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Pono nā gene vectors no ka mālama ʻana i ka cystic fibrosis lung maʻi e hoʻopaʻa i nā ala lele no ka mea ʻaʻole hāʻawi ka transduction lung peripheral i ka pōmaikaʻi therapeutic. ʻO ka pono o ka transduction viral pili pono i ka manawa noho vector. He mea koʻikoʻi akā paʻakikī ke hoʻokō. Gene carrier-conjugated magnetic particles i hiki ke kuhikuhi ʻia i ka ʻili o nā alaloa hiki ke hoʻomaikaʻi i ka huli ʻana o ka āpana. ʻO nā ʻiole anesthetized e nānā i ka dynamics a me nā hiʻohiʻona o kēlā me kēia ʻano a me ka nui o nā ʻāpana i loko o ka vivo. magnets, akā i ka wā o ka lawe ʻana, hoʻopaʻa ʻia nā waihona i ke kahua o ka ʻike kahi i ʻoi aku ka ikaika o ka pākuʻi.
ʻO ka cystic fibrosis (CF) ma muli o ka hoʻololi ʻana o ka gene hoʻokahi i kapa ʻia ʻo CF transmembrane conductance regulator (CFTR). ʻO ka protein CFTR he kaila ion i loaʻa i loko o nā epithelial cell a puni ke kino, e komo pū ana me nā ala ea, kahi kahua nui o CF pathogenesis. hoʻopilikia i ka hiki ʻana o ka ʻōnaehana mucociliary transport (MCT) e hoʻomaʻemaʻe i nā ʻāpana inhaled a me nā pathogens mai nā alaloa.
ʻO nā vectors LV kekahi o nā moho alakaʻi no ka CF airway gene therapy, no ka mea hiki iā lākou ke hoʻohui mau i ka gene therapeutic i loko o ke ea basal cell (airway stem cells). Hoʻomaka. ʻO ka lawe ʻana o ka vector i loko o ka māmā hiki ke hopena i ka transduction alveolar, akā ʻaʻohe pono therapeutic i CF. Akā naʻe, neʻe maoli nā wai e like me nā mea lawe gene i ka alveoli ma ka hoʻoulu ʻana ma hope o ka lawe ʻana3,4 a hoʻomaʻemaʻe koke ʻia nā ʻāpana therapeutic i loko o ka waha e MCT.LV transduction ʻana i ka manawa e pili pono ai ka vector i ka manawa e hiki ai ke hoʻomau i ka lōʻihi o ka lawe ʻana i ke kelepona. manawa "5 - hiki ke hoʻemi maʻalahi ʻia e ka ea ea maʻamau a me ka hopu ʻana i ka mucus particle coordinated a me MCT.
No ka lanakila ʻana i kēia keakea, manaʻo mākou e kōkua paha nā LV magnetic particles (MPs) i ʻelua ala hoʻohui. a me ASL) no ka neʻe ʻana i ka papa cell 6. Ua hoʻohana nui ʻia nā MP ma ke ʻano he mau kaʻa lawe lāʻau i hoʻopaʻa ʻia i ka wā e hoʻopaʻa ai lākou i nā antibodies, nā lāʻau chemotherapeutic, a i ʻole nā ​​molekele liʻiliʻi ʻē aʻe e pili ana i nā membrane cell a i ʻole e hoʻopaʻa ʻia i nā mea hoʻopaʻa ʻili pūnaewele pili a hōʻiliʻili ma nā kahua tumora i mua o ka uila paʻa. Māhele Mākēneki no ka Lapaʻau ʻana i ka maʻi maʻi 7. ʻO nā ʻenehana "hyperthermal" ʻē aʻe e manaʻo e hoʻomehana i nā MPs i ka wā e ʻike ʻia ai lākou i ka oscillating field magnetic, a laila e luku ai i nā cell tumor. ua hoʻokumu ʻia ka magnetotransfection, me ka hāʻawi ʻana in vitro o LV-MPs i ke kanaka bronchial epithelial cell line ma ke alo o kahi kahua magnetic static, e hoʻonui ana i ka pono o ka transduction e 186-fold i hoʻohālikelike ʻia me LV vector wale nō. Ua hoʻopili pū ʻia ʻo LV-MP i kahi hiʻohiʻona in vitro CF, kahi i hoʻonui ʻia ai ka transfection magnetic i ka transduction LV i ka air-liquid-fold moʻomeheu i ka 20. vivo magnetotransfection o nā kino ua loaʻa iki ka nānā 'ana a ua wale loiloi 'ia i loko o kekahi mau holoholona studies11,12,13,14,15, oi loa aku i loko o ka māmā16,17. Nonetheless, na manawa no ka magnetic transfection ma CF lung therapy ua maopopo. Tan et al. ʻO nā hoʻolālā inhalation CFTR e hoʻomaikaʻi i nā hopena lapaʻau i nā maʻi CF "6.
