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A ghaghị ileba anya na vectors gene vectors maka ọgwụgwọ cystic fibrosis nke akpa ume gaa n'okporo ụzọ ikuku na-eduzi, ebe ọ bụ na ntụgharị nku ume dị n'akụkụ enweghị mmetụta ọgwụgwọ.Ịrụ ọrụ nke ntụgharị nje na-emetụta kpọmkwem oge obibi nke onye na-ebu ya.Agbanyeghị, mmiri nnyefe dị ka ndị na-ebu mkpụrụ ndụ ihe nketa na-agbasa n'ime alveoli n'oge iku ume, a na-ewepụkwa ahụ ngwa ngwa nke ụdị ọ bụla site na njem mucociliary.Ịgbatị oge obibi nke ndị na-ebu mkpụrụ ndụ ihe nketa na traktị iku ume dị mkpa mana ọ siri ike nweta.Ihe ndọta jikọtara ndị na-ebubu nke enwere ike iduga n'elu traktị iku ume nwere ike imezi ebumnuche mpaghara.N'ihi nsogbu dị na in vivo imaging, omume nke obere ihe ndọta dị otú ahụ dị n'elu elu ikuku n'ihu ebe magnetik etinyere aghọtachaghị nke ọma.Ebumnuche nke ọmụmụ a bụ iji synchrotron imaging na-ahụ anya na vivo mmegharị nke usoro ihe ndọta dị na trachea nke oke anesthetized iji mụọ mgbanwe na usoro nke omume nke otu na nnukwu akụkụ na vivo.Anyị tụlekwara ma nnyefe nke ihe ndọta lentiviral n'ihu oghere ndọta ga-abawanye arụmọrụ nke ntụgharị na trachea oke.Onyonyo X-ray Synchrotron na-egosi omume nke irighiri ihe ndọta na ebe ndọta kwụ ọtọ na nke na-akpụ akpụ na vitro na na vivo.Enweghị ike ịdọrọ akụkụ dị mfe n'elu elu ụgbọ elu dị ndụ site na iji magnet, ma n'oge njem, ihe nkwụnye ego na-etinye uche n'ihe nlele, ebe oghere magnetik kacha sie ike.A na-abawanye arụmọrụ ntụgharị ugboro isii mgbe a na-ebuga ụmụ irighiri ihe ndọta lentiviral n'ihu oghere magnetik.N'ịchịkọta ọnụ, nsonaazụ ndị a na-egosi na ụmụ irighiri ihe ndọta lentiviral na oghere magnetik nwere ike bụrụ ụzọ bara uru iji kwalite ebumnuche mkpụrụ ndụ ihe nketa na ọkwa ntụgharị n'ime ụzọ ikuku na-eduzi na vivo.
Cystic fibrosis (CF) na-akpata site na ọdịiche dị na otu mkpụrụ ndụ ihe nketa a na-akpọ CF transmembrane conductance regulator (CFTR).Protein CFTR bụ ọwa ion nke dị na ọtụtụ mkpụrụ ndụ epithelial n'ime ahụ dum, gụnyere ikuku ikuku, ebe bụ isi na pathogenesis nke cystic fibrosis.Nkwarụ dị na CFTR na-eduga n'ụgbọ njem mmiri na-adịghị mma, akpịrị ịkpọ nkụ nke elu ụzọ ikuku, yana mbelata omimi mmiri elu ikuku (ASL).Ọ na-emebikwa ikike nke usoro njem mucociliary (MCT) iji kpochapụ ụzọ ikuku nke ụmụ irighiri inhalation na nje.Ebumnuche anyị bụ ịzụlite usoro ọgwụgwọ lentiviral (LV) iji wepụta ụdị mkpụrụ ndụ CFTR ziri ezi wee kwalite ahụike ASL, MCT na ngụgụ, yana ịga n'ihu na-emepụta teknụzụ ọhụrụ nwere ike tụọ parampat ndị a na vivo1.
Ndị na-ahụ maka LV bụ otu n'ime ndị na-ebute ụzọ maka ọgwụgwọ mkpụrụ ndụ cystic fibrosis, ọkachasị n'ihi na ha nwere ike itinye mkpụrụ ndụ ọgwụgwọ na-adịgide adịgide na sel basal ikuku.Nke a dị mkpa n'ihi na ha nwere ike iweghachi hydration nkịtị na mkpochapụ nke imi site n'ịwapụta n'ime mkpụrụ ndụ ikuku na-arụ ọrụ na-arụ ọrụ nke na-ejikọta ya na cystic fibrosis, na-ebute uru ogologo ndụ.A ghaghị iduzi vectors LV megide ụzọ ikuku na-eduzi, n'ihi na nke a bụ ebe ntinye aka na ngụgụ na CF na-amalite.Nnyefe nke vector miri emi n'ime ngụgụ nwere ike ibute ntụgharị alveolar, mana nke a enweghị mmetụta ọgwụgwọ na cystic fibrosis.Otú ọ dị, mmiri mmiri dị ka ndị na-ebu mkpụrụ ndụ ihe nketa na-akwaga na alveoli mgbe a na-eku ume mgbe a mụsịrị nwa3,4 na ihe ndị na-agwọ ọrịa na-awụpụ ngwa ngwa n'ime oghere ọnụ site na MCT.Ịrụ ọrụ nke ntụgharị LV metụtara kpọmkwem na ogologo oge vector na-anọ nso na sel ndị a na-eche ihu iji kwe ka ị nweta cellular - "oge obibi" 5 nke na-adị mfe mkpụmkpụ site na ikuku ikuku na-ahụkarị yana nchịkọta nchịkọta nke imi na MCT.Maka cystic fibrosis, ikike ịgbatị oge obibi LV n'ime ikuku dị mkpa iji nweta ọkwa dị elu nke ntụgharị na mpaghara a, mana ọ dị ugbu a ihe ịma aka.
