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Gene vectors maka ọgwụgwọ nke cystic fibrosis akpa ume ọrịa kwesịrị lekwasịrị eduzi airways n'ihi na peripheral nku ume transduction adịghị enye therapeutic benefit.Viral transduction arụmọrụ bụ kpọmkwem metụtara vector obibi time. Otú ọ dị, nnyefe mmiri mmiri dị ka gene-ebu ndammana diffuse n'ime alveoli n'oge n'ike mmụọ nsọ, na therapeutic particles n'ike n'ike nke oge obibi nke ọ bụla. gene-ebu na airways dị mkpa ma siri ike nweta.Gene ụgbọelu-conjugated magnetik ahụ nwere ike na-eduzi n'elu nke airways nwere ike imeziwanye mpaghara targeting.Due ka ihe ịma aka nke in vivo visualization, omume nke ndị dị otú ahụ obere magnetik ahụ n'elu airway elu na ọnụnọ nke etinyere magnetik ubi bụ emezighị aghọta.Ebumnobi nke synchrot nke a na-amụ a na-aghọtaghị nke a na-amụ na-eme ka a na-ahụ anya na-amụ ihe na-eme ka a na-ahụ anya na-amụ ihe na vivo. magnetik ahụ na trachea nke oke anesthetized na-enyocha ike na ihe nakawa etu esi nke onye na nnukwu urughuru omume na vivo.We wee enyocha ma nnyefe nke lentiviral magnetik ahụ n'ihu a magnetik ga-amụba transduction arụmọrụ na oke trachea. Synchrotron X-ray Onyonyo na-ekpughe na-akpali akpali na intro magnetik particles. vivo.Particles enweghị ike ịdọrọ n'ụzọ dị mfe n'akụkụ elu nke ikuku dị ndụ na magnets, ma n'oge njem, ihe nkwụnye ego na-etinye uche n'ọhịa nke ele ebe magnetik kasị ike. Transduction arụmọrụ na-amụbakwa okpukpu isii mgbe lentiviral magnetik ahụ na-anapụta na ọnụnọ nke a magnetik. Ọnụ, ndị a pụta na-atụ aro na particles nwere ike ịbụ ihe bara uru nke magnetik lekwasịrị anya ike ndọta. ma na-abawanye ọkwa ntụgharị na-eduzi ụzọ ikuku na vivo.
Cystic fibrosis (CF) na-akpata mgbanwe dị na otu mkpụrụ ndụ ihe nketa a na-akpọ CF transmembrane conductance regulator (CFTR) .Protein CFTR bụ ọwa ion nke dị n'ọtụtụ mkpụrụ ndụ epithelial n'ime ahụ dum, gụnyere ndị na-eduzi ikuku, ebe bụ isi nke CF pathogenesis. CFTR ntụpọ na-eduga na njem mmiri na-adịghị ahụkebe, na-eme ka mmiri dị omimi nke elu ụgbọ elu SLA. na-emebi ikike nke usoro njem mucociliary (MCT) iji kpochapụ ihe ndị na-eku ume na ihe ndị na-akpata site na ikuku. Ihe mgbaru ọsọ anyị bụ ịmepụta usoro ọgwụgwọ lentiviral (LV) iji nyefee akwụkwọ ziri ezi nke mkpụrụ ndụ CFTR ma melite ASL, MCT, na ahụike anụ ahụ, na ịnọgide na-emepụta nkà na ụzụ ọhụrụ nwere ike ịlele ihe ndị a na vivo1.
LV vectors bụ otu n'ime ndị na-eduga n'ihu maka CF airway gene therapy, tumadi n'ihi na ha nwere ike na-adịgide adịgide iwekota therapeutic gene n'ime airway basal cell (airway stem cell) .Nke a dị mkpa n'ihi na ha nwere ike iweghachi nkịtị hydration na mucus nwechapụ site iche n'ime functional gene-mgbaziri CF-metụtara airway elu sel, na-eme ka a na-eduzi na ndụ ogologo ndụ ikuku. bụ ebe ọrịa akpa ume CF na-amalite. Nnyefe nke vector miri emi n'ime ngụgụ nwere ike ịkpata transduction alveolar, ma nke a enweghị uru ọgwụgwọ na CF. Otú ọ dị, mmiri dị ka mkpụrụ ndụ ihe nketa na-ebugharị na-agagharị na alveoli n'ike mmụọ nsọ mgbe nnyefe3,4 na ọgwụgwọ ahụ na-ekpochapụ ngwa ngwa n'ime oghere ọnụ nke ogologo oge nke M. vector na-anọgide na-esote mkpụrụ ndụ lekwasịrị anya iji kwe ka nnabata cellular - "oge obibi" 5 - nke a na-ebelata ngwa ngwa site na ikuku ikuku nke mpaghara yana achikọtara ọnụ ọgụgụ mucus na MCT. Maka CF, ikike ịgbatị oge obibi nke LV n'ime ụgbọ elu dị mkpa iji nweta ọkwa dị elu nke transduction na mpaghara a, ma ọ dị ugbu a bụ ihe ịma aka.
