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Ya kamata a yi amfani da kwayoyin halittar da ke haifar da cutar huhu ta cystic fibrosis wajen magance hanyoyin numfashi domin fitar da huhu daga huhu ba ya samar da fa'ida ta magani. Ingancin fitar da kwayar cutar yana da alaƙa kai tsaye da lokacin zama a vector. Duk da haka, ruwan isarwa kamar masu ɗauke da kwayar halitta a zahiri yana yaɗuwa cikin alveoli yayin wahayi, kuma ƙwayoyin magani na kowane nau'i ana share su da sauri ta hanyar jigilar mucociliary. Tsawaita lokacin zama na masu ɗauke da kwayar halitta a cikin hanyoyin numfashi yana da mahimmanci amma yana da wahalar cimmawa. Ƙwayoyin maganadisu masu ɗauke da kwayar halitta waɗanda za a iya tura su zuwa saman hanyoyin numfashi na iya inganta maƙasudin yanki. Saboda ƙalubalen gani a cikin jiki, ba a fahimci halayen irin waɗannan ƙananan ƙwayoyin maganadisu a saman hanyoyin numfashi a gaban filin maganadisu da aka yi amfani da su ba. Manufar wannan binciken ita ce amfani da hoton synchrotron don hango motsin in vivo na jerin ƙwayoyin maganadisu a cikin trachea na beraye masu sa maye don bincika yanayin da tsarin halayen mutum da na babban ƙwayoyin cuta a cikin jiki. Sannan mun kuma tantance ko isar da ƙwayoyin maganadisu na lentiviral a gaban filin maganadisu zai ƙara ingancin transduction a cikin trachea na bera. Synchrotron Hoton X-ray yana nuna halayen ƙwayoyin maganadisu a cikin filayen maganadisu marasa motsi da marasa motsi a cikin vitro da kuma a cikin jiki. Ba za a iya jan ƙwayoyin cuta cikin sauƙi a saman hanyar iska mai rai tare da maganadisu ba, amma yayin jigilar su, ana tattara abubuwan da aka tara a cikin filin gani inda filin maganadisu ya fi ƙarfi. Hakanan ingancin watsawa ya ninka sau shida lokacin da aka kawo ƙwayoyin maganadisu na lentiviral a gaban filin maganadisu. Tare, waɗannan sakamakon sun nuna cewa ƙwayoyin maganadisu na lentiviral da filayen maganadisu na iya zama hanyoyi masu mahimmanci don inganta maƙasudin ƙwayoyin halitta da haɓaka matakan watsawa a cikin hanyar sadarwa ta hanyar rayuwa.
Ciwon cystic fibrosis (CF) yana faruwa ne sakamakon bambancin kwayar halitta guda ɗaya da ake kira CF transmembrane conductance regulator (CFTR). Sinadarin CFTR tashar ion ce da ke cikin ƙwayoyin epithelial da yawa a cikin jiki, gami da hanyoyin numfashi, babban wurin da ke haifar da cutar CF. Lalacewar CFTR tana haifar da jigilar ruwa mara kyau, bushewar saman hanyar iska da rage zurfin layin ruwan saman hanyar iska (ASL). Wannan kuma yana lalata ikon tsarin jigilar mucociliary (MCT) don share ƙwayoyin cuta da ƙwayoyin cuta da aka shaƙa daga hanyoyin iska. Manufarmu ita ce haɓaka maganin kwayar halitta ta lentiviral (LV) don isar da kwafin kwayar halittar CFTR daidai da inganta lafiyar ASL, MCT, da huhu, da kuma ci gaba da haɓaka sabbin fasahohi waɗanda ke iya auna waɗannan sigogi a cikin vivo1.
Vectors na LV suna ɗaya daga cikin manyan 'yan takarar maganin kwayoyin halittar iska ta CF, galibi saboda suna iya haɗa kwayar halittar magani cikin ƙwayoyin basal na iska (ƙwayoyin jijiyoyin iska). Wannan yana da mahimmanci saboda suna iya dawo da ruwa da kuma share majina ta hanyar bambanta su zuwa ƙwayoyin saman iska masu alaƙa da CF masu aiki, waɗanda ke haifar da fa'idodi na rayuwa. Ya kamata a mayar da vectors na LV zuwa ga hanyar iska mai gudana, domin a nan ne cutar huhu ta CF ta fara. Isarwa da vector cikin zurfi cikin huhu na iya haifar da transduction na alveolar, amma wannan ba shi da wani amfani na warkewa a cikin CF. Duk da haka, ruwa kamar masu ɗauke da kwayoyin halitta suna ƙaura zuwa alveoli bayan wahayi bayan haihuwa3,4 kuma ƙwayoyin magani suna ɓacewa cikin sauri zuwa cikin ramin baki ta hanyar MCT. Ingancin transduction na LV yana da alaƙa kai tsaye da tsawon lokacin da vector ɗin ya kasance kusa da ƙwayoyin da aka nufa don ba da damar ɗaukar ƙwayoyin halitta - "lokacin zama"5 - wanda sauƙin ragewa ta hanyar iskar iska ta yanki da kuma kama majina na barbashi da MCT. Ga CF, ikon tsawaita lokacin zama na LV a cikin hanyar iska yana da mahimmanci don cimma manyan matakan transduction a wannan yanki, amma har yanzu yana da ya kasance mai ƙalubale.