Iji merie nsogbu a, anyị na-atụ aro na irighiri ihe ndọta LV (MPs) nwere ike inye aka n'ụzọ abụọ.Nke mbụ, magnet nwere ike iduzi ha n'elu elu ikuku iji meziwanye ebumnuche ma nyere ụmụ irighiri ihe ndị na-ebu mkpụrụ ndụ aka ka ha nọrọ n'ebe kwesịrị ekwesị nke ikuku;na ASL) na-abanye n'ime oyi akwa cell 6. A na-eji MPs eme ihe dị ka ụgbọ ala ebufe ọgwụ ezubere iche mgbe ha na-ejikọta ọgwụ mgbochi, ọgwụ chemotherapy, ma ọ bụ obere ụmụ irighiri ihe ndị ọzọ na-ejikọta na membranes cell ma ọ bụ na-ejikọta na ndị na-anabata ihe elu cell ha ma na-agbakọta na saịtị tumor. ọnụnọ nke ọkụ eletrik.Oghere magnetik maka ọgwụgwọ ọrịa kansa 7. Ụzọ ndị ọzọ "hyperthermic" bụ iji gbuo mkpụrụ ndụ tumo site na kpo oku MPs mgbe ekpughere oghere magnetik na-emegharị.Ụkpụrụ nke nnyefe magnetik, bụ nke a na-eji oghere magnet dị ka onye na-ebugharị DNA iji kwalite nnyefe nke DNA n'ime sel, a na-ejikarị in vitro na-eji ụdị mkpụrụ ndụ na-abụghị nke nje na nje virus maka eriri mkpụrụ ndụ siri ike ịmegharị. ..Ịrụ ọrụ nke magnetotransfection LV na nnyefe nke LV MP in vitro n'ime eriri cell nke epithelium bronchial mmadụ na ọnụnọ nke oghere magnetik guzosiri ike, na-abawanye arụmọrụ nke ntụgharị site na ugboro 186 ma e jiri ya tụnyere LV vector naanị.A na-etinyekwa LV MT n'ụdị in vitro nke cystic fibrosis, ebe nfefe magnet na-abawanye ntụgharị LV na omenala ikuku mmiri site na ihe ruru 20 na ọnụnọ cystic fibrosis sputum10.Otú ọ dị, na vivo akụkụ magnetotransfection enwetala obere nlebara anya ma enyochala ya na ọmụmụ anụmanụ ole na ole11,12,13,14,15, karịsịa na ngụgụ16,17.Otú ọ dị, ohere nke nnyefe magnetik na ọgwụgwọ ngụgụ na cystic fibrosis doro anya.Tan et al.(2020) kwuru na "akwụkwọ nkwado maka nnyefe akpa ume dị mma nke magnetik nanoparticles ga-emeghe ụzọ maka atụmatụ iku ume CFTR n'ọdịnihu iji melite nsonaazụ ụlọ ọgwụ na ndị ọrịa nwere cystic fibrosis"6.
Omume nke obere ihe ndọta n'elu akụkụ iku ume na ọnụnọ nke oghere magnetik na-arụ ọrụ siri ike ịhụ anya na ọmụmụ ihe, ya mere a na-aghọtaghị ha.N'ọmụmụ ihe ndị ọzọ, anyị emepụtala usoro Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) maka eserese na-adịghị emerụ ahụ na ọnụọgụ nkeji na vivo mgbanwe na ASL18 omimi na omume MCT19,20 iji tụọ hydration elu gas channel. a na-ejikwa ya dị ka ihe ngosi mmalite ọgwụgwọ irè.Na mgbakwunye, usoro akara akara MCT anyị na-eji 10-35 µm dayameta irighiri ihe mejupụtara alumina ma ọ bụ iko ntụgharị dị elu dị ka akara MCT na-ahụ PB-PCXI21.Ụzọ abụọ a dabara adaba maka ịsepụta ụdị ihe dị iche iche, gụnyere MPs.
N'ihi mkpebi dị elu na nke nwa oge, nyocha ASL na MCT anyị dabeere na PB-PCXI dabara nke ọma iji mụọ ike na usoro omume nke otu na nnukwu ihe dị na vivo iji nyere anyị aka ịghọta na ebuli usoro nnyefe MP.Ụzọ anyị si eji ebe a dabere na ọmụmụ ihe anyị na-eji SPring-8 BL20B2 beamline, nke anyị na-ahụ anya mmegharị mmiri na-esote nnyefe nke vector dummy n'ime imi na pulmonary airway nke ụmụ oke iji nyere aka kọwaa usoro okwu mkpụrụ ndụ dị iche iche hụrụ. n'ime mkpụrụ ndụ ihe nketa anyị.ọmụmụ anụmanụ na-ebu dose nke 3.4 .
Ebumnuche nke ọmụmụ a bụ iji PB-PCXI synchrotron na-ahụ anya na mmegharị vivo nke usoro MP na trachea nke oke dị ndụ.Emebere ọmụmụ ihe onyonyo PB-PCXI iji nwalee usoro MP, ike ndọta, na ọnọdụ iji chọpụta mmetụta ha na mmegharị MP.Anyị chere na oghere ndọta dị n'èzí ga-enyere MF aka ịnọrọ ma ọ bụ kwaga na mpaghara ebumnuche.Ọmụmụ ihe ndị a kwekwara ka anyị chọpụta nhazi ndọta nke na-ebuli oke irighiri ihe fọdụrụ na trachea mgbe etinyere ya.N'ime usoro ọmụmụ nke abụọ, anyị bu n'obi iji nhazi a kachasị mma iji gosipụta usoro ntụgharị nke sitere na nnyefe vivo nke LV-MPs n'ọdụ ụgbọ elu oke, na-eche na nnyefe nke LV-MPs n'ọnọdụ nke ịchọta ụgbọ elu ga-arụpụta. na mmụba LV arụmọrụ..
Emere ọmụmụ anụmanụ niile dabere na ụkpụrụ nke Mahadum Adelaide (M-2019-060 na M-2020-022) na Kọmitii Ethics Animal SPring-8 Synchrotron kwadoro.Emere nnwale ndị a dịka ntụzịaka nke ARRIVE siri dị.
A na-ese foto x-ray niile na BL20XU beamline na SPring-8 synchrotron na Japan site na iji ntọala yiri nke a kọwara na mbụ21,22.Na nkenke, igbe nnwale ahụ dị 245 m site na mgbanaka nchekwa synchrotron.A na-eji ihe nlele-na-achọpụta anya nke 0.6 m maka ọmụmụ ihe onyonyo na 0.3 m maka ọmụmụ ihe onyonyo vivo iji mepụta mmetụta dị iche na ngalaba.A na-eji ọkụ monochromatic nwere ike nke 25 keV.Ejiri ihe ntụgharị X-ray dị elu (SPring-8 BM3) nweta ihe onyonyo a yana ihe nchọpụta sCMOS.Onye ntụgharị na-atụgharị X-ray ka ọ bụrụ ìhè a na-ahụ anya site na iji scintillator 10 µm gbara ọkpụrụkpụ (Gd3Al2Ga3O12), nke a na-eduzi ya na ihe mmetụta sCMOS site na iji ebumnuche microscope ×10 (NA 0.3).Ihe nchọpụta sCMOS bụ Orca-Flash4.0 (Hamamatsu Photonics, Japan) nwere nha n'usoro nke pikselụ 2048 × 2048 yana nha pikselụ arụrụ nke 6.5 × 6.5 µm.Ntọala a na-enye nha pikselụ isotropic dị irè nke 0.51 µm yana oghere nlele dị ihe dịka 1.1 mm × 1.1 mm.Ahọpụtara oge mkpughe nke 100 ms iji bulie ogo mgbama na mkpọtụ nke ụmụ irighiri ihe ndọta n'ime na n'èzí ụzọ ikuku ebe a na-ebelata ihe omume mmegharị nke iku ume kpatara.Maka ọmụmụ vivo, a na-etinye ihe mkpuchi X-ray ngwa ngwa n'ụzọ X-ray iji kpachie dose radieshon site na igbochi oghere X-ray n'etiti mkpughe.