Iji merie ihe mgbochi a, anyị na-atụ aro na ụmụ irighiri ihe ndọta LV (MPs) nwere ike inye aka n'ụzọ abụọ na-akwado ya. Nke mbụ, enwere ike iduzi ha n'ụzọ magnetik n'elu ikuku ikuku iji meziwanye ebumnuche ma nyere ndị na-ebu ihe na-ebu gene bi na mpaghara ikuku chọrọ; na ASL) ịkwaga na oyi akwa 6.MPs ejirila ya mee ihe dị ka ụgbọ ala na-ebuga ọgwụ ezubere iche mgbe ha na-ejikọta na mgbochi nje, ọgwụ chemotherapeutic, ma ọ bụ obere ụmụ irighiri ihe ndị ọzọ na-ejikọta na membranes cell ma ọ bụ na-ejikọta na ndị na-anabata sel dị mkpa ma na-agbakọta na saịtị tumor n'ihu ọkụ eletrik. Ubi magnetik maka ọgwụgwọ ọrịa cancer 7. Usoro ndị ọzọ "hyperthermal" na-achọ ikpo ọkụ MPs mgbe a na-ekpughere ha na oghere magnetik oscillating, si otú ahụ na-ebibi mkpụrụ ndụ tumor. Ụkpụrụ nke nnyefe magnetik, nke a na-eji oghere magnet mee ihe dị ka onye na-ebugharị ihe na-eme ka nnyefe nke DNA gaa na mkpụrụ ndụ, a na-ejikarị in vitro na-eji usoro siri ike nke mkpụrụ ndụ na-emepụta ihe na-emepụta ihe. nke LV magnetotransfection e guzosie ike, na in vitro nnyefe nke LV-MPs ka mmadụ bronchial epithelial cell akara na ọnụnọ nke a static magnetik ubi, na-amụba transduction arụmọrụ site 186-n'ogige atụrụ tụnyere LV vector naanị.LV-MP nwekwara e tinye n'ọrụ na ihe in vitro CF nlereanya, ebe magnetik transfection mụbara LV2d0 ọnụnọ nke ikuku n'ime ikuku-n'ikuku transduction na ikuku-n'ikuku ikuku. sputum10.Otú ọ dị, na vivo magnetotransfection nke akụkụ natara dịtụ nta anya na naanị e enyocha na a ole na ole anụmanụ ọmụmụ11,12,13,14,15, karịsịa na ngụgụ16,17. Otú o sina dị, ohere nke magnetik transfection na CF akpa ume ọgwụgwọ doro anya. Tan et al. nnyefe akpa ume ga-emeghe ụzọ maka atụmatụ iku ume CFTR n'ọdịnihu iji melite nsonaazụ ụlọ ọgwụ na ndị ọrịa CF"6.
Omume nke obere ihe ndọta magnetik n'elu elu ikuku na ọnụnọ magnetik etinyere na-esiri ike ịhụ anya na ọmụmụ ihe, ma si otú ahụ aghọtachaghị ya. N'ọmụmụ ihe ndị ọzọ, anyị mepụtara usoro synchrotron-propagation dabeere na oge-iche X-ray imaging (PB-PCXI) iji noninvasively na-ahụ anya na-atụle nkeji na vivo mgbanwe na ACT ruo omimi18 na Mgbanwe nke elu gas18. A na-eji ya dị ka ihe ngosi mmalite nke ịdị irè ọgwụgwọ. Tụkwasị na nke ahụ, usoro nyocha MCT anyị na-eji 10-35 µm dayameta ihe mejupụtara nke alumina ma ọ bụ iko index dị elu dị ka ihe nrịbama MCT a na-ahụ anya site na iji PB-PCXI21. Usoro abụọ ahụ dabara adaba maka ịhụ anya nke ụdị ihe dị iche iche, gụnyere MP.
N'ihi na ya elu gbasara ohere na temporal mkpebi, anyị PB-PCXI dabeere ASL na MCT analysis usoro na-ọma ruru eru maka enyocha ike na ihe nakawa etu esi nke otu na nnukwu urughuru omume na vivo na-enyere anyị aka ịghọta na ebuli MP gene nnyefe techniques.The obibia anyị na-arụ ọrụ ebe a na-enweta site na ọmụmụ anyị site na iji mmiri mmiri-8 BL20B22 na-esonụ na-eme ihe ngosi nke mmegharị ahụ na-esote site na iji mmiri mmiri-8 BL20B22. imi na pulmonary airway nke oke iji nyere aka kọwaa usoro okwu mkpụrụ ndụ anyị na-abụghị nke a na-ahụ n'ime mkpụrụ ndụ ihe ọmụmụ anụmanụ 3,4.
Ebumnuche nke ọmụmụ a bụ iji synchrotron PB-PCXI iji anya nke uche na mmegharị ahụ na vivo nke usoro MPs na trachea nke oke dị ndụ. A haziri ọmụmụ ihe ngosi PB-PCXI iji nwalee ọtụtụ MPs, ike ndọta, na ebe iji chọpụta mmetụta ha na mmegharị MP. Anyị chere na otu mpaghara ga-enyere aka na-etinye aka na mpaghara MP. anyị na-achọpụta nhazi magnetik nke na-eme ka ọnụ ọgụgụ nke ihe ndị dị na trachea na-eme ka ọ bụrụ na e deposition. N'ime usoro ọmụmụ nke abụọ, anyị na-achọ iji nhazi nhazi a kachasị mma iji gosipụta usoro ntụgharị nke na-esi na vivo nnyefe nke LV-MPs gaa n'ọdụ ụgbọ elu oke, dabere n'echiche ahụ na nnyefe nke LV-MPs n'ọnọdụ nke nleba anya ụgbọ elu ga-eme ka e nwee mgbanwe.
Emere ihe ọmụmụ anụmanụ niile dịka usoro iwu kwadoro nke Mahadum Adelaide (M-2019-060 na M-2020-022) na SPring-8 Synchrotron Animal Ethics Committee. A na-eme nnwale dịka ntuziaka ARRIVE si dị.
A na-eme ihe ngosi X-ray niile na BL20XU beamline na SPring-8 synchrotron na Japan, na-eji nhazi dị ka nke a kọwara na mbụ21,22. N'ụzọ dị nkenke, igbe nnwale ahụ dị 245 m site na mgbanaka nchekwa synchrotron. A na-eji ihe nlele-na-achọpụta anya nke 0.6 m na-eji ihe ọmụmụ ihe omimi nke 0.6 m mee ihe maka ọmụmụ ihe omimi nke vivo. A na-eji monochromatic beam energy nke 25 keV mee ihe. E weghaara ihe oyiyi site na iji ihe ntụgharị X-ray dị elu (SPring-8 BM3) tinyere ihe nchọpụta sCMOS. Onye ntụgharị na-atụgharị X-ray ka ọ bụrụ ìhè a na-ahụ anya site na iji 10 µm thick scintillator (Gd3Al2Ga3O12), nke a na-eduzi ya na ihe mmetụta 1CMOS0 na microscope. 0.3).Ihe nchọpụta sCMOS bụ Orca-Flash4.0 (Hamamatsu Photonics, Japan) nwere nha n'usoro nke 2048 × 2048 pikselụ yana nha pikselụ raw nke 6.5 × 6.5 µm. Ntọlite a na-enye nha pikselụ isotropic dị irè nke 0.51 µm na nlele ogologo × 1.1. A na-ahọrọ 100 ms iji mee ka ihe mgbaàmà na-eme ka ụda magnetik dị n'ime na n'èzí ụgbọ elu ahụ ka ọ na-ebelata ihe ndị na-eme ka iku ume na-eku ume. N'ime ọmụmụ ihe ọmụmụ vivo, a na-etinye ngwa ngwa X-ray shutter n'ụzọ X-ray iji gbochie dose radiation site na igbochi X-ray beam n'etiti ikpughe.