Domin shawo kan wannan cikas, muna ba da shawarar cewa ƙwayoyin maganadisu na LV (MPs) na iya taimakawa ta hanyoyi biyu masu dacewa. Da farko, ana iya shiryar da su ta hanyar maganadisu zuwa saman hanyar iska don inganta niyya da kuma taimaka wa ƙwayoyin da ke ɗauke da kwayoyin halitta su zauna a yankin hanyar iska da ake so; da kuma ASL) don matsawa zuwa matakin tantanin halitta 6. An yi amfani da MPs sosai a matsayin motocin isar da magunguna da aka yi niyya lokacin da suka ɗaure ga ƙwayoyin rigakafi, magungunan chemotherapeutic, ko wasu ƙananan ƙwayoyin halitta waɗanda ke haɗe da membranes na tantanin halitta ko kuma suka ɗaure ga masu karɓar saman tantanin halitta kuma suka taru a wuraren ƙari a gaban wutar lantarki mai tsauri. Filin Magnetic don Maganin Ciwon daji 7. Sauran dabarun "hyperthermal" suna nufin dumama MPs lokacin da suka fuskanci filayen maganadisu masu juyawa, ta haka ne suke lalata ƙwayoyin ƙari. Ka'idar transfection na magnetic, wanda ake amfani da filin maganadisu a matsayin wakilin transfection don haɓaka canja wurin DNA zuwa ƙwayoyin halitta, ana amfani da shi a cikin vitro ta amfani da nau'ikan vectors na kwayoyin halitta marasa ƙwayoyin cuta da ƙwayoyin cuta don layukan ƙwayoyin halitta masu wahalar transduce. An tabbatar da ingancin LV magnetotransfection, tare da isar da LV-MPs zuwa layin ƙwayoyin halittar bronchial na ɗan adam a gaban filin maganadisu mai tsauri, yana ƙara ingancin transduction sau 186 idan aka kwatanta da LV vector kaɗai. An kuma yi amfani da LV-MP ga samfurin CF na in vitro, inda transfection na magnetic ya ƙara transduction na LV a cikin al'adun haɗin gwiwa na iska da sau 20 a gaban sputum na CF10. Duk da haka, a cikin vivo magnetotransfection na gabobin jiki ba a sami kulawa sosai ba kuma an kimanta shi kawai a cikin wasu nazarin dabbobi 11,12,13,14,15, musamman a cikin huhu16,17. Duk da haka, damar da ake da ita na yin amfani da na'urar maganadisu a cikin maganin huhu na CF a bayyane take. Tan et al. (2020) sun bayyana cewa "binciken da aka yi na tabbatar da ingancin isar da na'urar maganadisu ta huhu zai share fagen dabarun shaƙa CFTR na gaba don inganta sakamakon asibiti ga marasa lafiya na CF"6.
Halayyar ƙananan ƙwayoyin maganadisu a saman iska a gaban filin maganadisu da aka yi amfani da shi yana da wahalar gani da nazari, don haka ba a fahimce su sosai ba. A wasu nazarin, mun ƙirƙiri hanyar amfani da fasahar X-ray mai kama da juna (PB-PCXI) don yin hangen nesa da ƙididdige canje-canje na minti a cikin zurfin ASL18 da MCT behavior19,20 ba tare da yin amfani da su ba don auna ruwan iskar gas kai tsaye a saman magudanar ruwa kuma ana amfani da shi azaman alamar farko ta ingancin magani. Bugu da ƙari, hanyar kimantawa ta MCT ɗinmu tana amfani da ƙwayoyin 10-35 µm diamita waɗanda aka haɗa da alumina ko gilashin mai haske mai yawa azaman alamun MCT da ake iya gani ta amfani da PB-PCXI21. Duk dabarun sun dace da ganin nau'ikan ƙwayoyin cuta iri-iri, gami da MP.
Saboda girman ƙudurin sarari da na ɗan lokaci, dabarun nazarin ASL da MCT na PB-PCXI sun dace sosai don bincika yanayin da yanayin halayen barbashi ɗaya da na babban jiki a cikin jiki don taimaka mana fahimtar da inganta dabarun isar da kwayoyin halitta na MP. Hanyar da muke amfani da ita a nan ta samo asali ne daga nazarinmu ta amfani da layin SPring-8 BL20B2, wanda muka hango motsin ruwa bayan isar da allurar vector a cikin hanyoyin hanci da huhu na beraye don taimakawa wajen bayyana yanayin bayyanar kwayoyin halitta marasa daidaituwa da aka lura a cikin nazarin dabbobi na maganin kwayoyin halitta namu 3,4.
Manufar wannan binciken ita ce amfani da synchrotron PB-PCXI don hango motsin in vivo na jerin MPs a cikin trachea na beraye masu rai. An tsara waɗannan nazarin hoton PB-PCXI don gwada nau'ikan MPs, ƙarfin filin maganadisu, da wurare don tantance tasirinsu akan motsin MP. Mun yi hasashen cewa filin maganadisu da aka yi amfani da shi a waje zai taimaka wa MP ɗin da aka kawo ya tsaya ko ya matsa zuwa yankin da aka nufa. Waɗannan nazarin sun kuma ba mu damar gano tsarin maganadisu wanda ke haɓaka adadin ƙwayoyin da aka riƙe a cikin trachea bayan an ajiye su. A cikin wani jerin bincike na biyu, mun yi ƙoƙarin amfani da wannan tsari mafi kyau don nuna tsarin transduction wanda ya samo asali daga isar da LV-MPs zuwa hanyar iska ta bera, bisa ga zato cewa isar da LV-MPs a cikin mahallin niyya ta hanyar iska zai haifar da ingantaccen aikin transduction na LV.
An gudanar da dukkan nazarin dabbobi bisa ga ka'idojin da Jami'ar Adelaide ta amince da su (M-2019-060 da M-2020-022) da Kwamitin Ɗabi'ar Dabbobin SPring-8 Synchrotron suka amince da su. An gudanar da gwaje-gwaje bisa ga ka'idojin ARRIVE.
An yi dukkan hotunan X-ray a layin haske na BL20XU a synchrotron na SPring-8 a Japan, ta amfani da saitin kama da wanda aka bayyana a baya21,22. A takaice, akwatin gwajin yana da nisan mita 245 daga zoben ajiya na synchrotron. Ana amfani da nisan samfurin-zuwa-na'urar ganowa na mita 0.6 don nazarin hotunan barbashi da kuma mita 0.3 don nazarin hotunan in vivo don samar da tasirin bambanci na lokaci. An yi amfani da kuzarin hasken monochromatic na 25 keV. An ɗauki hotuna ta amfani da mai canza hasken X-ray mai ƙuduri mai girma (SPring-8 BM3) wanda aka haɗa shi da mai gano sCMOS. Mai canza hasken yana canza hasken X zuwa haske mai gani ta amfani da mai cire haske mai kauri 10 µm (Gd3Al2Ga3O12), wanda daga nan aka tura shi zuwa firikwensin sCMOS ta amfani da maƙasudin microscope × 10 (NA 0.3). Mai gano sCMOS shine Orca-Flash4.0 (Hamamatsu Photonics, Japan) tare da girman jeri na 2048 × 2048 pixels da kuma girman pixel mai ƙarancin 6.5 × 6.5 µm. Wannan saitin yana samar da ingantaccen girman pixel isotropic na 0.51 µm da kuma filin kallon kusan 1.1 mm × 1.1 mm. An zaɓi tsawon fallasa na 100 ms don haɓaka rabon sigina-zuwa-amo na barbashi masu maganadisu a ciki da wajen hanyar iska yayin da ake rage tasirin motsin numfashi. Don nazarin a cikin vivo, an sanya rufe X-ray mai sauri a cikin hanyar X-ray don iyakance yawan radiation ta hanyar toshe hasken X-ray tsakanin fallasa.