He paʻakikī ke nānā ʻana a me ke aʻo ʻana i ke ʻano o nā ʻāpana magnetic liʻiliʻi ma nā ʻaoʻao o ke ea i ke alo o kahi kahua magnetic i hoʻohana ʻia, a no laila ʻaʻole maopopo. a hoʻohana ʻia ma ke ʻano he hōʻailona mua o ka pono o ka mālama ʻana. Eia kekahi, hoʻohana kā mākou ʻano loiloi MCT i nā ʻāpana 10-35 µm anawaena i haku ʻia me ka alumina a i ʻole ke aniani kikoʻī refractive kiʻekiʻe e like me nā māka MCT e ʻike ʻia me ka hoʻohana ʻana i ka PB-PCXI21. Ua kūpono nā ʻenehana ʻelua no ka nānā ʻana i ke ʻano o nā ʻano ʻāpana, me MP.
Ma muli o kāna hoʻonā kiʻekiʻe a me ke kino, ua kūpono kā mākou PB-PCXI-based ASL a me MCT no ka nānā ʻana i ka dynamics a me nā ʻano o ka ʻano pākahi a me ka nui i loko o vivo e kōkua iā mākou e hoʻomaopopo a hoʻomaikaʻi i nā ʻenehana lawe gene MP. pulmonary airways o nāʻiole e kōkua wehewehe i kā mākou Non-uniform gene expression hiʻohiʻona nānā 'ia i loko o kā mākou gene carrier dose noiʻi holoholona 3,4.
ʻO ka pahuhopu o kēia noiʻi ʻana ʻo ia ka hoʻohana ʻana i ka synchrotron PB-PCXI e ʻike i nā neʻe in vivo o kahi pūʻulu MP i ka trachea o nā ʻiole ola. I ka lua o nā haʻawina, ua ʻimi mākou e hoʻohana i kēia hoʻonohonoho hoʻonohonoho maikaʻi loa e hōʻike i ke ʻano transduction i hopena mai ka lawe ʻana i vivo o LV-MPs i ke ala ea ʻiole, e pili ana i ka manaʻo ʻo ka lawe ʻana o LV-MPs i ka pōʻaiapili o ka huli ʻana i ke ala e hoʻomaikaʻi ai i ka maikaʻi o LV.
Ua hanaʻia nā haʻawina holoholona a pau e like me nā protocols i'āponoʻia e ke Kulanui o Adelaide (M-2019-060 a me M-2020-022) a me ka SPring-8 Synchrotron Animal Ethics Committee. Ua hanaʻia nā ho'āʻo e like me nā kuhikuhi ARRIVE.
Ua hana ʻia nā kiʻi X-ray a pau ma ka BL20XU beamline ma SPring-8 synchrotron ma Iapana, me ka hoʻohana ʻana i kahi hoʻonohonoho e like me ka mea i hōʻike mua ʻia21,22. ʻO ka pōkole, aia ka pahu hoʻokolohua ma 245 m mai ke apo hoʻopaʻa synchrotron. Hoʻohana ʻia kahi laʻana-i-detector mamao o 0.6 m no nā haʻawina vivo imaging contrast. Ua hoʻohana ʻia kahi ikehu monochromatic beam o 25 keV. Ua kiʻi ʻia nā kiʻi me ka hoʻohana ʻana i ka mea hoʻololi X-ray kiʻekiʻe (SPring-8 BM3) i hui pū ʻia me kahi mea ʻike sCMOS. Hoʻololi ka mea hoʻololi i nā kukui X i ke kukui ʻike ʻia me ka hoʻohana ʻana i kahi scintillator mānoanoa 10 µm (Gd3Al2Ga3O12 kiʻekiʻe me ka hoʻohana ʻana i ka microscope objection sensor). (NA 0.3). ʻO Orca-Flash4.0 ka mea ʻike sCMOS (Hamamatsu Photonics, Iapana) me ka nui o 2048 × 2048 pixels a me ka nui pixel maka o 6.5 × 6.5 µm. Hāʻawi kēia hoʻonohonoho i ka nui isotropic pono nui o ka 0.51 µm × 1 ka lōʻihi. Ua koho ʻia ʻo 100 ms no ka hoʻonui ʻana i ka lākiō hōʻailona-a-noise o nā ʻāpana magnetic i loko a ma waho o ke alaloa ʻoiai e hōʻemi ana i nā mea hana hoʻoneʻe hanu.