Ejighị onye na-ebu LV mee ihe na ọmụmụ ihe ngosi SPring-8 PB-PCXI ọ bụla n'ihi na ụlọ ihe ngosi BL20XU abụghị Biosafety Level 2 kwadoro. Kama, anyị ahọrọla ọtụtụ MPs nke ọma sitere na ndị na-azụ ahịa azụmahịa abụọ-na-ekpuchi ọtụtụ nha, ihe, ntinye ígwè, na ngwa ngwa-mgbe ahụ na-eme ka ọ pụta ìhè n'ọhịa capilla MP. ụzọ ikuku. n'elu.MPs nso na size si 0.25 ka 18 μm na-mere si a dịgasị iche iche nke ihe (lee Isiokwu 1), ma mejupụtara nke ọ bụla sample, gụnyere size nke magnetik ahụ n'ime MP, bụ amaghị.Based na anyị nnukwu MCT ọmụmụ 19, 20, 21, 23, 24, anyị na-atụ anya na MPs dị ka obere dị ka 5 n'ikuku pụrụ ịhụ n'elu traμm n'elu 24. Mwepu okpokolo agba dị iche iche iji hụ nkwalite visibiliti nke mmegharị MP. Otu MP nha 0.25 μm pere mpe karịa mkpebi nke ngwaọrụ onyonyo, mana PB-PCXI na-atụ anya ịchọpụta ọdịiche olu ha na mmegharị nke mmiri dị elu nke etinyere ha mgbe etinyere ya.
A na-akwadebe ihe nlele maka MP ọ bụla na tebụl 1 na 20 μl iko capillaries (Drummond Microcaps, PA, USA) na n'ime ime nke 0.63 mm. A na-etinye ihe ndị dị na corpuscular na mmiri, ebe CombiMag dị na mmiri mmiri nke onye na-emepụta ihe. A na-etinye capillaries horizontally na sample ogbo ke imaging igbe, karị, na positioned nsọtụ nke fluid.A 19 mm dayameta (28 mm ogologo) nickel shei obere ụwa neodymium iron boron (NdFeB) magnet (N35, cat. No. LM1652, Jaycar Electronics, Australia) na a residual nweta ihe ndọta nke remotely 1. n'oge imaging. Ị nweta ihe oyiyi X-ray na-amalite mgbe magnet na-etinye ihe dịka 30 mm n'elu ihe nlele ahụ, a na-enwetakwa ihe oyiyi na ọnụego nke 4 okpokolo agba kwa nkeji.
Ntọlite onyonyo in vitro nwere ihe nlele MP na capillaries ugegbe n'ụdị ntụgharị ntụgharị xy. A na-eji eriri agbajiri ọbara ọbara mara okporo ụzọ nke beam X-ray.
Ozugbo e guzobere vitro vitro nke MPs, a nwalere akụkụ nke ha na vivo na ụdị anụ ọhịa ụmụ nwanyị albino Wistar oke (~ 12 izu, ~ 200 g) .0.24 mg / kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg / kg midazolam (Dormicum®, mg / Japan) butere P. (Vetorphale®, Meiji Seika) A na-eji ngwakọta nke Pharma mee ka oke na-egbu ahụ ahụ, Japan) site na injection intraperitoneal. Mgbe a gwọchara ya, a kwadebere ha maka ịse foto site n'iwepụ ajị anụ gburugburu trachea, na-etinye tube endotracheal (ET; 16 Ga iv cannula, Terumo BCT) na na-eme ka anụ ahụ na-eme ka ahụ dịkwuo mma iji mee ka ha nwee ike ime ka ahụ ha nwee ike ime ka ahụ ha nwee ike ime ka ha dị iche iche. 22 .A na-ejikọta efere ihe oyiyi ahụ na nhazi ntụgharị ihe nlele na igbe ihe ngosi na ntakịrị akụkụ iji kwado trachea n'ahịrị na foto X-ray, dị ka egosiri na foto 2a.
(a) Na vivo imaging setup na SPring-8 igbe onyonyo, a na-eji ụzọ X-ray beam akara akara akara na-acha uhie uhie.
Usoro mgbapụta sirinji na-achịkwa nke dịpụrụ adịpụ (UMP2, World Precision Instruments, Sarasota, FL) na-eji sirinji 100 μl iko 100 μl jikọtara ya na PE10 tubing (OD 0.61 mm, ID 0.28 mm) site na 30 Ga agịga. Mark na tube iji hụ na n'ọnụ bụ na n'ọnụ dị na ziri ezi tube ET mgbe ọnọdụ na micropusing trache. syringe plunger e wepụrụ mgbe ọnụ nke tube na-emikpu n'ime MP sample ga-ebunye.The loaded nnyefe tube e wee tinye n'ime endotracheal tube, na-etinye n'ọnụ n'ime akụkụ kasị ike nke anyị na-atụ anya na-etinyere magnetik.Image nnweta e jikwaa site na iji respiration detector ejikọrọ na anyị Arduino dabeere oge igbe, na ihe niile na-egosi na-eji na-emepe ihe ngosi okpomọkụ / shutter okpomọkụ, e dekọrọ ihe ngosi okpomọkụ / ihe oyiyi okpomọkụ. Powerlab na LabChart (AD Instruments, Sydney, Australia) 22. Mgbe ịse foto Mgbe ogige ahụ enweghị ike ịnweta, a na-etinye igwefoto IP abụọ (Panasonic BB-SC382) na ihe dịka 90 Celsius na ibe ya ma jiri ya nyochaa ọnọdụ nke magnet n'akụkụ trachea n'oge eserese (Fig 2b, c) iji nweta njedebe nke ihe oyiyi n'oge ọ bụla na-eme ka ọ na-eme ihe nkiri. plateau.