Ba a yi amfani da na'urar ɗaukar hoto ta LV a cikin wani nazarin hotunan SPring-8 PB-PCXI ba saboda ɗakin daukar hoto na BL20XU ba shi da takardar shaidar Biosafety Level 2. Madadin haka, mun zaɓi nau'ikan 'yan majalisa masu kyau daga masu samar da kayayyaki biyu na kasuwanci - waɗanda suka ƙunshi girma dabam-dabam, kayan aiki, yawan ƙarfe, da aikace-aikace - da farko don fahimtar yadda filayen maganadisu ke shafar motsin MP a cikin ƙwayoyin gilashi, sannan kuma a cikin hanyoyin iska masu rai. a saman. Girman MPs ya bambanta daga 0.25 zuwa 18 μm kuma an yi su ne daga nau'ikan kayan aiki daban-daban (duba Tebur 1), amma ba a san abun da ke cikin kowane samfurin ba, gami da girman ƙwayoyin maganadisu a cikin MP. Dangane da nazarin MCT mai zurfi 19, 20, 21, 23, 24, muna tsammanin za a iya ganin MPs masu ƙanƙanta kamar 5 μm a saman hanyar iska ta tracheal, misali ta hanyar cire firam ɗin a jere don ganin ingantaccen ganuwa na motsi na MP. MP guda ɗaya mai girman 0.25 μm ya fi ƙanƙanta fiye da ƙudurin na'urar daukar hoto, amma ana sa ran PB-PCXI zai gano bambancin girman su da kuma motsin ruwan saman da aka ajiye su bayan an ajiye su.
An shirya samfura ga kowane MP a cikin Tebur 1 a cikin ƙananan gilashin μl 20 (Drummond Microcaps, PA, Amurka) tare da diamita na ciki na 0.63 mm. Ƙananan ƙwayoyin jiki suna samuwa a cikin ruwa, yayin da ƙananan ƙwayoyin CombiMag suna samuwa a cikin ruwan mallakar masana'anta. Kowace bututu an cika ta da ruwa (kimanin 11 μl) kuma an sanya ta a kan mariƙin samfurin (duba Hoto na 1). An sanya ƙananan ƙwayoyin gilashi a kwance a kan matakin samfurin a cikin akwatin hoto, bi da bi, kuma an sanya gefunan ruwan. An haɗa wani ƙarfe mai siffar nickel mai diamita 19 mm (tsawon 28 mm) mai siffar nickel mai siffar neodymium iron boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Ostiraliya) tare da ragowar magnetization na 1.17 Tesla zuwa wani mataki na fassara daban don cimma Canza matsayinsa daga nesa yayin daukar hoto. Samun hotunan X-ray yana farawa lokacin da aka sanya magnet ɗin kusan 30 mm sama da samfurin, kuma ana samun hotuna a cikin saurin firam 4 a kowace daƙiƙa. A lokacin daukar hoto, an kawo magnet kusa da bututun gilashin (kimanin milimita 1) sannan a juya shi tare da bututun don tantance tasirin ƙarfin filin da matsayinsa.
Saitin hotunan in vitro wanda ke ɗauke da samfuran MP a cikin ƙananan ƙwayoyin gilashi a kan matakin fassarar samfurin xy. Hanyar hasken X-ray an yi masa alama da layi ja mai lanƙwasa.
Da zarar an gano ganuwa ta 'yan majalisar a cikin vitro, an gwada wani ɓangare na su a cikin in vivo a cikin berayen Wistar na mace masu kama da daji (kimanin makonni 12, ~200 g).0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Japan) da 4 mg/kg butorphale (Vetorphale®, Meiji Seika) An yi wa berayen maganin sa barci da cakuda Pharma), Japan) ta hanyar allurar ciki. Bayan maganin sa barci, an shirya su don daukar hoto ta hanyar cire gashin da ke kewaye da trachea, saka bututun endotracheal (ET; 16 Ga iv cannula, Terumo BCT) sannan aka kwantar da su a kan farantin hoto na musamman wanda ke ɗauke da jakar zafi don kiyaye zafin jiki 22. Daga nan aka haɗa farantin hoton zuwa matakin fassarar samfurin a cikin akwatin hoto a ɗan kusurwa don daidaita trachea a kwance a cikin hoton X-ray, kamar yadda aka nuna. a cikin Hoto na 2a.
(a) Saita hoton cikin jiki a cikin akwatin hoton SPring-8, hanyar hasken X-ray an yi mata alama da layin ja mai lanƙwasa. (b,c) An yi amfani da kyamarorin IP guda biyu da aka ɗora a kan bututun numfashi daga nesa. A gefen hagu na hoton allo, ana iya ganin madaurin waya da ke riƙe da kan, kuma ana iya ganin madaurin isar da saƙo a wurinsa a cikin bututun ET.
An haɗa tsarin famfon sirinji mai sarrafawa daga nesa (UMP2, World Precision Instruments, Sarasota, FL) ta amfani da sirinji gilashi 100 μl zuwa bututun PE10 (OD 0.61 mm, ID 0.28 mm) ta hanyar allurar Ga 30. Yi alama a bututun don tabbatar da cewa ƙarshen yana cikin madaidaicin matsayi a cikin trachea lokacin saka bututun ET. Ta amfani da micropump, an cire bututun sirinji yayin da aka nutsar da ƙarshen bututun a cikin samfurin MP da za a kawo. Daga nan aka saka bututun isar da kaya da aka ɗora a cikin bututun endotracheal, inda aka sanya ƙarshen a cikin mafi ƙarfi na filin maganadisu da ake tsammani. An sarrafa samun hoto ta amfani da na'urar gano numfashi da aka haɗa da akwatin lokaci na Arduino ɗinmu, kuma an yi rikodin duk sigina (misali zafin jiki, numfashi, buɗewa/rufewa da ɗaukar hoto) ta amfani da Powerlab da LabChart (AD Instruments, Sydney, Australia) 22. Lokacin ɗaukar hoto Lokacin da aka kasa samun damar shiga cikin akwatin, kyamarorin IP guda biyu (Panasonic BB-SC382) an sanya su a kusan 90° ga juna. kuma an yi amfani da su don sa ido kan matsayin maganadisu dangane da trachea yayin daukar hoto (Hoto na 2b, c). Don rage abubuwan da ke motsa jiki, an sami hoto ɗaya a kowace numfashi a lokacin da ake fitar da ruwa daga ƙarshen teku.