ʻAʻole hoʻohana ʻia ka mea lawe LV i nā haʻawina kiʻi SPring-8 PB-PCXI no ka mea ʻaʻole i hōʻoia ʻia ke keʻena kiʻi BL20XU Biosafety Level 2. Akā, ua koho mākou i kahi ʻano MP i hōʻike maikaʻi ʻia mai nā mea kūʻai aku ʻelua-e uhi ana i ka nui o ka nui, nā mea waiwai, nā hao hao, a me nā noi-e hoʻomaopopo mua i ka pili ʻana o ka MP a me nā aniani i loko o ke aniani. ma luna o ka surface.MPs i ka nui mai 0.25 a hiki i 18 μm a ua hana 'ia mai na mea like 'ole (e nana Papa 1), akā, i ka haku mele 'ana o kēlā me kēia hāpana, me ka nui o ka magnetic particles i loko o ka MP, i ike. ka unuhi ʻana i nā kiʻi ʻaoʻao e ʻike i ka ʻike hoʻonui ʻia o ka neʻe MP. ʻO kahi MP 0.25 μm nui ka liʻiliʻi ma mua o ka hoʻonā ʻana o ka mea kiʻi kiʻi, akā manaʻo ʻia ʻo PB-PCXI e ʻike i ko lākou ʻokoʻa leo a me ka neʻe ʻana o ka wai o ka ʻili i waiho ʻia ma hope o ka waiho ʻana.
Ua hoʻomākaukauʻia nā laʻana no kēlā me kēia MP ma ka Papa 1 i loko o 20 μl aniani capillaries (Drummond Microcaps, PA, USA) me ke anawaena o loko o 0.63 mm. Loaʻa nā mea kino kino i loko o ka wai,ʻoiai e loaʻa ana nā'āpana CombiMag i loko o ka waiwai waiwai o ka mea hana. Hoʻopihaʻia kēlā me kēia paipu me ka wai (e like me ka 11 μl holder). hoʻonoho ʻia ma ke kahua hoʻohālike i loko o ka pahu kiʻi, kēlā me kēia, a hoʻonoho i nā ʻaoʻao o ka wai. Hoʻomaka ka loaʻa ʻana o nā kiʻi X-ray i ka wā e hoʻonoho ʻia ai ka magnet ma kahi o 30 mm ma luna o ka hāpana, a loaʻa nā kiʻi ma ka wikiwiki o 4 mau kiʻi i kekona.
In vitro imaging setup loaʻa nā laʻana MP i nā capillaries aniani ma ke kahua unuhi xy hāpana. Ua kaha ʻia ke ala o ka kukuna X-ray me kahi laina ʻulaʻula.
I ka manawa i hoʻokumu ʻia ai ka ʻike in vitro o nā MP, ua hoʻāʻo ʻia kahi ʻāpana o lākou i loko o ka vivo i nā ʻiole Wistar albino wahine ʻano hihiu (~ 12 mau wiki, ~ 200 g). Seika) Ua hoʻomaʻamaʻa ʻia nā ʻiole me ka hui ʻana o Pharma), Iapana) e ka intraperitoneal injection. Ma hope o ka anesthesia, ua mākaukau lākou no ke kiʻi ʻana ma o ka wehe ʻana i ka hulu a puni ka trachea, e hoʻokomo i kahi paipu endotracheal (ET; 16 Ga iv cannula, Terumo BCT) a me ka hoʻopaʻa ʻole ʻana iā lākou ma luna o kahi ʻeke kiʻi wela i hana ʻia a mālama ʻia ka ʻeke kiʻi wela. i hoʻopili ʻia i ka pae unuhi hāpana ma ka pahu kiʻi ma kahi kihi liʻiliʻi e hoʻopololei i ka trachea ma ke kiʻi X-ray, e like me ka mea i hōʻike ʻia ma ke Kiʻi 2a.
(a) In vivo imaging setup in the SPring-8 imaging box, the path of the X-ray beam is marked with a red dashed line.(b,c) Magnet localization on the trachea was remotely using two orthogonally mounted IP cameras.