A na-ejikọta magnet na ọkwa nke abụọ nke nwere ike ịnọ n'ebe dịpụrụ adịpụ site n'èzí ụlọ ịse foto. A nwalere ọnọdụ magnet dị iche iche na nhazi, gụnyere: A na-agbanye n'akụkụ ihe dịka 30 Celsius n'elu trachea (nhazi nke egosiri na Figures 2a na 3a); otu ndọta n'elu anụmanụ na nke ọzọ dị n'okpuru ebe a, nke nwere okporo osisi na-adọta (Fig 3b); otu ndọta n'elu anụmanụ na nke ọzọ dị n'okpuru ebe a, na okporo osisi ndị e debere iji chụpụ (Ọnọdụ 3c); na otu magnet n'elu na perpendicular na trachea (Figure 3d) . Ozugbo a na-ahazi anụmanụ na magnet na MP a ga-anwale na-ebuba n'ime mgbapụta sirinji, na-ebuga 50 μl dose na ọnụego 4 μl / sec mgbe ị na-enweta ihe oyiyi. A na-emegharị magnet azụ azụ ma ọ bụ na-aga n'ihu na trachea mgbe ọ na-aga n'ihu iji nweta ihe oyiyi.
Nhazi ndọta maka in vivo imaging (a) otu magnet n'elu trachea na akụkụ nke ihe dịka 30 °, (b) magnet abụọ setịpụrụ ịdọta, (c) magnet abụọ ka a ga-atụgharị, (d) otu magnet n'elu na perpendicular na trachea. Onye na-ekiri anya na-ele anya site n'ọnụ gaa na ngụgụ site na trachea, na X-ray na-agafe n'akụkụ aka nri ma ọ bụ n'akụkụ aka ekpe. na-agagharị n'ogologo ụzọ ikuku ma ọ bụ aka ekpe na aka nri n'elu trachea n'ụzọ nke eriri X-ray.
Anyị na-achọkwa ịchọpụta visibiliti na omume nke ụmụ irighiri ihe na ikuku ikuku na-enweghị mgbagwoju anya ume iku ume na obi obi. Ya mere, na njedebe nke oge eserese, a na-egbu ụmụ anụmanụ na-egbu egbu maka pentobarbital overdose (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg ip n'elu ikpo okwu, mgbe ụfọdụ anụmanụ na-akwụsị na-eku ume na ikpo okwu). A na-emegharị usoro onyonyo ọzọ, na-agbakwunye dose ọzọ nke MP ma ọ bụrụ na ọ nweghị MP ahụghị n'elu elu ikuku.
A na-edozi ihe onyonyo ndị enwetara n'ọhịa dị larịị na oghere gbara ọchịchịrị wee gbakọta na fim (Freek 20 kwa nkeji; 15-25 × ọsọ ọsọ dabere na ọnụego iku ume) site na iji edemede ọdịnala edere na MATLAB (R2020a, The Mathworks).
A na-eduzi ọmụmụ ihe ọmụmụ vector niile nke LV na Laboratory Animal Research Facility na Mahadum Adelaide na ebumnuche iji nsonaazụ nke nnwale SPring-8 iji chọpụta ma nnyefe LV-MP n'ihu oghere magnet nwere ike ịkwalite mbufe mkpụrụ ndụ na vivo. Iji chọpụta mmetụta nke MP na magnetik, a na-emeso otu ìgwè abụọ: otu ìgwè e nyere LV-MP na otu LV nke ọzọ na-anata magnet, na-enwetakwa otu magnet.
A na-emepụta vectors LV gene vectors site na iji ụzọ akọwara mbụ 25, 26 .The LacZ vector na-egosipụta mkpụrụ ndụ beta-galactosidase nke anụ ahụ nuklia nke a na-ebugharị site na onye na-akwado MPSV (LV-LacZ), nke na-emepụta ngwaahịa mmeghachi omume na-acha anụnụ anụnụ na mkpụrụ ndụ transduced, nke a na-ahụ anya na ngụgụ anụ ahụ n'ihu na akụkụ anụ ahụ.Titration na-arụ ọrụ nke ọma na mkpụrụ ndụ mkpụrụ ndụ Lac. hemocytometer iji gbakọọ titer na TU / ml. A na-echekwa ndị na-ebu ibu na -80 Celsius C, thawed tupu ejiri ya, na-ejikọta ya na CombiMag site na ịgwakọta na 1: 1 ratio na incubating na ice maka ọ dịkarịa ala 30 nkeji tupu nnyefe.
Nkịtị Sprague Dawley oke (n = 3 / otu, ~ 2-3 bụ ndị aneshetized intraperitoneally na ngwakọta nke 0.4 mg/kg medetomidine (Domitor, Ilium, Australia) na 60 mg / kg ketamine (Ilium, Australia) ọnwa-ochie) ip) injection na ndị na-abụghị ogwugwo n'ọnụ nwere ike ịhụ na ikuku 16 Gaiv. anụ ahụ na-enweta LV transduction, ọ bụ oyi na-eji anyị na mbụ kọwara n'ibu perturbation protocol, nke tracheal airway elu e ji agwụ axially na a waya nkata (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Tracheal nchekwa nkeji mgbe e mere kabinet dị iche iche nke L. mmekpa ahụ.