An haɗa maganadisu zuwa mataki na biyu wanda za a iya samunsa daga nesa daga wajen wurin ɗaukar hoto. An gwada matsayi da tsare-tsare daban-daban na maganadisu, waɗanda suka haɗa da: An ɗora su a kusurwar kusan 30° sama da trachea (tsari da aka nuna a Hotuna 2a da 3a); maganadisu ɗaya sama da dabbar ɗayan kuma a ƙasa, tare da sandunan da aka saita don jawo hankali (Hoto na 3b); maganadisu ɗaya sama da dabbar ɗayan kuma a ƙasa, tare da sandunan da aka saita don korar su (Hoto na 3c); da maganadisu ɗaya a sama kuma a tsaye ga trachea (Hoto na 3d). Da zarar an daidaita dabbar da maganadisu kuma an ɗora MP ɗin da za a gwada a cikin famfon sirinji, a ba da allurar 50 μl a ƙimar 4 μl/sec yayin da ake samun hotuna. Daga nan sai a motsa maganadisu gaba da gaba ko a gefe a cikin trachea yayin da ake ci gaba da samun hotuna.
Tsarin maganadisu don ɗaukar hoton in vivo (a) maganadisu guda ɗaya a saman trachea a kusurwar kusan 30°, (b) maganadisu guda biyu da aka saita don jawo hankali, (c) maganadisu guda biyu da aka saita don korar su, (d) maganadisu guda ɗaya a sama da kuma a tsaye a cikin trachea. Mai lura ya kalli ƙasa daga baki zuwa huhu ta cikin trachea, kuma hasken X-ray ya ratsa ta gefen hagu na bera ya fita daga gefen dama. Ana motsa maganadisu tare da tsawon hanyar iska ko hagu da dama sama da trachea a cikin hanyar hasken X-ray.
Mun kuma nemi sanin yadda ƙwayoyin cuta ke bayyana a cikin hanyoyin numfashi ba tare da numfashi mai rikitarwa da motsin zuciya ba. Saboda haka, a ƙarshen lokacin ɗaukar hoton, an kashe dabbobi da gangan saboda yawan shan pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, Amurka; ~65 mg/kg ip). An bar wasu dabbobi a kan dandamalin ɗaukar hoton, kuma da zarar numfashi da bugun zuciya suka tsaya, an maimaita aikin ɗaukar hoton, ana ƙara ƙarin MP idan babu MP a saman hanyar numfashi.
An gyara hotunan da aka samu a cikin filin da aka yi amfani da shi da kuma cikin duhu sannan aka haɗa su cikin fim (firam 20 a kowace daƙiƙa; 15-25 × saurin da ya dace ya danganta da saurin numfashi) ta amfani da rubutun da aka rubuta a cikin MATLAB (R2020a, The Mathworks).
An gudanar da dukkan nazarin isar da kwayoyin halittar LV a Cibiyar Binciken Dabbobi ta Dakin Gwaji a Jami'ar Adelaide kuma an yi nufin amfani da sakamakon gwajin SPring-8 don tantance ko isar da LV-MP a gaban filin maganadisu zai iya haɓaka canja wurin kwayoyin halitta a cikin jiki. Don tantance tasirin MP da filin maganadisu, an yi wa ƙungiyoyi biyu na dabbobi magani: an ba wa rukuni ɗaya LV-MP tare da maganadisu da aka sanya, ɗayan rukunin kuma ya sami rukunin sarrafawa tare da LV-MP ba tare da maganadisu ba.
An samar da vectors na kwayoyin halittar LV ta amfani da hanyoyin da aka bayyana a baya 25, 26. Vector na LacZ yana bayyana kwayar halittar beta-galactosidase da aka gano a cikin kwayar halittar da ke cikin kwayar halittar MPSV (LV-LacZ) ke jagoranta, wacce ke samar da samfurin amsawar shuɗi a cikin ƙwayoyin da aka canza, waɗanda ake iya gani a gaban kyallen huhu da sassan nama. An gudanar da titration a cikin al'adun ƙwayoyin halitta ta hanyar ƙidaya adadin ƙwayoyin LacZ masu kyau da hannu tare da na'urar auna hemocytometer don ƙididdige titer a cikin TU/ml. Ana adana masu ɗaukar kaya a -80 °C, ana narke su kafin amfani, kuma ana ɗaure su zuwa CombiMag ta hanyar haɗawa a rabo na 1:1 kuma a saka su a kan kankara na akalla mintuna 30 kafin haihuwa.
An yi wa berayen Sprague Dawley na yau da kullun sabulun sabulu (n = 3/rukuni, ~2-3) allurar allurar 0.4 mg/kg medetomidine (Domitor, Ilium, Ostiraliya) da allurar ketamine (Ilium, Ostiraliya) mai watanni 60) da allurar baki ba tare da tiyata ba tare da tiyata ba tare da allurar 16 Ga iv. Don tabbatar da cewa kyallen hanyar iska ta tracheal ta sami transduction na LV, an yi mata gyaran fuska ta amfani da tsarin mu na inji da aka bayyana a baya, inda aka goge saman hanyar iska ta tracheal a hankali da kwandon waya (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, Amurka) 30 s28. Daga nan aka yi allurar LV-MP ta tracheal a cikin kabad na aminci na halitta kimanin mintuna 10 bayan an yi ta.
An tsara filin maganadisu da aka yi amfani da shi a wannan gwajin ta hanyar da ta dace da nazarin hoton X-ray na in vivo, tare da maganadisu iri ɗaya da aka riƙe a saman trachea ta amfani da maɓallan stent na distillation (Hoto na 4). An aika da ƙarar μl 50 (2 × 25 μl aliquots) na LV-MP zuwa cikin trachea (n = dabbobi 3) ​​ta amfani da pipette mai ɗauke da tip ɗin gel kamar yadda aka bayyana a baya. Ƙungiyar sarrafawa (n = dabbobi 3) ​​sun karɓi LV-MP iri ɗaya ba tare da amfani da maganadisu ba. Bayan an gama jiko, ana cire cannula daga bututun ET kuma ana fitar da dabbar. Magnetin yana nan a wurin na tsawon mintuna 10, sannan a cire shi. Beraye sun sami allurar meloxicam (1 ml/kg) ta ƙarƙashin fata (Ilium, Ostiraliya) sannan aka mayar da maganin sa barci ta hanyar allurar ip na 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Ostiraliya). An ajiye berayen da ɗumi kuma ana sa ido a kansu har sai sun warke gaba ɗaya daga maganin sa barci.