Ejighị mgbasa ozi LV mee ihe na ọmụmụ ihe onyonyo SPring-8 PB-PCXI ọ bụla n'ihi na ụlọ onyonyo BL20XU abụghị ọkwa Biosafety Level 2.Kama, anyị na-ahọrọ a nso nke ọma mara MPs si abụọ azụmahịa mgbere na-ekpuchi a nso nke nha, ihe, ígwè ịta, na ngwa, - mbụ iji ghọta otú magnetik ubi emetụta ije nke MPs na iko capillaries, na mgbe ahụ na. ụzọ ikuku ndụ.elu.Ogo MP dị iche site na 0.25 ruo 18 µm ma mee ya site na ihe dị iche iche (lee Tebụl 1), mana ihe mejupụtara nke ihe nlele ọ bụla, gụnyere nha nke ihe ndọta dị na MP, amabeghị.Dabere na nnukwu ọmụmụ MCT anyị 19, 20, 21, 23, 24, anyị na-atụ anya na ndị omeiwu gbadara 5 µm nwere ike ịhụ n'elu elu ikuku tracheal, dịka ọmụmaatụ, site n'iwepụ okpokolo agba na-esochi iji hụ ọganihu nke mmegharị MP.Otu MP nke 0.25 µm pere mpe karịa mkpebi nke ngwaọrụ onyonyo, mana a na-atụ anya PB-PCXI ịchọpụta ọdịiche volumetric ha na mmegharị nke mmiri mmiri dị n'elu ebe edobere ha mgbe etinyechara ya.
Ihe nlele maka MP ọ bụla na tebụl.A kwadebere 1 na 20 μl iko capillaries (Drummond Microcaps, PA, USA) nke nwere dayameta dị n'ime nke 0.63 mm.Anụ ahụ dị n'ime mmiri, ebe CombiMag dị na mmiri mmiri nke onye nrụpụta.Ọkara ọ bụla jupụtara na mmiri mmiri (ihe dị ka 11 µl) tube ọ bụla ma tinye ya na njide nlele (lee foto 1).A na-etinye capillaries iko ahụ n'ahịrị na ogbo n'ime ụlọ ihe ngosi, n'otu n'otu, ma tinye ya na nsọtụ nke mmiri mmiri.Akara 19 mm dayameta (28 mm ogologo) nickel-shell magnet mere nke ụwa na-adịghị ahụkebe, neodymium, iron na boron (NdFeB) (N35, pusi no. LM1652, Jaycar Electronics, Australia) na remanence nke 1.17 T jikọtara ya na a. Tebụl mbufe dị iche iche iji nweta gbanwee ọnọdụ gị mgbe ị na-enye ihe.Onyonyo X-ray na-amalite mgbe ndọta edobere ihe dịka 30 mm n'elu ihe nlele ahụ wee nweta onyonyo na okpokolo agba anọ kwa sekọnd.N'oge eserese, a na-ebute magnetik nso tube capillary iko (n'ebe dị anya nke ihe dịka 1 mm) wee gaa n'akụkụ tube ahụ iji chọpụta mmetụta nke ike na ọnọdụ ubi.
Ntọlite ​​onyonyo in vitro nwere ihe nlele MP na capillaries ugegbe n'oge ntụgharị asụsụ xy.Ụzọ nke oghere X-ray ka ejiri eriri nwere ntụpọ uhie ka akara.
Ozugbo e hiwere vitro vitro nke MPs, a nwalere akụkụ nke ha na vivo na ụdị anụ ọhịa Wistar albino ụmụ nwanyị (~ 12 izu, ~ 200 g).Medetomidine 0.24 mg/kg (Domitor®, Zenoaq, Japan), midazolam 3.2 mg/kg (Dormicum®, Astellas Pharma, Japan) na butorphanol 4 mg/kg (Vetorphale®, Meiji Seika).Ejiri ngwakọta Pharma (Japan) were gbasie oke oke ahụ n'ọnụ site na ntụtụ intraperitoneal.Mgbe mgbanyụ ahụ gasịrị, a kwadebere ha maka ịse foto site n'iwepụ ajị anụ gburugburu trachea, na-etinye tube endotracheal (ET; 16 Ga intravenous cannula, Terumo BCT), ma mee ka ha nọrọ n'ọnọdụ dị elu na efere ihe ngosi omenala nke nwere akpa ọkụ. iji nọgide na-enwe okpomọkụ ahụ.22. A na-ejikọta efere ihe oyiyi ahụ na ọkwa nlele na igbe ihe ngosi na ntakịrị akụkụ iji kwado trachea n'ahịrị na foto x-ray dị ka egosiri na foto 2a.
(a) Ntọlite ​​onyonyo vivo na ngalaba onyonyo SPring-8, okporo ụzọ beam X-ray nke akara akara akara uhie.(b,c) A na-eme ebe ndọta tracheal n'ebe dịpụrụ adịpụ site na iji igwefoto IP abụọ etinyere na orthogonally.N'akụkụ aka ekpe nke onyonyo na ihuenyo, ị nwere ike ịhụ loop waya na-ejide isi na cannula nnyefe arụnyere n'ime tube ET.
Sistemụ mgbapụta sirinji a na-achịkwa nke ọma (UMP2, World Precision Instruments, Sarasota, FL) na-eji sirinji iko 100 µl jikọọ na tubing PE10 (0.61 mm OD, 0.28 mm ID) na-eji agịga 30 Ga.Kaa akara na tube ahụ iji hụ na ọnụ ahụ dị n'ọnọdụ ziri ezi na trachea mgbe ị na-etinye tube endotracheal.N'iji micropump, e wepụrụ plunger sirinji wee mikpuo ọnụ ọnụ tube n'ime ihe nlele MP nke a ga-ebuga.A na-etinyezi tube nnyefe a na-ebunye n'ime tube endotracheal, na-etinye ọnụ n'akụkụ kachasị ike nke oghere magnetik anyị na-atụ anya ya.A na-achịkwa nnweta onyonyo site na iji ihe nchọta iku ume jikọtara na igbe oge dabere na Arduino, yana akara ngosi niile (dịka ọmụmaatụ, okpomọkụ, iku ume, oghere meghere/mmechi, na nnweta onyonyo) ka edekọtara site na iji Powerlab na LabChart (AD Instruments, Sydney, Australia) 22 Mgbe Onyonyo Mgbe ụlọ na-adịghị, igwefoto IP abụọ (Panasonic BB-SC382) ka edobere na ihe dịka 90 Celsius n'otu n'otu ma jiri ya na-achịkwa ọnọdụ magnet n'akụkụ trachea n'oge eserese (Nyocha 2b, c).Iji wedata arịa mmegharị ahụ, enwetara otu onyonyo n'otu ume n'oge oke ikuku iku ume.
A na-ejikọta ndọta ahụ na ọkwa nke abụọ, nke nwere ike ịdị na-adị anya na mpụga nke ahụ onyonyo.A nwalere ọnọdụ dị iche iche na nhazi nke magnet, gụnyere: etinye ya n'akụkụ ihe dịka 30 Celsius n'elu trachea (nhazi na-egosi na Figures 2a na 3a);otu magnet n'elu anụmanụ na nke ọzọ dị n'okpuru ebe a, nke nwere okporo osisi ndị e debere maka mmasị (Fig 3b)., otu magnet n'elu anụmanụ na nke dị n'okpuru, na okporo osisi ndị e debere maka ịkpọasị (Ọnọdụ 3c), na otu magnet n'elu na perpendicular na trachea (Figure 3d).Mgbe ịtọlitechara anụ ahụ na ndọta wee tinye MP n'okpuru ule n'ime mgbapụta sirinji, wepụta dose nke 50 µl na ọnụego 4 µl / sk mgbe ị nwetara onyonyo.A na-ebugharị ndọta ahụ azụ na gaba n'akụkụ ma ọ bụ gafee trachea ka ọ na-aga n'ihu inweta onyonyo.