Ua hoʻopili ʻia kahi ʻōnaehana pamu syringe mamao (UMP2, World Precision Instruments, Sarasota, FL) me ka hoʻohana ʻana i ka syringe aniani 100 μl i ka paipu PE10 (OD 0.61 mm, ID 0.28 mm) ma o ka nila 30 Ga. Hoʻihoʻi ʻia ʻo ka piko o ka paipu i hoʻokomo ʻia i loko o ka hāpana MP e hoʻouna ʻia. (AD Instruments, Sydney, Australia) 22. I ke kiʻi ʻana I ka hiki ʻole ke komo ʻana o ka pā, ua hoʻonoho ʻia ʻelua mau kamera IP (Panasonic BB-SC382) ma kahi o 90 ° kekahi i kekahi a ua hoʻohana ʻia e nānā i ke kūlana o ka magnet e pili ana i ka trachea i ka wā kiʻi (Fig. 2b, c).
Hoʻopili ʻia kahi magnet i kahi pae ʻelua e hiki ke loaʻa mamao mai waho o ka hale kiʻi. hoʻokahi magnet ma luna o ka holoholona a ʻo kekahi ma lalo, me nā pou i hoʻonohonoho ʻia e huki (Figure 3b ); hoʻokahi magnet ma luna o ka holoholona a ʻo kekahi ma lalo, me nā lāʻau i hoʻonohonoho ʻia e kipaku (Figure 3c); a hoʻokahi magnet ma luna a kū pololei i ka trachea (Figure 3d). Ke hoʻonohonoho ʻia ka holoholona a me ka magnet a hoʻokomo ʻia ka MP e hoʻāʻo ʻia i loko o ka pauma syringe, e hāʻawi i kahi 50 μl dose ma ka wikiwiki o 4 μl/sec i ka loaʻa ʻana o nā kiʻi.
ʻO ka hoʻonohonoho hoʻonohonoho no ka in vivo imaging (a) hoʻokahi magnet ma luna o ka trachea ma kahi kihi o kahi o 30 °, (b) ʻelua mau magnet i hoʻonohonoho ʻia e huki, (c) ʻelua mau magnet i hoʻonohonoho ʻia e kipi, (d) hoʻokahi magnet ma luna a kū pololei i ka trachea. Nānā ka mea nānā mai ka waha a i ka māmā ma o ka trachea, a hele ka ʻaoʻao ʻaoʻao ma ka ʻaoʻao X-ray' beam. hoʻoneʻe ʻia ka magnet ma ka lōʻihi o ke alaloa a i ʻole hema a ʻākau paha ma luna o ka trachea ma ke kuhikuhi o ka kukuna X-ray.
Ua ʻimi pū mākou e hoʻoholo i ka ʻike a me ke ʻano o nā mea i loko o nā alaloa me ka ʻole o ka hanu hoʻohaunaele a me ka neʻe ʻana o ka naʻau. Ua hana hou ʻia ke kaʻina hana, me ka hoʻohui ʻana i ka nui o MP inā ʻaʻohe MP i ʻike ʻia ma ka ʻili o ke ea.
ʻO nā kiʻi i kiʻi ʻia he kahua pālahalaha a hoʻoponopono ʻia ka māla ʻeleʻele a laila hui ʻia i kahi kiʻiʻoniʻoni (20 mau kiʻi i kekona; 15-25 × ka wikiwiki maʻamau e pili ana i ka hanu hanu) me ka hoʻohana ʻana i kahi palapala maʻamau i kākau ʻia ma MATLAB (R2020a, The Mathworks).
Ua mālama ʻia nā haʻawina hoʻouna ʻana o LV gene vector ma ka Laboratory Animal Research Facility ma ke Kulanui o Adelaide a manaʻo e hoʻohana i nā hopena o ka hoʻokolohua SPring-8 e ʻike ai inā hiki i ka lawe ʻana o LV-MP i mua o kahi kahua magnetic ke hoʻonui i ka hoʻololi ʻana o ka gene i vivo.
LV gene vectors ua hanaia me ka ho'ohana 'ana i ka mea i wehewehe mua 'ia 25, 26 . The LacZ vector expresses the nuclear-localized beta-galactosidase gene driven by the constitutive MPSV promoter (LV-LacZ), which produces a blue reaction product in transduced cells, visible in lung tissue fronts and tissue sections. titer i TU/ml. Hoʻopaʻa ʻia nā mea lawe ma -80 °C, hoʻoheheʻe ʻia ma mua o ka hoʻohana ʻana, a hoʻopaʻa ʻia iā CombiMag ma ka hui ʻana ma kahi ratio 1: 1 a me ka incubating ma ka hau no ka liʻiliʻi o 30 mau minuke ma mua o ka lawe ʻana.