Na'urar isar da sako ta LV-MP a cikin kabad mai kariya daga halittu. Ana iya ganin cibiyar Luer mai launin toka mai haske ta bututun ET tana fitowa daga baki kuma an saka ƙarshen gel na bututun da aka nuna a hoton ta bututun ET zuwa zurfin da ake so a cikin bututun.
Mako guda bayan an yi amfani da LV-MP wajen allurar LV, an kashe dabbobi ta hanyar shaƙa iskar CO2 100% kuma an tantance yanayin LacZ ta amfani da maganinmu na yau da kullun na X-gal. An cire zoben caudal mafi yawan cartilaginous guda uku don tabbatar da cewa duk wani lalacewar injiniya ko riƙe ruwa daga wurin da aka sanya bututun endotracheal ba a haɗa su cikin binciken ba. An yanke kowace trachea a tsayi don ƙirƙirar rabi biyu don bincike, kuma an ɗora su a cikin kwano mai ɗauke da robar silicone (Sylgard, Dow Inc) ta amfani da allurar Minutien (Fine Science Tools) don ganin saman haske. An tabbatar da rarrabawa da tsarin ƙwayoyin da aka canza ta hanyar ɗaukar hoto na gaba ta amfani da na'urar hangen nesa ta Nikon (SMZ1500) tare da kyamarar DigiLite da software na TCapture (Tucsen Photonics, China). An samo hotunan a girman 20x (gami da mafi girman saitin cikakken faɗin trachea), tare da ɗaukar hoton tsawon trachea mataki-mataki, yana tabbatar da isasshen haɗuwa tsakanin kowane hoto don ba da damar "dinka" hoto. Hotuna daga kowane trachea An haɗa su cikin hoto ɗaya ta amfani da Editan Haɗa Hoto v2.0.3 (Microsoft Research) ta amfani da tsarin motsi na planar. An ƙididdige yankunan bayyanar LacZ a cikin hotunan haɗaɗɗen trachea daga 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, Saturation > 0.15, da Value < 0.7. Ta hanyar bin diddigin yanayin nama, an samar da abin rufe fuska da hannu a cikin GIMP v2.10.24 don kowane hoton haɗaɗɗen don gano yankin nama da hana duk wani gano ƙarya daga wajen kyallen trachea. An tattara wuraren da aka yi wa fenti daga duk hotunan haɗaɗɗen daga kowace dabba don samar da jimlar yankin da aka yi wa fenti ga wannan dabba. Sannan an raba yankin da aka yi wa fenti da jimlar yankin abin rufe fuska don samar da yankin da aka daidaita.
An saka kowace hanyar numfashi a cikin paraffin kuma an yanke sassan 5 μm. An yi wa sassan fenti da ja mai kauri na tsawon minti 5 sannan aka samo hotuna ta amfani da na'urar hangen nesa ta Nikon Eclipse E400, kyamarar DS-Fi3 da kuma manhajar ɗaukar abubuwan NIS (sigar 5.20.00).
An gudanar da dukkan nazarin kididdiga a cikin GraphPad Prism v9 (GraphPad Software, Inc.). An saita mahimmancin ƙididdiga a p ≤ 0.05. An tabbatar da daidaito ta amfani da gwajin Shapiro-Wilk, kuma an tantance bambance-bambancen da ke cikin tabo na LacZ ta amfani da gwajin t-test mara haɗin kai.
An duba 'yan majalisar guda shida da aka bayyana a cikin Jadawali na 1 ta amfani da PCXI, kuma an bayyana ganuwa a cikin Jadawali na 2. Ba a ganuwa ga 'yan majalisar polystyrene guda biyu (MP1 da MP2; 18 μm da 0.25 μm, bi da bi) a ƙarƙashin PCXI ba, amma sauran samfuran an iya gane su (misalai an nuna su a Hoto na 5). MP3 da MP4 (10-15% Fe3O4; 0.25 μm da 0.9 μm, bi da bi) suna da ɗan bayyane. Duk da cewa suna ɗauke da wasu ƙananan ƙwayoyin da aka gwada, MP5 (98% Fe3O4; 0.25 μm) shine mafi bayyana. Samfurin CombiMag MP6 yana da wuya a gano. A duk lokuta, ikonmu na gano MP ya inganta sosai ta hanyar fassara maganadisu a baya da gaba daidai da capillary. Lokacin da maganadisu suka motsa daga capillary, ƙwayoyin sun faɗaɗa a cikin dogon igiyoyi, amma yayin da maganadisu suka yi kusa kuma ƙarfin filin maganadisu ya ƙaru, igiyoyin barbashi sun gajarta yayin da ƙwayoyin suka yi ƙaura zuwa saman saman capillary (duba Ƙarin Bidiyo S1: MP4), yana ƙaruwa Yawan barbashi na saman. A akasin haka, lokacin da aka cire maganadisu daga capillary, ƙarfin filin yana raguwa kuma MPs sun sake tsara su zuwa dogayen igiyoyi waɗanda suka fito daga saman capillary (duba Ƙarin Bidiyo S2:MP4). Bayan maganadisu ya daina motsi, barbashi suna ci gaba da motsawa na ɗan lokaci bayan sun isa matsayin daidaito. Yayin da MP ke motsawa zuwa da nesa da saman capillary, barbashi masu maganadisu galibi suna jan tarkace ta cikin ruwan.
Ganuwa ta MP a ƙarƙashin PCXI ta bambanta sosai tsakanin samfura.(a) MP3, (b) MP4, (c) MP5 da (d) MP6. Duk hotunan da aka nuna a nan an ɗauke su da maganadisu wanda ke kusan mm 10 kai tsaye sama da capillary. Manyan da'irori da ke bayyane kumfa ne na iska da aka makale a cikin capillaries, wanda a bayyane yake nuna fasalin gefen baƙi da fari na hoton bambancin lokaci. Akwatin ja yana ɗauke da girman haɓaka bambanci. Lura cewa diamita na zane-zanen maganadisu a cikin duk siffofi ba su da girma kuma sun fi kusan sau 100 girma fiye da yadda aka nuna.