Nhazi ndọta maka in vivo imaging (a) otu magnet n'elu trachea n'akụkụ ihe dịka 30º, (b) magnetik abụọ ahaziri maka mma, (c) magnetik abụọ ahaziri maka ịsọ oyi, (d) otu magnet dị n'elu na perpendicular n'akụkụ ahụ. trachea.Onye na-ekiri ya lere anya site n'ọnụ ruo na ngụgụ site na trachea na oghere X-ray gafere n'akụkụ aka ekpe nke oke ahụ wee pụọ n'akụkụ aka nri.A na-ebugharị ndọta n'ogologo ụzọ ikuku ma ọ bụ aka ekpe na aka nri n'elu trachea n'akụkụ ụzọ X-ray beam.
Anyị chọkwara ịchọpụta visibiliti na omume nke ụmụ irighiri ihe na ikuku ikuku na enweghị ngwakọta nke iku ume na obi obi.Ya mere, na njedebe nke oge eserese ahụ, a na-eme ka ụmụ anụmanụ nwee mmetụta nke mmadụ n'ihi nkwụsị nke pentobarbital (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~ 65 mg/kg ip).A na-ahapụ ụfọdụ anụmanụ n'elu ikpo okwu onyonyo, na mgbe nkwụsị nke iku ume na nkụchi obi, a na-emeghachi usoro ihe ngosi ahụ, na-agbakwunye mgbakwunye ọzọ nke MP ma ọ bụrụ na ọ dịghị MP na-ahụ anya n'elu elu ikuku.
A na-edozi ihe onyonyo a rụpụtara maka oghere dị larịị na nke gbara ọchịchịrị wee kpọkọta ya na ihe nkiri (Freek 20 kwa nkeji; 15–25 × ọsọ ọsọ dabere na ọnụego iku ume) site na iji edemede omenala edere na MATLAB (R2020a, The Mathworks).
Emere ọmụmụ ihe niile gbasara nnyefe vector LV gene na Mahadum Adelaide Laboratory Animal Research Center na ebumnuche iji nsonaazụ nke nnwale SPring-8 chọpụta ma nnyefe LV-MP n'ihu oghere ndọta nwere ike ịkwalite mbufe mkpụrụ ndụ na vivo. .Iji nyochaa mmetụta nke MF na magnetik, a na-emeso ìgwè abụọ nke anụmanụ: otu ìgwè na-agbanye LV MF na ntinye magnet, na ndị ọzọ na-agbanye otu njikwa na LV MF na-enweghị magnet.
A na-emepụta vectors nke LV site na iji ụzọ akọwara mbụ 25, 26 .Vector LacZ na-egosipụta mkpụrụ ndụ beta-galactosidase nke mpaghara mpaghara nuklia nke MPSV mebere nkwalite (LV-LacZ), nke na-emepụta ngwaahịa mmeghachi omume na-acha anụnụ anụnụ n'ime mkpụrụ ndụ atụgharị, nke a na-ahụ anya n'ihu na akụkụ anụ ahụ ngụgụ.Emere titration na omenala cell site na iji aka na-agụ ọnụ ọgụgụ nke mkpụrụ ndụ LacZ-positive site na iji hemocytometer iji gbakọọ titer na TU/ml.A na-echekwa ndị na-ebu ya na -80 Celsius C, gbazee tupu ejiri ya, ma kee ya na CombiMag site na ịgwakọta 1: 1 na incubating na ice maka opekata mpe 30 nkeji tupu ebuga ya.
Normal Sprague Dawley oke (n = 3 / otu, ~ 2-3 ip anesthetized na ngwakọta nke 0.4mg / kg medetomidine (Domitor, Ilium, Australia) na 60mg / kg ketamine (Ilium, Australia) na 1 ọnwa) ip. ) ogwu ogbugba na nke na-abụghị nke ịwa ahụ site n'ọnụ ọnụ nwere 16 Ga intravenous cannula.Iji hụ na anụ ahụ ikuku tracheal na-enweta ntụgharị LV, a na-edozi ya site na iji usoro mgbagwoju anya nke anyị kọwara na mbụ bụ nke a na-eji nkata waya na-ehichapụ tracheal airway elu nke axially na nkata waya (N-Circle, nitinol stone extractor without tip NTSE-022115 ) -UDH , Cook Medical, USA) 30 p28.Mgbe ahụ, ihe dị ka nkeji 10 ka ọ gbasasịrị na kabinet biosafety, a na-eme nlekọta tracheal nke LV-MP.
A haziri oghere ndọta ejiri mee nnwale a ka ọ dị ka ihe ọmụmụ x-ray in vivo, yana otu ihe ndọta ahụ ejiri n'elu trachea nwere mgbachi stent distillation (Nyocha 4).A na-ebunye olu 50 µl (2 x 25 µl aliquots) nke LV-MP na trachea (n = anụmanụ 3) site na iji pipette gel-tipped dị ka akọwara na mbụ.Otu njikwa (n = 3 anụmanụ) nwetara otu LV-MP n'ejighị magnetik.Mgbe emechara infusion, a na-ewepụ cannula na tube endotracheal ma na-ekpochapụ anụ ahụ.Igwe ndọta ahụ na-anọ ebe nkeji iri tupu ewepụ ya.Ejiri meloxicam (1 ml/kg) were meloxicam (1 ml/kg) were were were mebie oke oke n'ime ala nke na-esote ya na mwepụ anesthesia site na intraperitoneal injection nke 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia).A na-ekpo ọkụ ma hụ oke oke ruo mgbe mgbake zuru oke site na nrịanrịa.
Ngwa nnyefe LV-MP n'ime kabinet nchekwa ndụ.Ị nwere ike ịhụ na aka uwe Luer-mkpọchi isi awọ nke ET tube na-esi n'ọnụ pụta, a na-etinyekwa n'ọnụ gel pipette na ọnụ ọgụgụ ahụ site na tube ET ruo omimi a chọrọ n'ime trachea.
