɓangarorin LV bai bayyana suna yin illa ga lafiyar dabbobi ba, kamar yadda ƙa'idar isar da kayan aikin mu ta LV ta yi.Hotunan gaba na yankin tracheal da aka fuskanci matsala ta injiniya (Ƙarin Hoto 1) sun nuna cewa ƙungiyar da aka yi wa LV-MP tana da matakan da suka fi girma a gaban magnet (Fig. 9a).Ƙaramin ƙananan launin shuɗi LacZ ya kasance a cikin ƙungiyar kulawa (Hoto 9b).Ƙididdigar yankunan da aka saba da su na X-Gal sun nuna cewa gudanar da LV-MP a gaban filin maganadisu ya haifar da haɓaka kusan sau 6 (Fig. 9c).
Misalin hotuna masu haɗaka da ke nuna fassarar tracheal tare da LV-MP (a) a gaban filin maganadisu da (b) in babu maganadisu.(c) Ƙididdigar ƙididdiga mai mahimmanci a cikin al'ada na fassarar LacZ a cikin trachea tare da yin amfani da magnet (* p = 0.029, t-test, n = 3 a kowace ƙungiya, yana nufin ± daidaitaccen kuskure na ma'ana).
Sassan ja-ja-jaja masu saurin tsaka-tsaki (misali da aka nuna a Ƙarin Fig. 2) ya nuna cewa ƙwayoyin LacZ da aka lalata sun kasance a cikin samfurin guda ɗaya kuma a wuri ɗaya kamar yadda aka ruwaito a baya.
Babban ƙalubalen a cikin jiyya ta hanyar iska ya kasance daidaitaccen yanki na ɓangarori masu ɗaukar hoto a cikin wuraren da ake sha'awa da kuma samun nasarar babban matakin haɓakawa a cikin huhu ta hannu a gaban kwararar iska da cirewar gamsai mai aiki.Ga masu ɗaukar LV waɗanda aka yi niyya don maganin cututtukan numfashi a cikin cystic fibrosis, haɓaka lokacin zama na barbashi mai ɗaukar hoto a cikin hanyoyin iska ya kasance makasudin da ba za a iya cimma ba.Kamar yadda Castellani et al ya nuna, yin amfani da filayen maganadisu don haɓaka transduction yana da fa'ida akan sauran hanyoyin isar da kwayoyin halitta kamar su electroporation saboda yana iya haɗa sauƙi, tattalin arziki, isar da gida, haɓaka haɓakawa, da ɗan gajeren lokacin shiryawa.kuma mai yiwuwa ƙananan adadin abin hawa10.Duk da haka, a cikin vivo ƙaddamarwa da halayen ƙwayoyin maganadisu a cikin hanyoyin iska a ƙarƙashin tasirin ƙarfin maganadisu na waje ba a taɓa yin bayanin ba, kuma a gaskiya ba a nuna ikon wannan hanyar don ƙara matakan bayyanar da kwayoyin halitta a cikin madaidaicin hanyoyin iska ba a cikin vivo.
Gwaje-gwajenmu na in vitro akan PCXI synchrotron ya nuna cewa duk barbashi da muka gwada, ban da MP polystyrene, suna bayyane a cikin saitin hoto da muka yi amfani da su.A gaban filin maganadisu, filayen maganadisu suna yin kirtani, tsawonsu yana da alaƙa da nau'in barbashi da ƙarfin filin maganadisu (watau kusanci da motsi na maganadisu).Kamar yadda aka nuna a hoto na 10, igiyoyin da muke gani suna samuwa yayin da kowane ɓangarorin mutum ya zama magnetized kuma yana haifar da filin maganadisu na gida.Waɗannan ɓangarorin daban-daban suna haifar da wasu ɓangarorin makamantansu don tattarawa da haɗawa da motsin kirtani na rukuni saboda sojojin gida daga rundunonin jan hankali da ture wasu barbashi.
Zane yana nuna (a, b) sarƙoƙi na barbashi da ke samuwa a cikin masu cike da ruwa da (c, d) mai cike da iska.Lura cewa ba a zana capillaries da trachea zuwa sikelin.Panel (a) kuma ya ƙunshi bayanin MF mai ɗauke da barbashi Fe3O4 da aka shirya cikin sarƙoƙi.