A haziri oghere magnetik eji mee nnwale a n'otu ụzọ ahụ na ọmụmụ ihe onyonyo X-ray na vivo, yana otu ndọta ahụ ejidere n'elu trachea site na iji vidiyo stent distillation (Njirimara 4) .A 50 μl olu (2 × 25 μl aliquots) nke LV-MP na-enyefe n'ime trachea (n = 3 μl) nke a na-ebufe n'ime trachea (n = 3 tip) na mbụ. 3 anụmanụ) natara otu LV-MPs na-enweghị iji magnet. Mgbe infusion zuru ezu, a na-ewepụ cannula na tube ET ma na-ekpochapụ anụ ahụ. magnet na-anọgide na ebe maka nkeji 10, wee wepụ ya. Rats natara dose nke meloxicam subcutaneous (1 ml / kg) (Ilium, Australia) na-esote na reversal of mg /nespazol. (Antisedan, Zoetis, Australia) .A na-ekpo ọkụ ma na-enyocha oke oke ruo mgbe mgbake zuru oke site na anesthesia.
Ngwa nnyefe LV-MP n'ime ụlọ nchekwa nchekwa ndụ. Enwere ike ịhụ ìhè isi awọ Luer hub nke ET tube na-esi n'ọnụ pụta ma tinye n'ọnụ gel nke pipette dị na foto a site na tube ET ruo omimi a chọrọ n'ime trachea.
Otu izu mgbe usoro ọgwụgwọ LV-MP gasịrị, a na-egbu ụmụ anụmanụ site na 100% CO2 inhalation na okwu LacZ na-enyocha site na iji usoro ọgwụgwọ X-gal anyị. A na-ewepụ ihe mgbanaka atọ nke ọtụtụ cartilaginous iji hụ na ihe ọ bụla n'ibu mmebi ma ọ bụ njigide mmiri site na ntinye tube endotracheal adịghị etinye ya na nyocha. A na-egbutu trachea ọ bụla iji mee ka ọ bụrụ ihe na-emepụta ihe na-edozi ogologo oge, na-eme ka ha nwee ike ịmepụta ogologo oge. silicone roba (Sylgard, Dow Inc) na-eji Minutien agịga (Fine Science Tools) iji anya nke uche n'elu luminal. A na-akwado nkesa na ụkpụrụ nke mkpụrụ ndụ transduced site na foto ihu ihu site na iji microscope Nikon (SMZ1500) na igwefoto DigiLite na TCapture software (Tucsen Photonics, China) na-enweta ihe oyiyi 20 kachasị elu na obosara nke obosara. trachea), na ogologo trachea dum na-ese onyinyo nzọụkwụ-site-nzọụkwụ, na-eme ka nkwụsịtụ zuru oke n'etiti ihe oyiyi ọ bụla iji nye ohere maka ihe oyiyi "stitching" .A na-ejikọta ihe oyiyi sitere na trachea ọ bụla n'ime otu ihe oyiyi mejupụtara site na iji Image Composite Editor v2.0.3 (Microsoft Research) tinye n'ọrụ a planar ngagharị algọridim nke anụmanụ. MATLAB script (R2020a, MathWorks) dị ka akọwara na mbụ, na-eji ntọala nke 0.35
A na-etinye trachea ọ bụla n'ime paraffin na akụkụ 5 μm. A na-ekpuchi akụkụ na-acha uhie uhie na-anọpụ iche maka 5 min ma nweta ihe oyiyi site na iji Nikon Eclipse E400 microscope, DS-Fi3 igwefoto na NIS element capture software (mbipute 5.20.00).
A na-eme nyocha ndekọ ọnụ ọgụgụ niile na GraphPad Prism v9 (GraphPad Software, Inc.) .Edebere ihe ndekọ ọnụ ọgụgụ dị na p ≤ 0.05. Achọpụtara ụkpụrụ nkịtị site na iji ule Shapiro-Wilk, na ọdịiche dị na LacZ staining na-atụle site na iji t-ule na-enweghị atụ.
A na-enyocha ndị MP isii a kọwara na tebụl 1 site na iji PCXI, a na-akọwakwa visibiliti na Tebụl 2. Abụọ polystyrene MPs (MP1 na MP2; 18 μm na 0.25 μm, n'otu n'otu) adịghị ahụ anya n'okpuru PCXI, ma ihe ndị ọzọ a na-ahụ anya bụ ndị a na-amata (ihe atụ na-egosi na 5% MP3-2 na Figure 5 MP3-5). μm na 0.9 μm n'otu n'otu) bụ nke a na-ahụ anya nke ọma. Ọ bụ ezie na enwere ụfọdụ n'ime ụmụ irighiri ihe ndị a nwalere, MP5 (98% Fe3O4; 0.25 μm) bụ ihe a na-akpọkarị. CombiMag ngwaahịa MP6 siri ike ịhụ. N'ọnọdụ niile, ike anyị ịchọpụta MP na-abawanye nke ọma site n'ịsụgharị ihe ndọta ahụ na-atụgharị azụ. capillary, ihe ndị ahụ na-agbatị ogologo eriri ogologo, ma ka magnets na-abịaru nso na ike magnetik na-abawanye, eriri ahụ na-ebelata ka ihe ndị ahụ na-akwaga n'elu elu nke capillary (lee ihe mgbakwunye Video S1: MP4), na-abawanye Njupụta nke elu. elu elu nke capillary (lee ihe mgbakwunye Video S2: MP4) .Mgbe magnet kwụsịrị ịkwagharị, ihe ndị ahụ na-aga n'ihu na-aga n'ihu obere oge mgbe ha rutere n'ọnọdụ nhata. Ka MP na-aga n'ebe dị elu nke elu nke capillary, ihe ndọta na-adọrọkarị ihe irighiri ihe site na mmiri.
The visibiliti nke MP n'okpuru PCXI dịgasị iche iche nke ukwuu n'etiti samples.(a) MP3, (b) MP4, (c) MP5 na (d) MP6. All oyiyi gosiri ebe a na-ewere na a magnet dị ihe dị ka 10 mm kpọmkwem n'elu capillary. The pụtara nnukwu okirikiri bụ ikuku egosipụta tọrọ atọ na capillaries, na-egosi n'ụzọ doro anya na oji na-acha ọcha n'ọnụ atụmatụ nke acha ọbara ọbara dị iche iche nwere. Dayameta nke schematics magnet n'ọnụọgụ niile agaghị abawanye ma buru ihe dịka 100 ugboro karịa egosiri.