Otu izu ka usoro nchịkwa LV-MP gasịrị, a na-eji obi mmadụ chụọ anụmanụ site na iku ume nke 100% CO2 na okwu LacZ ka a na-enyocha site na iji ọgwụgwọ X-gal anyị.E wepụrụ mgbanaka cartilage atọ kachasị mma iji hụ na ihe ọ bụla mebiri emebi ma ọ bụ njide mmiri n'ihi ntinye tube endotracheal agaghị etinye na nyocha.A na-egbutu trachea ọ bụla n'ogologo iji nweta ọkara abụọ maka nyocha ma tinye ya n'ime iko nwere roba silicone (Sylgard, Dow Inc) na-eji agịga Minutien (Fine Science Tools) iji hụ anya n'elu luminal.E gosipụtara nkesa na agwa nke mkpụrụ ndụ ndị a gbanwere site na foto ihu ihu site na iji microscope Nikon (SMZ1500) nwere igwefoto DigiLite na TCapture software (Tucsen Photonics, China).Enwetara ihe oyiyi na 20x na-ebuwanye ibu (gụnyere ntọala kachasị maka obosara zuru oke nke trachea), na ogologo ogologo trachea ahụ gosipụtara nzọụkwụ site na nzọụkwụ, na-enye nkwụsịtụ zuru oke n'etiti ihe oyiyi ọ bụla iji mee ka ihe oyiyi "kpachie".A na-ejikọta foto ndị sitere na trachea nke ọ bụla n'ime otu ihe oyiyi mejupụtara site na iji Composite Image Editor version 2.0.3 (Microsoft Research) na-eji usoro mmegharị ahụ. Mpaghara okwu LacZ dị n'ime tracheal mejupụtara onyonyo sitere na anụmanụ ọ bụla ka ejiri ederede MATLAB akpaaka (R2020a, MathWorks) dị ka akọwara na mbụ28, na-eji ntọala 0.35 <Hue <0.58, Saturation> 0.15, na Uru <0.7. A na-atụle mpaghara okwu LacZ n'ime tracheal ihe oyiyi sitere na anụmanụ ọ bụla site na iji edemede MATLAB akpaaka (R2020a, MathWorks) dị ka akọwara na mbụ28, na-eji ntọala 0.35 <Hue <0.58, Saturation> 0.15, na Uru <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. A na-atụle mpaghara okwu LacZ na tracheal ihe oyiyi sitere na anụmanụ ọ bụla site na iji edemede MATLAB akpaaka (R2020a, MathWorks) dị ka akọwara na mbụ28 site na iji ntọala 0.35.0.15 na uru <0 .7.如, 述 自动 自动 自动 自动 (R2020a, Mathworks): 来自 的 lacz 表达 的 lacz 表达 进行 进行 0,15, 和值 <0.7 的 的.前所, 述 自动 Matlab 脚本 ((R2020a, MathWorts)) 每 使用 使用 使用 使用 使用 使用 使用 色调 <0.15 和值 ..... …………………………………………………………………………………… 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 . A na-edepụta mpaghara okwu LacZ na ihe oyiyi nke trachea nke anụmanụ ọ bụla site na iji edemede MATLAB na-akpaghị aka (R2020a, MathWorks) dị ka akọwara na mbụ site na iji ntọala 0.35 < hue <0.58, saturation> 0.15 na uru <0.7.Site n'ịchọgharị anụ ahụ na GIMP v2.10.24, ejiri aka mee ihe nkpuchi maka ihe oyiyi ọ bụla mejupụtara iji chọpụta mpaghara anụ ahụ ma gbochie nchọpụta ụgha ọ bụla n'èzí tracheal anụ ahụ.Achịkọtara ebe ntụpọ sitere na ihe oyiyi niile mejupụtara nke anụmanụ ọ bụla iji nye mkpokọta ntụpọ maka anụmanụ ahụ.E kewara ebe a na-ese ihe site na mkpokọta nkpuchi ahụ iji nweta ebe a na-ahazi.
A gbanyere trachea nke ọ bụla n'ime paraffin wee kewaa akụkụ 5 µm n'obosara.A na-eji igwe na-acha uhie uhie na-anọpụ iche kpuchie akụkụ ndị ahụ maka nkeji 5 wee nweta onyonyo site na iji microscope Nikon Eclipse E400, igwefoto DS-Fi3 na ngwa NIS mmewere na-ejide ngwa ngwa (ụdị 5.20.00).
Emere nyocha ndekọ aha niile na GraphPad Prism v9 (GraphPad Software, Inc.).Edebere mkpa ndekọ aha na p ≤ 0.05.A nwalere ụkpụrụ site na iji nyocha Shapiro-Wilk na ọdịiche dị na ntụpọ LacZ ka a na-enyocha site na iji t-ule na-enweghị atụ.
Ndị MP isii a kọwara na Tebụl 1 bụ PCXI nyochara ya, a kọwakwara visibiliti na Tebụl 2. Ndị MP abụọ polystyrene (MP1 na MP2; 18 µm na 0.25 µm, n'otu n'otu) enweghị ike ịhụ site na PCXI, mana enwere ike ịchọpụta ihe atụ ndị fọdụrụnụ. (ihe atụ na-egosi na foto nke 5).MP3 na MP4 na-adịghị ahụkebe (10-15% Fe3O4; 0.25 µm na 0.9 µm, n'otu n'otu).Ọ bụ ezie na MP5 (98% Fe3O4; 0.25 µm) nwere ụfọdụ n'ime ụmụ irighiri ihe kacha nta nwalere, ọ bụ nke kacha akpọpụta.Ngwaahịa CombiMag MP6 siri ike ịmata.N'ọnọdụ niile, ike anyị ịchọpụta MF ka emelitere nke ukwuu site n'ịkwagharị magnet azụ na azụ n'otu aka ahụ na capillary.Ka ihe ndọta ahụ na-esi na capillary pụọ, a dọpụtara ihe ndị ahụ n'agbụ dị ogologo, mana ka ndọta na-abịaru nso na ike ndọta na-abawanye, agbụ ndị ahụ na-ebelata ka ụmụ irighiri ihe na-akwaga n'elu elu nke capillary (lee vidiyo mgbakwunye S1). : MP4), ịba ụba urughuru njupụta n'elu.N'aka nke ọzọ, mgbe e wepụrụ magnet na capillary, ike ubi na-ebelata ma ndị MP na-edozigharị n'agbụ dị ogologo na-esi na elu elu nke capillary (lee Video S2: MP4).Mgbe magnet kwụsịrị ịkwagharị, ihe ndị ahụ na-aga n'ihu ruo oge ụfọdụ mgbe ha rutere n'ọnọdụ nha.Ka MP na-aga n'ihu na pụọ ​​na elu elu nke capillary, ihe ndọta na-adọta irighiri ihe site na mmiri mmiri.
Ọhụụ nke MP n'okpuru PCXI dịgasị iche iche n'etiti ihe nlele.(a) MP3, (b) MP4, (c) MP5 na (d) MP6.Ewere ihe ndọta edobere ihe dịka 10 mm ozugbo n'elu capillary.Okirikiri buru ibu pụtara ìhè bụ ikuku ikuku tọrọ n'ime capillaries, na-egosi n'ụzọ doro anya njirimara akụkụ ojii na nke ọcha nke foto ọdịiche nke oge.Igbe uhie na-egosi mmụba nke na-eme ka ọdịiche ahụ dịkwuo mma.Rịba ama na dayameta nke sekit magnetik n'ọnụọgụ niile agaghị abawanye ma buru ihe dịka 100 ugboro karịa egosiri.