Lokacin da maganadisu ya motsa a kan capillary, kusurwar kirtani na barbashi ya kai matsayi mai mahimmanci don MP3-5 mai dauke da Fe3O4, bayan haka igiyar barbashi ba ta ci gaba da kasancewa a matsayinsa na asali ba, amma ya motsa tare da saman zuwa sabon matsayi.maganadisu.Wataƙila wannan tasirin yana faruwa saboda saman gilashin capillary yana da santsi don ƙyale wannan motsi ya faru.Abin sha'awa shine, MP6 (CombiMag) ba ya yin wannan hanya, watakila saboda barbashi sun kasance ƙanana, suna da nau'i daban-daban ko cajin saman, ko kuma ruwan jigilar kaya ya shafi ikon motsi.Bambanci a cikin hoton barbashi na CombiMag shima ya fi rauni, yana nuni da cewa ruwa da barbashi na iya samun yawa iri daya sabili da haka ba sa iya matsawa juna cikin sauki.Har ila yau, barbashi za su iya makale idan magnet ɗin yana motsawa da sauri, yana nuna cewa ƙarfin maganadisu ba zai iya shawo kan rikice-rikice tsakanin barbashi a cikin ruwa koyaushe ba, yana nuna cewa ƙarfin filin maganadisu da nisa tsakanin maganadisu da wurin da ake niyya bai kamata ya zo a matsayin mamaki.muhimmanci.Wadannan sakamakon sun kuma nuna cewa ko da yake maganadiso na iya kama da yawa microparticles da ke gudana ta wurin da aka yi niyya, yana da wuya a iya dogara da maganadisu don motsa barbashin CombiMag tare da saman trachea.Don haka, mun yanke shawarar cewa a cikin vivo LV binciken MF yakamata ya yi amfani da filayen maganadisu na tsaye don kai hari ga takamaiman wuraren bishiyar iska.
Da zarar an isar da ɓangarorin cikin jiki, suna da wahalar ganewa a cikin mahallin hadadden ƙwayar motsi na jiki, amma an inganta ƙarfin gano su ta hanyar matsar da maganadisu a kwance akan trachea don “juya” igiyoyin MP.Yayin da hoto na ainihi zai yiwu, yana da sauƙi don gane motsin barbashi bayan an kashe dabbar ta hanyar mutuntaka.Matsakaicin MP ya kasance mafi girma a wannan wurin lokacin da aka sanya magnet akan wurin hoto, kodayake ana samun wasu barbashi a ƙasan trachea.Ba kamar nazarin in vitro ba, ba za a iya jan barbashi ƙasa da trachea ta motsin maganadisu ba.Wannan binciken ya yi daidai da yadda ɗigon da ke rufe saman trachea yakan aiwatar da ɓangarorin shaƙa, yana kama su a cikin gamji sannan ya share su ta hanyar hanyar cire muco-ciliary.
Mun yi hasashe cewa yin amfani da maganadisu a sama da ƙasa da trachea don jan hankali (Fig. 3b) zai iya haifar da ƙarin filin maganadisu iri ɗaya, maimakon filin maganadisu wanda ke da hankali sosai a lokaci ɗaya, mai yuwuwar haifar da rarraba iri ɗaya na barbashi..Duk da haka, bincikenmu na farko bai sami tabbataccen hujja da za ta goyi bayan wannan hasashe ba.Hakazalika, saita nau'ikan maganadisu don tunkuɗewa (Fig. 3c) bai haifar da ƙarin daidaitawar barbashi a cikin hoton ba.Wadannan binciken guda biyu sun nuna cewa saitin-magnet mai dual-magnet baya inganta kulawar gida na nunin MP, kuma cewa sakamakon karfi da karfin maganadisu yana da wahalar kunnawa, yana mai da wannan hanyar ba ta da amfani.Hakazalika, daidaita ma'aunin maganadisu a sama da ko'ina cikin trachea (Hoto na 3d) shima bai kara adadin barbashi da suka rage a wurin da aka zana ba.Wasu daga cikin waɗanan madadin jeri na ƙila ba za su yi nasara ba saboda suna haifar da raguwa a cikin ƙarfin filin maganadisu a cikin yankin ajiya.Don haka, saitin maganadisu ɗaya a digiri 30 (Fig. 3a) ana ɗaukarsa mafi sauƙi kuma mafi inganci a cikin hanyar gwaji ta vivo.
Binciken na LV-MP ya nuna cewa lokacin da aka haɗu da vectors na LV tare da CombiMag kuma an ba da su bayan an damu da jiki a gaban filin maganadisu, matakan watsawa ya karu sosai a cikin trachea idan aka kwatanta da sarrafawa.Dangane da nazarin hotunan synchrotron da sakamakon LacZ, filin maganadisu ya bayyana yana iya kiyaye LV a cikin trachea kuma ya rage adadin ƙwayoyin vector waɗanda nan da nan suka shiga zurfi cikin huhu.Irin wannan ci gaban da aka yi niyya na iya haifar da ingantaccen aiki yayin da ake rage masu isar da titers, fassarar da ba a yi niyya ba, illa masu kumburi da rigakafi, da farashin canja wurin kwayoyin halitta.Mahimmanci, bisa ga masana'anta, CombiMag za a iya amfani da su a hade tare da sauran hanyoyin canja wurin kwayoyin halitta, ciki har da sauran ƙwayoyin cuta (irin su AAV) da kuma nucleic acid.