Ka a na-atụgharị magnet n'aka ekpe na aka nri n'akụkụ elu nke 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, a na-adọkpụrụ ihe ndị ahụ n'akụkụ elu elu nke capillary. Nke a na-ebutekarị n'ime MPs dị iche iche na-adọrọ adọrọ na-adọrọ adọrọ na-adọrọ adọrọ na-adọrọ adọrọ n'ime nnukwu vidiyo na-adọrọ adọrọ. S3: MP5) .Nke a na-apụtakwa ìhè karịsịa mgbe ị na-ese onyinyo dị nso na njedebe capillary, nke na-eme ka ndị MPs chịkọta ma tinye uche na ikuku mmiri-ikuku interface. Akụkụ dị na MP6, bụ ndị siri ike ịghọta karịa MP3-5, adịghị adọta dị ka magnet na-agagharị na capillary, ma eriri MP na-ekewa, na-ahapụ akụkụ nke vidiyo 6). N'ọnọdụ, mgbe oghere magnetik etinyere na-ebelata site na ịkwaga magnet ahụ n'ebe dị anya site na ebe a na-ese foto, ndị omeiwu ọ bụla fọdụrụ ji nwayọọ nwayọọ na-agbada n'elu ala nke tube site na ike ndọda mgbe ọ na-anọgide na eriri (lee Video S5: MP3).
Akuku nke eriri MP na-agbanwe ka a na-atụgharị magnet n'aka nri n'elu capillary.(a) MP3, (b) MP4, (c) MP5 na (d) MP6. Igbe uhie nwere ihe dị iche iche na-eme ka mmụba dị elu. Rịba ama na vidiyo ndị na-agbakwụnye na-enye ihe ọmụma ka ha na-ekpughe usoro ihe dị mkpa na ozi dị ike nke a na-apụghị ịhụ anya na foto ndị a static.
Nnwale anyị gosipụtara na ịkwagharị magnet ahụ ji nwayọọ nwayọọ na-agaghachi na trachea na-eme ka anya nke MP na ọnọdụ mgbagwoju anya na vivo.In vivo test adịghị arụ ọrụ dị ka polystyrene beads (MP1 na MP2) na-adịghị ahụ anya na capillary. A na-anwale onye ọ bụla n'ime MPs anọ fọdụrụ na vivo na magnet ogologo axis ahaziri n'elu trachea nke 3 ruo 3 na ọnụ ọgụgụ vertical. 3a), n'ihi na nke a mere ka agbụ MP ogologo oge ma dị irè karịa nhazi magnet akwụsị. MP3, MP4 na MP6 achọpụtaghị na trachea nke anụmanụ ọ bụla dị ndụ. Mgbe a na-ese onyinyo ikuku nke oke ikuku mgbe e gburu anụmanụ ahụ n'ụzọ mmadụ, ihe ndị ahụ nọgidere na-adịghị ahụ anya ọbụna mgbe agbakwunyere olu ọzọ site na iji mgbapụta sirinji.MP5 nwere akụkụ kachasị elu, ya mere, a na-eji ígwè oxidize mee ihe na-eme ka ọ bụrụ naanị ihe a na-ahụ anya. na vivo omume nke MP.
Ịtụkwasị magnet n'elu trachea n'oge MP nnyefe mere ka ọtụtụ ndị, ma ọ bụghị ihe niile, MPs na-etinye uche n'ọhịa nke echiche. Iberibe na-abanye trachea na-kasị mma hụrụ na humanely àjà anụmanụ. Foto 7 na Supplementary Video S6: MP5 na-egosi ngwa ngwa magnetik njide na alignment nke irighiri n'elu nke ventral trachea, nwere ike na-egosi na trache a chọrọ n'ógbè. distally tinyere trachea mgbe MP nnyefe, ụfọdụ MPs hụrụ nso na carina, na-atụ aro na magnetik ike ezughị ezu na-anakọta ma na-ejigide ndị MP niile, dị ka a napụtara ha site na mpaghara nke kacha magnetik ike n'oge usoro mmiri. Otú o sina dị, postpartum MP mkpokọta dị elu na gburugburu ebe oyiyi, na-atụ aro na ọtụtụ MPs nọgidere na ọdụ ụgbọ elu mpaghara ebe a na-etinyere magnetik.
Foto sitere na (a) tupu na (b) mgbe nnyefe nke MP5 n'ime trachea nke oke euthanized n'oge na-adịbeghị anya na magnet na-etinye ya kpọmkwem n'elu ebe a na-ese foto. Ebe a na-ese onyinyo dị n'etiti mgbanaka cartilage abụọ. Tupu nnyefe MP, enwere mmiri mmiri n'ime ikuku. Igbe na-acha uhie uhie nwere ihe dị iche iche na-ebuli elu. Ihe oyiyi ndị a sitere na vidiyo 5 .
Ịsụgharị magnet n'akụkụ trachea na vivo mere ka agbụ MP gbanwee akụkụ n'ime elu ikuku n'ụzọ yiri nke a na-ahụ na capillaries (lee Figure 8 na Supplementary Video S7: MP5) Otú ọ dị, na ọmụmụ ihe anyị, MPs enweghị ike ịdọrọ n'elu elu nke ikuku dị ndụ dị ka ha nwere ike na capillaries. N'ọnọdụ ụfọdụ, MP yinye ga-adị ogologo aka ekpe na nke ziri ezi. na-egosi na ọ na-agbanwe omimi nke elu mmiri mmiri oyi akwa mgbe magnet na-ebugharị ogologo oge n'akụkụ trachea, na-agbasawanye mgbe magnet na-ebugharị ozugbo n'elu na eriri irighiri ahụ na-atụgharị gaa n'ọnọdụ kwụ ọtọ (lee S7 Video Supplementary). : MP5 na 0: 09, ala aka nri) .Ụdị njirimara nke mmegharị ahụ gbanwere mgbe a sụgharịrị magnet n'ofe elu nke trachea n'akụkụ (ya bụ, n'aka ekpe ma ọ bụ aka nri nke anụmanụ karịa n'ogologo trachea) . A ka na-ahụkwa ihe ndị ahụ n'ụzọ doro anya ka ha na-akwaga, ma mgbe e wepụrụ magnet na trachea, ndụmọdụ nke urughuru eriri: Mgbakwunye 5 pụtara anya. 0: 08) .Nke a kwekọrọ na omume MP anyị hụrụ n'okpuru ebe ndọta etinyere na capillary iko.