Ka magnet na-aga n'aka ekpe na aka nri n'elu capillary, akụkụ nke eriri MP na-agbanwe ka ọ kwekọọ na magnet (lee foto 6), si otú a na-akọwapụta ahịrị oghere magnet.Maka MP3-5, mgbe ụda ahụ rutere n'akụkụ ọnụ ụzọ, irighiri ihe ndị ahụ na-adọkpụrụ n'elu elu nke capillary.Nke a na-ebutekarị ndị omeiwu na-agbakọta n'ìgwè buru ibu n'akụkụ ebe magnetik kacha sie ike (lee Vidiyo S3: MP5).Nke a na-apụtakwa ìhè karịsịa mgbe ị na-ese onyinyo dị nso na njedebe nke capillary, nke na-eme ka MP na-agbakọta ma tinye uche na mmiri mmiri-ikuku interface.Ụmụ irighiri ihe dị na MP6, bụ ndị siri ike ịmata ọdịiche karịa ndị dị na MP3-5, adọpụghị mgbe magnet ahụ na-aga n'akụkụ capillary, mana eriri MP kewara, na-ahapụ ihe ndị ahụ na-ele anya (lee Video S4: MP6).N'ọnọdụ ụfọdụ, mgbe oghere ndọta etinyere belatara site na ịkwaga magnet n'ebe dị anya site na saịtị onyonyo, ndị omeiwu ọ bụla fọdụrụ ji nwayọọ nwayọọ na-agbada n'elu ala nke tube site na ike ndọda, na-anọgide na eriri (lee Video S5: MP3). .
Akụkụ nke eriri MP na-agbanwe ka magnet na-aga n'aka nri n'elu capillary.(a) MP3, (b) MP4, (c) MP5 na (d) MP6.Igbe uhie na-egosi mmụba nke na-eme ka ọdịiche ahụ dịkwuo mma.Biko mara na vidiyo ndị agbakwunyere bụ maka ebumnuche ozi ka ha na-ekpughe ụkpụrụ dị mkpa na ozi dị ike nke enweghị ike ịhụ anya na onyonyo ndị a kwụ ọtọ.
Nnwale anyị egosila na iji nwayọọ nwayọọ na-ebugharị magnet na azụ na trachea na-eme ka anya nke MF na ọnọdụ nke mgbagwoju anya na vivo.Enweghị ule in vivo n'ihi na a naghị ahụ beads polystyrene (MP1 na MP2) na capillary.A nwalere nke ọ bụla n'ime MF anọ fọdụrụ na vivo na ogologo axis nke magnet ahụ etinyere n'elu trachea n'akụkụ ihe dịka 30 ° na vetikal (lee foto 2b na 3a), n'ihi na nke a mere ka agbụ MF dị ogologo ma dị irè karị. karịa magnet..nhazi akwụsị.MP3, MP4 na MP6 ahụbeghị na trachea nke anụmanụ ọ bụla dị ndụ.Mgbe ị na-ahụ anya traktị iku ume nke oke ka ha gbuchara ụmụ anụmanụ n'ụzọ mmadụ, ihe ndị ahụ ka na-adịghị ahụ anya ọbụlagodi mgbe agbakwunyere olu ọzọ site na iji mgbapụta sirinji.MP5 nwere ọdịnaya ígwè oxide kachasị elu ma bụrụ naanị ihe a na-ahụ anya, ya mere ejiri ya nyochaa ma kọwaa omume MP na vivo.
Idonye ndọta n'elu trachea n'oge ntinye MF mere ka ọtụtụ, ma ọ bụghị ihe niile, MF na-etinye uche na mpaghara nlele.A na-ahụta ntinye tracheal nke ụmụ irighiri ihe n'ime anụmanụ ndị euthanized na-adịghị mma.Onyonyo 7 na Video S6 mgbakwunye: MP5 na-egosi njide ngwa ngwa magnetik na nhazi nke ihe dị n'elu trachea ventral, na-egosi na ndị MP nwere ike irube n'akụkụ ebe a chọrọ nke trachea.Mgbe a na-achọgharị anya n'akụkụ trachea mgbe nnyefe MF gasịrị, ahụrụ ụfọdụ MF nso na carina, nke na-egosi enweghị ike ndọta zuru oke iji nakọta ma jide MF niile, ebe ọ bụ na a napụtara ha site na mpaghara ike ndọta kachasị n'oge nchịkwa mmiri.usoro.Agbanyeghị, nleba anya MP mgbe ọmụmụ nwa dị elu karịa mpaghara onyonyo a, na-atụ aro na ọtụtụ ndị omeiwu ka nọ na mpaghara ụgbọ elu ebe ike ndọta etinyere dị elu.
Onyonyo nke (a) tupu na (b) mgbe ebufesịrị MP5 n'ime trachea nke oke euthanized na nso nso a nwere magnet etinyere n'elu ebe onyonyo.Ebe a gosipụtara dị n'etiti mgbanaka cartilaginous abụọ.Enwere mmiri mmiri n'ụzọ ikuku tupu ebupụta MP.Igbe uhie na-egosi mmụba nke na-eme ka ọdịiche ahụ dịkwuo mma.A na-ese foto ndị a na vidiyo e gosipụtara na S6: MP5 Mgbakwụnye vidiyo.
Ịkwagharị magnet n'akụkụ trachea na vivo mere ka mgbanwe dị na akụkụ nke yinye MP n'elu elu ụgbọ elu, yiri nke a na-ahụ na capillaries (lee foto 8 na Video S7: MP5).Otú ọ dị, n'ime ọmụmụ ihe anyị, a pụghị ịdọrọ ndị MP n'elu traktị iku ume ndụ, dị ka capillaries nwere ike ime.Mgbe ụfọdụ, agbụ MP na-agbatịkwu ka magnet na-aga n'aka ekpe na aka nri.N'ụzọ na-akpali mmasị, anyị chọpụtakwara na agbụ ahụ na-agbanwe omimi nke elu oyi akwa nke mmiri mmiri mgbe magnet na-emegharị ogologo oge n'akụkụ trachea, na-agbasawanye mgbe magnet na-ebugharị ozugbo n'elu na agbụ ahụ na-atụgharị gaa na vetikal (lee anya). Video mgbakwunye S7).: MP5 na 0:09, ala aka nri).Ụdị mmegharị ahụ e ji mara ya gbanwere mgbe a na-ebugharị magnet n'akụkụ n'akụkụ elu nke trachea (ya bụ, n'aka ekpe ma ọ bụ aka nri nke anụmanụ, karịa n'ogologo trachea).A ka na-ahụ ụmụ irighiri ihe ndị ahụ nke ọma n'oge mmegharị ha, mana mgbe e wepụrụ magnet na trachea, a na-ahụ ntụmadị nke eriri irighiri ahụ (lee vidiyo S8: MP5, malite na 0:08).Nke a na-ekwenye na omume a na-ahụ anya nke oghere magnetik n'okpuru ọrụ nke oghere magnetik etinyere na capillary ugegbe.
Ihe onyonyo onyonyo na-egosi MP5 n'ime trachea nke oke na-egbu egbu.(a) A na-eji magnet nweta ihe oyiyi n'elu na n'aka ekpe nke trachea, mgbe ahụ (b) mgbe ọ na-ebugharị magnet n'aka nri.Igbe uhie na-egosi mmụba nke na-eme ka ọdịiche ahụ dịkwuo mma.Ihe onyonyo ndị a sitere na vidiyo egosipụtara na vidiyo mgbakwunye S7: MP5.