Yayin da aka fassara maganadisu a hagu da dama a saman murfin, kusurwar igiyar MP tana canzawa don daidaitawa da maganadisu (duba Hoto na 6), don haka tana fayyace layukan filin maganadisu. Don MP3-5, bayan da murfin ya kai kusurwar ƙofar, ana jan barbashi a saman saman murfin. Wannan yakan haifar da tarin MPs zuwa manyan rukuni kusa da inda filin maganadisu ya fi ƙarfi (duba Bidiyon Ƙarin S3:MP5). Wannan kuma yana bayyana musamman lokacin da aka ɗauki hoto kusa da ƙarshen murfin, wanda ke sa MPs su taru kuma su mai da hankali a mahaɗin ruwa-iska. Barbashi a cikin MP6, waɗanda suka fi wahalar ganewa fiye da MP3-5, ba a ja su yayin da maganadisu ke motsawa tare da murfin ba, amma igiyoyin MP sun rabu, suna barin barbashi a cikin filin kallo (duba Bidiyon Ƙarin S4:MP6). A wasu lokuta, lokacin da aka rage filin maganadisu da aka yi amfani da shi ta hanyar motsa maganadisu a babban nisa daga wurin hoton, duk wani MP da ya rage ya sauko a hankali zuwa ƙasan bututun ta hanyar nauyi yayin da yake ci gaba da kasancewa a cikin zaren (duba Bidiyon Ƙarin S5: MP3).
Kusurwar igiyar MP tana canzawa yayin da aka fassara maganadisu zuwa dama a saman capillary.(a) MP3, (b) MP4, (c) MP5 da (d) MP6. Akwatin ja yana ɗauke da girman da ke ƙara bambanci. Lura cewa ƙarin bidiyon suna da bayanai masu amfani domin suna bayyana mahimman tsarin barbashi da bayanai masu ƙarfi waɗanda ba za a iya gani a cikin waɗannan hotunan ba.
Gwaje-gwajenmu sun nuna cewa motsa maganadisu a hankali a kan hanyar trachea yana sauƙaƙa ganin MP a cikin mahallin motsi mai rikitarwa a cikin vivo. Ba a yi gwajin In vivo ba saboda ba a ga beads na polystyrene (MP1 da MP2) a cikin capillary ba. An gwada kowanne daga cikin sauran 'yan majalisar guda huɗu a cikin vivo tare da dogon axis na maganadisu da aka saita a sama da trachea a kusurwar kusan 30° zuwa tsaye (duba Hotuna 2b da 3a), saboda wannan ya haifar da tsawon sarƙoƙi na MP kuma ya fi tasiri fiye da yadda aka dakatar da tsarin maganadisu. Ba a gano MP3, MP4 da MP6 a cikin trachea na kowace dabba mai rai ba. Lokacin da aka ɗauki hoton hanyoyin iska na bera bayan an kashe dabbobin da ɗan adam, ƙwayoyin ba su ganuwa ko da an ƙara ƙarin girma ta amfani da famfon sirinji. MP5 yana da mafi girman abun ciki na ƙarfe oxide kuma shine kawai ƙwayar da ake gani, don haka ana amfani da shi don tantancewa da kuma siffanta halayen MP na in vivo.
Sanya maganadisu a kan trachea yayin haihuwa ta MP ya sa 'yan majalisar da yawa, amma ba duka ba, suka taru a fagen gani. Ana iya ganin barbashi da ke shiga trachea a cikin dabbobin da aka yanka da ɗan adam. Hoto na 7 da Bidiyo na Ƙarin S6: MP5 yana nuna kama maganadisu cikin sauri da daidaita barbashi a saman trachea na ventral, yana nuna cewa ana iya tura 'yan majalisar zuwa yankunan da ake so na trachea. Lokacin da ake bincike nesa da trachea bayan haihuwa ta MP, an gano wasu 'yan majalisar kusa da carina, yana nuna cewa ƙarfin filin maganadisu bai isa ya tattara da riƙe dukkan 'yan majalisar ba, saboda an isar da su ta yankin ƙarfin filin maganadisu mafi girma yayin aikin ruwa. Duk da haka, yawan MP bayan haihuwa ya fi girma a kusa da yankin da aka ɗauka hoton, yana nuna cewa 'yan majalisar da yawa sun kasance a yankunan hanyar iska inda ƙarfin filin maganadisu da aka yi amfani da shi ya fi girma.
Hotuna daga (a) kafin da (b) bayan isar da MP5 zuwa cikin trachea na bera da aka kashe kwanan nan tare da maganadisu da aka sanya kai tsaye a saman yankin daukar hoto. Yankin da aka nuna yana tsakanin zoben guringuntsi guda biyu. Kafin isar da MP, akwai wani ruwa a cikin hanyar iska. Akwatin ja yana dauke da girman da ke kara bambanci. Waɗannan hotunan sun fito ne daga bidiyon da aka nuna a cikin Ƙarin Bidiyo S6:MP5.
Fassara maganadisu tare da trachea a cikin jiki ya sa sarkar MP ta canza kusurwa a cikin saman hanyar iska ta hanyar kama da wacce aka gani a cikin capillaries (duba Hoto na 8 da Bidiyon Ƙarin S7: MP5). Duk da haka, a cikin bincikenmu, ba za a iya jan MPs tare da saman hanyar iska mai rai kamar yadda suka iya tare da capillaries ba. A wasu lokuta, sarkar MP za ta yi tsayi yayin da maganadisu ke motsawa hagu da dama. Abin sha'awa, mun kuma gano cewa igiyar barbashi ta bayyana tana canza zurfin layin ruwa na saman lokacin da aka motsa maganadisu a tsayi tare da trachea, kuma tana faɗaɗa lokacin da aka motsa maganadisu kai tsaye a sama kuma an juya igiyar barbashi zuwa matsayi a tsaye (duba Bidiyon Ƙarin S7). : MP5 a 0:09, ƙasan dama). Tsarin motsin ya canza lokacin da aka fassara maganadisu a saman trachea a gefe (wato, zuwa hagu ko dama na dabbar maimakon a kan tsawon trachea). Har yanzu ana iya ganin barbashin yayin da suke motsawa, amma lokacin da aka cire maganadisu daga trachea, ƙarshen igiyoyin barbashi sun bayyana (duba Ƙarin Bidiyo S8:MP5, farawa daga 0:08). Wannan ya yi daidai da ɗabi'ar MP da muka lura a ƙarƙashin filin maganadisu da aka yi amfani da shi a cikin babban gilashin.