Ihe atụ onyonyo na-egosi MP5 na trachea nke oke anụ ahụ dị ndụ.
Mgbe a na-ahazi okporo osisi abụọ ahụ n'ebe ugwu-n'ebe ndịda n'elu na n'okpuru trachea (ya bụ na-adọta; Fig. 3b), MP chords pụtara ogologo oge ma dị na sidewall nke trachea kama na dorsal tracheal elu (lee S9 Video S9: MP5) . Otú ọ dị, ọnụ ọgụgụ dị elu nke ụmụ irighiri ihe n'otu ebe a na-enweta mmiri mmiri (ie) adịghị achọpụta ihe dị elu n'elu ebe a na-ebufe mmiri. A na-eji ngwaọrụ abụọ-magnet eme ihe, nke na-emekarị mgbe a na-eji ngwaọrụ otu magnet eme ihe. Mgbe ahụ, mgbe a na-ahazi otu magnet ka ọ ghara ịkwanye ogwe osisi ndị ahụ na-atụgharị (Fig 3c), ọnụ ọgụgụ nke ihe ndị a na-ahụ anya na-ahụ anya na-apụtaghị na ọ na-abawanye mgbe nnyefe. Nhazi nke ma abụọ-magnet nhazi bụ ihe ịma aka n'ihi na elu ma ọ bụ nkwanye ùgwù ndọta na-eme ka ihe ndọtị ahụ gbanwee. yiri nke ikuku ma na-agafe n'okporo ụzọ ikuku na 90 degrees nke mere na ahịrị ubi gafere tracheal mgbidi orthogonally (Fig 3d), nhazi nke e mere iji chọpụta ma ọ bụrụ na enwere ike ịhụ nchịkọta mkpụrụ ndụ na mgbidi n'akụkụ. Otú ọ dị, na nhazi a, ọ dịghị ihe ọ bụla a na-amata nke mkpokọta MP ma ọ bụ mmegharị magnet. Dabere na nhazi nhazi 3 niile, nhazi nhazi nke 3. 3a) ahọpụtara maka ọmụmụ ihe ọmụmụ mkpụrụ ndụ ihe nketa vivo.
Mgbe a na-esere anụmanụ ahụ ugboro ugboro ozugbo e gbuchara mmadụ, enweghị mmegharị anụ ahụ na-agbagwoju anya pụtara na enwere ike ịhụ ahịrị ahụ dị mma na nke dị mkpụmkpụ n'ọhịa interchondral doro anya, "na-ama jijiji" kwekọrọ na ntụgharị ntụgharị nke magnet. Otú o sina , ka na-enweghị ike ịhụ n'ụzọ doro anya na ọnụnọ na ngagharị nke MP6 ahụ.
The LV-LacZ titer bụ 1.8 × 108 TU/ml, na mgbe 1: 1 na-agwakọta na CombiMag MP (MP6), ụmụ anụmanụ natara a 50 μl tracheal dose nke 9 × 107 TU/ml LV ụgbọ (ie 4.5 × 106 TU / oke). .N'ime ọmụmụ ihe ndị a, kama ịsụgharị magnet n'oge ọrụ, anyị na-edozi magnet n'otu ọnọdụ iji chọpụta ma mgbanwe LV (a) nwere ike imeziwanye ma e jiri ya tụnyere nnyefe vector na enweghị oghere magnetik, na (b) nwere ike ilekwasị anya na cell Airway na-atụgharị gaa na mpaghara magnetik lekwasịrị anya n'ógbè nke elu ụgbọ elu.
Ọnụnọ nke magnets na iji CombiMag jikọtara ya na LV vectors apụtaghị na ọ nwere mmetụta ọjọọ na ahụike anụmanụ, dị ka ọkọlọtọ LV vector nnyefe protocol.Onyonyo n'ihu nke tracheal mpaghara doro n'ibu perturbation (Mgbakwunye Fig. 1) gosiri na e nwere budata elu etoju nke transduction n'ime ìgwè anụmanụ na-emeso na a obere-acha anụnụ anụnụ ego nke LV-MP (Fig. LacZ staining dị na njikwa njikwa (Fig 9b) .Nkọwa nke mpaghara X-Gal nkịtị na-egosi na nchịkwa nke LV-MP n'ihu ebe ndọta na-emepụta ihe dị ka 6-fold mmelite (Fig 9c).
Ihe atụ nke ihe atụ mejupụtara transduction tracheal site na LV-MP (a) na ọnụnọ nke magnetik na (b) na enweghị magnet.
Akụkụ ndị na-acha uhie uhie na-anọpụ iche (ihe atụ egosipụtara na Fig. 2) gosiri sel ndị nwere ntụpọ LacZ dị n'ụdị na ọnọdụ yiri nke ahụ dịka akọwara na mbụ.
Ihe ịma aka dị mkpa maka ọgwụgwọ mkpụrụ ndụ ihe nketa ikuku na-anọgide na-edezi ebe ziri ezi nke ihe ndị na-ebu ụgbọelu gaa na mpaghara mmasị na ịnweta ọkwa dị elu nke transduction arụmọrụ na nku ume na-akpụ akpụ na ọnụnọ nke ikuku na-arụ ọrụ na-arụ ọrụ. magnetik ubi ka mma transduction nwere uru tụnyere ndị ọzọ gene nnyefe ụzọ dị ka electroporation, dị ka ọ nwere ike ikpokọta mfe, na-eri-irè, nnyefe localization, mụbara arụmọrụ, na mkpumkpu incubation ugboro , na ikekwe a nta ụgbọelu dose10. Otú ọ dị, na vivo deposition na omume nke magnetik ahụ na airways mgbe a kọwara n'ezie fesibility ụmụ irighiri n'ime ikuku, nke a na-akọwaghị n'ezie usoro nke ike ndọta. egosipụtara na vivo iji kwalite ọkwa mkpụrụ ndụ ihe nketa n'ụzọ ikuku na-emebibeghị.