Mgbe a na-emegharị okporo osisi abụọ a na mgbago ugwu-ndịda n'elu na n'okpuru trachea (ya bụ, na-adọta; Fig. 3b), ụda MP pụtara ogologo oge ma dị na mgbidi mpụta nke trachea kama ịnọ n'elu azụ azụ nke trachea. trachea (lee ihe odide ntụkwasị).Vidiyo S9: MP5).Otú ọ dị, achọpụtaghị mmụkọ dị elu nke ụmụ irighiri ihe n'otu saịtị (ya bụ, ihu azụ nke trachea) mgbe nchịkwa mmiri gasịrị site na iji ngwaọrụ magnet abụọ, nke na-emekarị site na otu ngwaọrụ magnet.Mgbe ahụ, mgbe a haziri otu magnet ka ọ chụghachite okporo osisi ndị na-abụghị (Njirimara 3c), ọnụ ọgụgụ ụmụ irighiri ihe a na-ahụ anya n'ọhụụ amụbaghị mgbe ebufechara ya.Ịtọlite ​​​​ma ọ bụ nhazi magnet abụọ bụ ihe ịma aka n'ihi ike ndọta dị elu nke na-adọta ma ọ bụ na-akwagharị magnetik n'otu n'otu.A gbanwere ntọala ahụ ka ọ bụrụ otu magnet dị na ọdụ ụgbọ elu ma na-agafe n'okporo ụzọ ikuku na ogo 90 nke mere na ahịrị ike gafere mgbidi tracheal n'ụzọ ziri ezi (Ọnọdụ 3d), nghazi nke e bu n'obi iji chọpụta ohere nke nchịkọta ahụ. mgbidi n'akụkụ.hụ.Agbanyeghị, na nhazi a, enweghị mmegharị mkpokọta MF nwere ike ịmata ma ọ bụ mmegharị magnet.Dabere na nsonaazụ ndị a niile, a họọrọ nhazi nke nwere otu magnet na nhazi ọkwa 30 maka ọmụmụ vivo nke ndị na-ebu mkpụrụ ndụ (Fig 3a).
Mgbe a na-ese onyinyo anụmanụ ahụ ọtụtụ oge ozugbo e jirichara mmadụ chụọ àjà, enweghị mmegharị anụ ahụ na-etinye aka pụtara na a ga-ahụta ahịrị ahụ dị mkpụmkpụ nke dị mkpụmkpụ n'ọhịa intercartilaginous doro anya, 'na-amagharị' dịka ntụgharị ntụgharị nke magnet.hụ n'ụzọ doro anya na ọnụnọ na ije nke MP6 ahụ.
Ọnụ ọgụgụ nke LV-LacZ bụ 1.8 x 108 IU/mL, na mgbe ịgwakọta 1: 1 na CombiMag MP (MP6), a na-agbanye ụmụ anụmanụ na 50 µl nke tracheal dose nke 9 x 107 IU / ml nke ụgbọ LV (ya bụ 4.5). x 106 TU / oke).)).N'ime ọmụmụ ihe ndị a, kama ịkwagharị magnet n'oge ọrụ, anyị na-edozi magnet n'otu ọnọdụ iji chọpụta ma ọ bụrụ na ntụgharị LV nwere ike (a) ka ọ dịkwuo mma ma e jiri ya tụnyere nnyefe vector na enweghị oghere magnetik, na (b) ma ọ bụrụ na ụgbọ elu nwere ike. lekwasị anya.A na-atụgharị mkpụrụ ndụ ndị ahụ n'akụkụ ebe ebumnuche magnetik nke elu akụkụ iku ume.
Ọnụnọ nke magnets na ojiji CombiMag yana vectors LV egosighi na ọ na-emetụta ahụike anụmanụ n'ụzọ dị njọ, dị ka ọkọlọtọ nnyefe vector LV ọkọlọtọ anyị si mere.Onyonyo dị n'ihu nke mpaghara tracheal nke etinyere n'ọrụ arụrụ arụ (Mgbakwunye Fig. 1) gosiri na otu LV-MP mesoro nwere ọkwa ntụgharị dị elu nke ukwuu na ọnụnọ magnet (Fig 9a).Naanị obere ntụpọ LacZ na-acha anụnụ anụnụ dị na otu njikwa (Foto 9b).Ọnụ ọgụgụ nke mpaghara X-Gal na-eme ka ọ dị mma gosiri na nchịkwa nke LV-MP n'ihu ebe magnetik mere ka ọ dịkwuo ihe dịka 6 ugboro (Fig 9c).
Ọmụmaatụ nke onyonyo agwakọtara na-egosi ntụgharị tracheal na LV-MP (a) n'ihu oghere ndọta yana (b) na enweghị ndọta.(c) Mgbanwe dị ịrịba ama na mpaghara nkịtị nke ntụgharị LacZ na trachea site na iji magnet (* p = 0.029, t-test, n = 3 kwa otu, pụtara ± njehie ọkọlọtọ nke pụtara).
Akụkụ ndị na-acha uhie uhie na-anọpụ iche (ihe atụ egosipụtara na Fig. 2) gosiri na sel ndị nwere ntụpọ LacZ dị n'otu nlele na n'otu ebe dịka akọwara na mbụ.
Ihe ịma aka dị mkpa na usoro ọgwụgwọ mkpụrụ ndụ ikuku na-anọgide na-abụ kpọmkwem nhazi nke ihe ndị na-ebu ụgbọelu na mpaghara mmasị na mmezu nke ọkwa dị elu nke mmepụta n'ime akpa ume mkpanaka n'ihu ikuku ikuku na mkpochapụ na-arụ ọrụ.Maka ndị na-ebu LV ezubere maka ọgwụgwọ ọrịa iku ume na cystic fibrosis, ịba ụba oge obibi nke ụmụ irighiri ihe na-ebu n'ime ụzọ ikuku na-eduzi ugbu a bụ ihe mgbaru ọsọ a na-apụghị iru.Dị ka Castellani et al kwuru, iji oghere ndọta na-eme ka ntụgharị nwere uru karịa ụzọ nnyefe mkpụrụ ndụ ihe nketa ndị ọzọ dị ka electroporation n'ihi na ọ nwere ike ijikọta mfe, akụ na ụba, nnyefe mpaghara, ịba ụba arụmọrụ, na oge ntinye oge dị mkpirikpi.na ikekwe obere dose nke ụgbọ ala10.Otú ọ dị, na vivo deposition na omume nke magnetik particles na airways n'okpuru mmetụta nke mpụga magnetik ike ọ dịtụghị mgbe a kọwara, na n'ezie ike nke usoro a na-amụba mkpụrụ ndụ okwu larịị na emebibeghị emebi ikuku ụzọ ndụ na vivo.