Misalin hotuna da ke nuna MP5 a cikin trachea na bera mai rai da aka yi wa sabulun wanka.(a) Ana amfani da maganadisu don samun hotuna a sama da hagu na trachea, sannan (b) bayan an motsa maganadisu zuwa dama. Akwatin ja yana ɗauke da girman da ke ƙara bambanci. Waɗannan hotunan sun fito ne daga bidiyon da aka nuna a cikin Ƙarin Bidiyo S7:MP5.
Lokacin da aka tsara sandunan biyu a yanayin arewa-kudu a sama da ƙasa da trachea (watau jawo hankali; Hoto na 3b), waƙoƙin MP sun bayyana sun fi tsayi kuma suna kan gefen trachea maimakon a saman trachea na baya (duba Bidiyon Ƙarin Bayani S9:MP5). Duk da haka, ba a gano yawan ƙwayoyin cuta a wuri ɗaya ba (watau, saman trachea) bayan isar da ruwa lokacin da aka yi amfani da na'urar maganadisu biyu, wanda yawanci yakan faru lokacin da aka yi amfani da na'urar maganadisu ɗaya. Sannan lokacin da aka tsara maganadisu ɗaya don korar sandunan da aka juya (Hoto na 3c), adadin ƙwayoyin da ake gani a fagen kallo bai yi kama da sun ƙaru ba bayan isarwa. Saitin saitin duka saitunan maganadisu biyu yana da ƙalubale saboda ƙarfin filin maganadisu mai yawa wanda ke jan ko tura maganadisu, bi da bi. Sai an canza saitin zuwa maganadisu ɗaya daidai da hanyar iska amma yana ratsa hanyar iska a digiri 90 don layukan filin su ratsa bangon trachea ta hanyar da ta dace (Hoto na 3d), hanyar da aka tsara don tantance ko za a iya lura da tarin ƙwayoyin cuta a bangon gefe. Duk da haka, A cikin wannan tsari, babu wani motsi da za a iya gane shi na tarin MP ko motsin maganadisu. Dangane da duk waɗannan sakamakon, an zaɓi tsarin daidaitawa na maganadisu ɗaya, digiri 30 (Hoto na 3a) don nazarin masu ɗaukar kwayar halitta a cikin jiki.
Lokacin da aka yi ta ɗaukar hoton dabbar akai-akai bayan kisan kai na ɗan adam, rashin motsin nama mai rikitarwa yana nufin cewa ana iya gano layukan barbashi masu ƙanƙanta da gajeru a cikin fili mai haske na interchondral, "mai rawar jiki" daidai da motsin fassara na maganadisu. Duk da haka, har yanzu ba za a iya ganin kasancewar da motsi na barbashi na MP6 a sarari ba.
Titer ɗin LV-LacZ shine 1.8 × 108 TU/ml, kuma bayan an haɗa shi da CombiMag MP (MP6) da 1:1, dabbobi sun sami maganin tracheal na 9 × 107 TU/ml LV (watau 4.5 × 106 TU/rat). A cikin waɗannan nazarin, maimakon fassara maganadisu yayin naƙuda, mun gyara maganadisu a wuri ɗaya don tantance ko za a iya inganta transduction na LV (a) idan aka kwatanta da isar da vector idan babu filin maganadisu, kuma (b) za a iya mayar da hankali kan ƙwayoyin iskar iska ana canza su zuwa yankunan da ake nufi da maganadisu na hanyar iskar sama.
Kasancewar maganadisu da amfani da CombiMag tare da vectors na LV bai bayyana yana da mummunan tasiri ga lafiyar dabbobi ba, kamar yadda tsarin isar da vector na LV na yau da kullun ya yi. Hotunan gaba na yankin tracheal da aka fuskanci matsala ta injiniya (Hoto na 1) sun nuna cewa akwai matakan transduction mafi girma a cikin rukunin dabbobin da aka yi wa magani da LV-MP lokacin da magnet ɗin yake (Hoto na 9a). Ƙananan adadin fenti na LacZ shuɗi ne kawai ke nan a cikin rukunin sarrafawa (Hoto na 9b). Ƙididdige wuraren da aka yi wa fenti na X-Gal sun nuna cewa gudanar da LV-MP a gaban filin maganadisu ya haifar da ci gaba kusan ninki 6 (Hoto na 9c).
Misalin hotunan da aka haɗa da aka nuna transduction na tracheal ta hanyar LV-MP (a) a gaban filin maganadisu da kuma (b) a rashin maganadisu. (c) Ingantaccen ƙididdiga a yankin transduction na LacZ da aka daidaita a cikin trachea lokacin amfani da maganadisu (*p = 0.029, gwajin t, n = 3 ga kowace ƙungiya, matsakaicin ± SEM).
Sassan da aka yi wa fenti ja mai sauri (misali da aka nuna a cikin Ƙarin Hoto na 2) sun nuna ƙwayoyin da aka yi wa fenti LacZ da ke cikin tsari da wuri iri ɗaya kamar yadda aka ruwaito a baya.
Babban ƙalubale ga maganin kwayoyin halittar iska shine daidaita wurin da ƙwayoyin cuta masu ɗaukar jini ke zuwa yankunan da ake sha'awa da kuma cimma manyan matakan ingancin watsawa a cikin huhu mai motsi a gaban kwararar iska da kuma share majina mai aiki. Ga masu ɗaukar LV waɗanda aka tsara don magance cututtukan hanyar iska ta CF, ƙara lokacin zama na ƙwayoyin cuta masu ɗaukar jini a cikin hanyoyin sadarwa na numfashi ya kasance babban buri da har yanzu ba a iya warwarewa ba. Kamar yadda Castellani et al. suka nuna, amfani da filayen maganadisu don inganta watsawa yana da fa'idodi idan aka kwatanta da sauran hanyoyin isar da kwayoyin halitta kamar electroporation, domin yana iya haɗa sauƙi, inganci, wurin isarwa, ƙaruwar inganci, da gajerun lokutan shiryawa, da kuma wataƙila ƙaramin adadin jigilar jini. Duk da haka, ba a taɓa bayyana ajiyar ƙwayoyin maganadisu a cikin hanyoyin sadarwa a ƙarƙashin tasirin ƙarfin maganadisu na waje ba, kuma ba a nuna yuwuwar wannan hanyar a cikin jiki don haɓaka matakan bayyanar kwayoyin halitta a cikin hanyoyin sadarwa masu rai ba.