Nnwale anyị in vitro synchrotron PCXI gosiri na ihe niile anyị nwalere, ewezuga polystyrene MP, bụ ndị a na-ahụ anya na nhazi onyonyo nke anyị ji mee ihe. N'ihu oghere magnetik, MPs na-emepụta eriri nke ogologo ya metụtara ụdị urughuru na ike magnetik (ya bụ nso nso na mmegharị nke magnet). mpaghara magnetik. Ndị a dị iche iche ubi na-eme ka ndị ọzọ yiri ahụ na-agbakọta na jikọọ, na otu eriri-dị ka mmegharị n'ihi obodo agha si obodo mara mma na-asọ oyi ndị ọzọ ahụ.
Ngosipụta atụmatụ (a,b) ụgbọ oloko nke a na-emepụta n'ime capillaries jupụtara na mmiri na (c,d) ikuku jupụtara na trachea. Rịba ama na capillaries na trachea adịghị adọta na nha.
Mgbe a na-ebugharị magnet n'elu capillary, akụkụ nke eriri eriri ahụ ruru ebe dị oke egwu maka MP3-5 nwere Fe3O4, mgbe nke ahụ gasịrị, eriri ahụ adịkwaghị n'ọnọdụ mbụ, mana ọ na-aga n'ihu na ọnọdụ ọhụrụ.magnet. Nke a nwere ike ime n'ihi na iko capillary elu dị nro iji kwe ka mmegharị a mee. ihe ndị ahụ dị ntakịrị, nwere mkpuchi dị iche iche ma ọ bụ ụgwọ elu, ma ọ bụ mmiri mmiri na-ebu ibu na-emetụta ikike ha nwere ike ịkwaga. Ihe oyiyi dị iche iche nke CombiMag particles dịkwa ike karị, na-atụ aro na mmiri na ihe ndị ahụ nwere ike inwe njupụta dị otú ahụ na ya mere ọ naghị adị mfe ịkwaga ibe ha. Ọ bụghị ihe mgbagwoju anya na ike ndọta na anya dị n'etiti magnet na ebe e lekwasịrị anya dị ezigbo mkpa. Na-ejikọta ọnụ, nsonaazụ ndị a na-atụkwa aro na, mgbe magnetik nwere ike ijide ọtụtụ MPs na-asọfe ebe a na-eche ihu, ọ bụ ezie na magnet nwere ike ịdabere na ya ịkwaga CombiMag particles tinyere n'elu trachea. Ya mere, anyị kwubiri na na vivo LV-MP ọmụmụ kwesịrị iji mee ihe kpọmkwem n'ọhịa nke osisi magnetik.
Mgbe ahụ na-anapụta n'ime ahụ, ha na-esi ike ịmata na onodu nke mgbagwoju na-akpụ akpụ anụ anụ ahụ, ma ike ịchọpụta ha na-enwekwukwa site n'ịsụgharị magnet horizontally n'elu trachea ka "wiggle" na MP ụdọ. N'adịghị ka ọmụmụ in vitro, a pụghị ịdọrọ ụmụ irighiri ihe n'akụkụ trachea site n'ịsụgharị magnet. Nchọpụta a kwekọrọ n'otú imi nke na-ekpuchi elu nke trachea na-esi emepụta ihe ndị dị n'ime trachea, ọnyà ha na imi ma mesịa kpochapụ site na usoro mkpochapụ nke mucociliary.
Anyị na-eche na iji magnetik maka mma n'elu na n'okpuru trachea (Fig 3b) nwere ike ime ka oghere magnetik dị iche iche dị iche iche, karịa ebe magnetik nke na-etinye uche n'otu oge, nke nwere ike iduga na nkesa nkesa. urughuru deposition na imaged area.These abụọ nchoputa na-egosi na sọrọ-magnet setup adịghị budata mma mpaghara akara nke MP ezubere iche, na na n'ihi ike magnetik agha ndị siri ike hazi, na-eme ka nke a obibia na-erughị ihe bara uru. N'otu aka ahụ, orienting magnet n'elu na site trachea (Fig. 3d) nakwa emeghị dịkwuo ọnụ ọgụgụ nke ahụ-magnet setup adịghị budata mma mpaghara akara nke MP ezubere iche, na na n'ihi na ike magnetik ike na-esi ike hazi, na-eme ka nke a obibia obere ihe bara uru. ike ndọta n'ime ebe nkwụnye ego. Ya mere, a na-ewere nhazi magnetik otu ogo 30-ogo (Njirimara 3a) dịka usoro kachasị mfe na nke kachasị mma maka nyocha vivo.
The LV-MP ọmụmụ gosiri na mgbe LV vectors na-ejikọta na CombiMag na nyefee mgbe anụ ahụ perturbation na ọnụnọ nke a magnetik, transduction etoju abawanyela nke ukwuu na trachea ma e jiri ya tụnyere njikwa.Dabere na synchrotron imaging ọmụmụ na LacZ results, magnetik oko doro anya na-enwe ike ichekwa LV n'ime trachea ma belata ọnụ ọgụgụ nke vector na-abanye n'ime oghere ozugbo. nwere ike iduga n'ịrụ ọrụ dị elu ka ị na-ebelata titers ndị a na-enyefe, ntụgharị ihe na-apụ apụ, mmetụta ndị na-egbuke egbuke na mgbochi, na ụgwọ ndị na-ebu mkpụrụ ndụ ihe nketa. N'ụzọ dị mkpa, dị ka onye nrụpụta si kwuo, CombiMag nwere ike iji ya na usoro ndị ọzọ na-ebufe mkpụrụ ndụ ihe nketa, gụnyere ya na vectors viral ndị ọzọ (dị ka AAV) na nucleic acid.
Oge nzipu: Jul-16-2022