Nnwale in vitro anyị na PCXI synchrotron gosiri na ihe niile anyị nwalere, ewezuga MP polystyrene, na-ahụ anya na nhazi onyonyo anyị ji.N'ihu oghere magnetik, oghere ndọta na-etolite eriri, ogologo ya na-ejikọta ya na ụdị irighiri ihe na ike nke oghere magnetik (ya bụ, nso na mmegharị nke magnet).Dị ka egosiri na eserese 10, eriri ndị anyị na-ahụ na-etolite ka akụkụ nke ọ bụla na-aghọ magnetized na-akpalite oghere magnetik nke ya.Ndị a dị iche iche ubi na-eme ka ndị ọzọ yiri ahụ na-anakọta na jikọọ na otu eriri mmegharị n'ihi obodo agha si obodo ike nke mma na ichupu nke ndị ọzọ ahụ.
Eserese na-egosi (a, b) agbụ nke ụmụ irighiri ihe na-etolite n'ime capillaries jupụtara na mmiri na (c, d) trachea jupụtara n'ikuku.Rịba ama na a naghị adọta capillaries na trachea na ọnụ ọgụgụ.Ogwe (a) nwekwara nkọwa nke MF nwere ụmụ irighiri ihe Fe3O4 ahaziri n'agbụ.
Mgbe magnet ahụ kwagara n'elu capillary, akụkụ nke eriri ahụ ruru ebe dị oke egwu maka MP3-5 nwere Fe3O4, mgbe nke ahụ gasịrị, eriri ahụ adịkwaghị n'ọnọdụ mbụ ya, kama ọ kwagara n'elu gaa n'ọnọdụ ọhụrụ.magnet.O yikarịrị ka mmetụta a na-eme n'ihi na elu nke capillary enyo dị mma nke ọma iji kwe ka mmegharị a mee.N'ụzọ na-akpali mmasị, MP6 (CombiMag) emeghị omume otú a, ikekwe n'ihi na ụmụ irighiri ihe ndị ahụ dị ntakịrị, nwere mkpuchi dị iche iche ma ọ bụ ụgwọ elu, ma ọ bụ mmiri na-ebu ibu na-emetụta ikike ha nwere ike ịkwaga.Ọdịiche dị na ihe oyiyi ahụ CombiMag na-esikwa ike, na-atụ aro na mmiri mmiri na irighiri ihe nwere ike inwe otu njupụta ma ya mere enweghị ike ịkwaga ibe ha ngwa ngwa.Akụkụ ahụ nwekwara ike ịrapagidesi ike ma ọ bụrụ na magnet na-agba ọsọ ngwa ngwa, na-egosi na ike ndọta enweghị ike imeri esemokwu dị n'etiti ihe ndị dị na mmiri ahụ mgbe niile, na-atụ aro na ike magnetik na anya dị n'etiti magnet na ebe e lekwasịrị anya ekwesịghị ịbịa dị ka a. ihe ijuanya.mkpa.Nsonaazụ ndị a na-egosikwa na ọ bụ ezie na magnets nwere ike ijide ọtụtụ microparticles na-asọfe ebe a na-achọsi ike, o yighị ka a ga-adabere na ndọta ibugharị ihe CombiMag n'akụkụ elu nke trachea.Ya mere, anyị kwubiri na na vivo LV ọmụmụ MF kwesịrị iji oghere ndọta static iji lekwasị anya n'akụkụ ụfọdụ nke osisi ikuku.
Ozugbo a na-ebunye ihe ndị ahụ n'ime ahụ, ọ na-esiri ha ike ịmata n'ọnọdụ dị mgbagwoju anya nke anụ ahụ na-akpụ akpụ nke anụ ahụ, ma a na-eme ka ike nchọpụta ha dịkwuo mma site n'ịkwagharị magnet n'akụkụ trachea iji "na-emegharị" eriri MP.Ọ bụ ezie na ịse foto n'ezie ga-ekwe omume, ọ dị mfe ịghọta mmegharị ahụ mgbe e gbuchara anụmanụ ahụ n'ụzọ mmadụ.Ọnụnọ MP na-adịkarị elu n'ebe a mgbe a na-etinye magnet n'elu ebe a na-ese foto, ọ bụ ezie na a na-ahụkarị ihe ụfọdụ n'ime trachea.N'adịghị ka ọmụmụ in vitro, a pụghị ịdọrọ irighiri ihe n'ime trachea site na mmegharị nke magnet.Nchọpụta a kwekọrọ n'otú imi nke na-ekpuchi elu trachea na-esikarị amụba ụmụ irighiri ihe, na-akụnye ha n'ime imi ma mesịa kpochapụ ha site na usoro mkpochapụ muco-ciliary.
Anyị chere na iji magnet n'elu na n'okpuru trachea na-adọrọ mmasị (Fig 3b) nwere ike ime ka oghere magnetik dị n'otu, karịa ebe ndọta na-etinye uche nke ukwuu n'otu oge, nwere ike ịkpata nkesa ihe dị iche iche..Agbanyeghị, ọmụmụ mmalite anyị ahụghị ihe akaebe doro anya iji kwado echiche a.N'otu aka ahụ, ịtọ otu ụzọ ndọta iji chụsasịa (Fig 3c) emeghị ka ihe na-edozi ahụ na mpaghara ihe oyiyi.Nchọpụta abụọ a na-egosi na ntọlite ​​​​magnet anaghị eme ka njikwa mpaghara MP na-arụ ọrụ nke ọma, yana na ike ndọta siri ike na-esi na ya pụta na-esiri ike ịmegharị, na-eme ka usoro a ghara ịdị irè.N'otu aka ahụ, idobe ndọta n'elu na n'ofe trachea (Njirimara 3d) emebeghịkwa ọnụ ọgụgụ nke ụmụ irighiri ihe fọdụrụ na mpaghara onyonyo.Ụfọdụ n'ime nhazi ndị ọzọ nwere ike ọ gaghị aga nke ọma ka ha na-ebute mbelata ike ndọta na mpaghara nkwụnye ego.Ya mere, a na-ewere nhazi otu magnet na 30 degrees (Fig 3a) dị ka ihe kachasị mfe na nke kachasị mma na usoro nyocha vivo.
Ọmụmụ ihe ọmụmụ LV-MP gosiri na mgbe ejikọtara vectors LV na CombiMag ma nyefee ya mgbe ọ gbasasịrị n'anụ ahụ n'ihu oghere magnetik, ọkwa ntụgharị abawanyela nke ukwuu na trachea ma e jiri ya tụnyere njikwa.Dabere na ọmụmụ ihe onyonyo synchrotron na nsonaazụ LacZ, oghere ndọta pụtara na ọ nwere ike idobe LV n'ime trachea wee belata ọnụọgụ vector nke batara n'ime ngụgụ ozugbo.Mgbanwe ndị dị otú ahụ ezubere iche nwere ike iduga nrụmọrụ dị elu ma na-ebelata titers, ntụgharị na-abụghị nke ezubere iche, mmetụta mkpali na mgbochi mgbochi, yana ụgwọ mbufe mkpụrụ ndụ ihe nketa.N'ụzọ dị mkpa, dị ka onye nrụpụta si kwuo, enwere ike iji CombiMag yana ụzọ mbufe mkpụrụ ndụ ihe nketa ndị ọzọ, gụnyere vectors viral ndị ọzọ (dị ka AAV) na acid nucleic.