Gwaje-gwajenmu na in vitro synchrotron PCXI sun nuna cewa duk barbashi da muka gwada, ban da polystyrene MP, an gansu a cikin saitin hoton da muka yi amfani da shi. A gaban filin maganadisu, MPs suna samar da igiyoyi waɗanda tsayinsu 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 muka gani an samar da su ne saboda kowace barbashi tana maganadisu kuma tana haifar da nata filin maganadisu na gida. Waɗannan filayen daban-daban suna haifar da wasu barbashi masu kama da juna su haɗu da juna, tare da motsin kamar igiya na rukuni saboda ƙarfin gida daga ƙarfin da ke jan hankali da ƙyama na sauran barbashi.
Tsarin da ke nuna (a,b) jiragen ƙasa na barbashi da aka samar a cikin ƙwayoyin jini masu cike da ruwa da kuma (c,d) hanyoyin jini masu cike da iska. Lura cewa ba a jawo ƙwayoyin jini da hanyoyin jini zuwa sikelin ba. Faifan (a) kuma ya ƙunshi bayanin MP, wanda ya ƙunshi ƙwayoyin Fe3O4 da aka shirya a cikin igiyoyi.
Lokacin da aka motsa maganadisu sama da capillary, kusurwar igiyar barbashi ta kai ga maƙasudin MP3-5 wanda ke ɗauke da Fe3O4, bayan haka igiyar barbashi ba ta sake tsayawa a matsayinta na asali ba, amma ta koma saman zuwa wani sabon matsayi.magnet. Wannan tasirin yana iya faruwa ne saboda saman murfin gilashi yana da santsi don barin wannan motsi ya faru. Abin sha'awa, MP6 (CombiMag) bai yi irin wannan hali ba, wataƙila saboda ƙwayoyin sun yi ƙanƙanta, suna da launi daban-daban ko cajin saman, ko kuma wani ruwa mai ɗaukar kaya na musamman ya shafi ikonsu na motsawa. Bambancin hoton barbashi na CombiMag shi ma ya yi rauni, yana nuna cewa ruwa da barbashi na iya samun irin wannan yawa don haka ba sa motsawa cikin sauƙi zuwa ga juna. Barbashi kuma na iya makale idan maganadisu ta yi sauri sosai, yana nuna cewa ƙarfin filin maganadisu ba zai iya shawo kan gogayya tsakanin barbashi a cikin ruwan ba, yana nuna cewa wataƙila ba abin mamaki bane cewa ƙarfin filin maganadisu da nisan da ke tsakanin maganadisu da yankin da aka nufa Yana da mahimmanci sosai. Idan aka haɗa su, waɗannan sakamakon kuma suna nuna cewa, yayin da maganadisu na iya kama MPs da yawa waɗanda ke gudana ta yankin da aka nufa, da wuya a dogara da maganadisu don motsa ƙwayoyin CombiMag a saman trachea. Saboda haka, mun kammala da cewa a cikin vivo nazarin LV-MP ya kamata ya yi amfani da filayen maganadisu masu tsauri don kai hari ga takamaiman yankuna na bishiyar hanyar iska.
Idan aka kawo ƙwayoyin cuta a cikin jiki, yana da wuya a gane su a cikin mahallin ƙwayoyin cuta masu rikitarwa, amma an ƙara ƙarfin gano su ta hanyar fassara maganadisu a kwance a saman trachea don "juya" igiyoyin MP. Duk da cewa hoton kai tsaye yana yiwuwa, yana da sauƙin gane motsin ƙwayoyin cuta da zarar an kashe dabbar da kyau. Yawan MP gabaɗaya ya fi yawa a wannan wurin lokacin da aka sanya maganadisu a saman yankin hoton, kodayake ana samun wasu ƙwayoyin cuta a kusa da trachea. Sabanin nazarin in vitro, ba za a iya jan ƙwayoyin cuta tare da trachea ta hanyar fassara maganadisu ba. Wannan binciken ya yi daidai da yadda majina da ke rufe saman trachea yawanci ke sarrafa ƙwayoyin da aka shaƙa, yana kama su a cikin majina sannan daga baya ya share ta hanyar hanyar share mucociliary.
Mun yi hasashen cewa amfani da maganadisu don jan hankali a sama da ƙasan trachea (Hoto na 3b) na iya haifar da filin maganadisu mai daidaito, maimakon filin maganadisu wanda ke da ƙarfi sosai a wani lokaci, wanda hakan zai iya haifar da rarraba ƙwayoyin cuta iri ɗaya. Duk da haka, bincikenmu na farko bai sami wata hujja bayyananniya da za ta goyi bayan wannan hasashe ba. Haka nan, daidaita maganadisu biyu don korar su (Hoto na 3c) bai haifar da ƙarin ajiyar ƙwayoyin cuta a yankin da aka nuna ba. Waɗannan binciken guda biyu sun nuna cewa saitin maganadisu biyu bai inganta ikon sarrafa MP na gida ba, kuma ƙarfin maganadisu mai ƙarfi da aka samu yana da wahalar saitawa, wanda hakan ya sa wannan hanyar ba ta da amfani. Hakazalika, daidaita maganadisu a sama da ta hanyar trachea (Hoto na 3d) kuma bai ƙara yawan ƙwayoyin da aka riƙe a yankin da aka nuna ba. Wasu daga cikin waɗannan saitunan madadin ba za su yi nasara ba saboda suna haifar da ƙarancin ƙarfin filin maganadisu a cikin yankin ajiya. Saboda haka, ana ɗaukar tsarin maganadisu na kusurwar digiri 30 guda ɗaya (Hoto na 3a) a matsayin hanya mafi sauƙi kuma mafi inganci don gwajin in vivo.
Binciken LV-MP ya nuna cewa lokacin da aka haɗa vectors na LV tare da CombiMag kuma aka kawo su bayan rikicewar jiki a gaban filin maganadisu, matakan transduction sun ƙaru sosai a cikin trachea idan aka kwatanta da sarrafawa. Dangane da nazarin hotunan synchrotron da sakamakon LacZ, filin maganadisu ya sami damar adana LV a cikin trachea da rage adadin barbashi na vector waɗanda suka shiga cikin huhu nan da nan. Irin waɗannan ci gaban da aka yi niyya na iya haifar da ingantaccen aiki yayin da ake rage adadin da aka bayar, transduction daga manufa, tasirin kumburi da na rigakafi, da farashin mai ɗaukar kwayar halitta. Abu mafi mahimmanci, a cewar masana'anta, ana iya amfani da CombiMag tare da sauran hanyoyin canja wurin kwayar halitta, gami da sauran vectors na ƙwayoyin cuta (kamar AAV) da nucleic acid.