ʻO nāʻiole Sprague Dawley maʻamau (n = 3 / hui, ~ 2-3 i hoʻopaʻaʻia intraperitoneally me ka hui pūʻana o 0.4 mg / kg medetomidine (Domitor, Ilium, Australia) a me 60 mg / kg ketamine (Ilium, Australia) mahina) ip) injection a me ka non-surgical oral cannulation me ka 16 Ga ivduction hiki ke loaʻa i ka tissue transnula. ua hoʻopili ʻia me ka hoʻohana ʻana i kā mākou protocol perturbation mechanical i wehewehe mua ʻia, kahi i hoʻopili ʻia ai ka tracheal airway surface axially me ka uea hīnaʻi (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.
Ua hoʻonohonoho ʻia ke kahua magnetic i hoʻohana ʻia i kēia hoʻokolohua ma ke ʻano like me ka in vivo X-ray imaging study, me nā magnet like i paʻa ma luna o ka trachea me ka hoʻohana ʻana i nā stent distillation clips (Figure 4). 3 holoholona) loaʻa nā LV-MP like me ka hoʻohana ʻole ʻana i kahi magnet. Ma hope o ka hoʻopau ʻana o ka infusion, ua wehe ʻia ka cannula mai ka paipu ET a hoʻokuʻu ʻia ka holoholona. Noho mau ka magnet ma kahi o 10 mau minuke, a laila wehe ʻia. (Antisedan, Zoetis, Australia) Ua mālama ʻia nā ʻiole i mahana a nānā ʻia a hiki i ka hoʻōla piha ʻana mai ka maʻi maʻi.
ʻO ka mea lawe LV-MP i loko o kahi keʻena palekana olaola. Hiki ke ʻike ʻia ka pahu Luer hina hina o ka paipu ET e puka mai ana mai ka waha a hoʻokomo ʻia ka piko gel o ka pipette i hōʻike ʻia ma ke kiʻi ma o ka paipu ET i ka hohonu i makemake ʻia i loko o ka trachea.
Hoʻokahi pule ma hope o ke kaʻina hana dosing LV-MP, ua pepehi kanaka ʻia nā holoholona e ka inhalation 100% CO2 a ua loiloi ʻia ka ʻōlelo LacZ me ka hoʻohana ʻana i kā mākou X-gal maʻamau. (Sylgard, Dow Inc) me ka Minutien needle (Fine Science Tools) e nānā i ka luminal surface.The distribution and pattern of transduced cells was confirmed by frontal photographing using a Nikon microscope (SMZ1500) with a DigiLite camera and TCapture software (Tucsen Photonics, China). ʻO ka lōʻihi o ka trachea i kiʻi ʻia i kēlā me kēia kiʻi, e hōʻoia i ka lawa ʻana ma waena o kēlā me kēia kiʻi e ʻae ai i ke kiʻi "hili ʻia". (R2020a, MathWorks) e like me ka mea i hōʻike mua ʻia, me ka hoʻohana ʻana i nā hoʻonohonoho o 0.35 0.15, a me ka waiwai <0.7. Ma ka ʻimi ʻana i nā ʻāpana o ka ʻiʻo, ua hana lima ʻia kahi mask ma GIMP v2.10.24 no kēlā me kēia kiʻi composite i mea e pale ai i nā wahi kikoʻī mai waho aku. Ua hōʻuluʻulu ʻia nā kiʻi i hoʻohui ʻia mai kēlā me kēia holoholona e hoʻohua i ka nui o ka ʻāpana ʻeleʻele no kēlā holoholona.
Hoʻokomo ʻia kēlā me kēia trachea i loko o ka paraffin a ua ʻoki ʻia nā ʻāpana 5 μm. Ua counterstained nā ʻāpana me ka ʻulaʻula wikiwiki kūʻokoʻa no 5 min a ua loaʻa nā kiʻi me ka microscope Nikon Eclipse E400, kāmela DS-Fi3 a me NIS element capture software (version 5.20.00).
Ua hanaʻia nāʻikepili helu a pau ma GraphPad Prism v9 (GraphPad Software, Inc.). Ua hoʻonohonohoʻia ka nui o ka helu ma ka p ≤ 0.05. Ua hōʻoiaʻia ka maʻamau me ka ho'āʻo Shapiro-Wilk, a ua loiloiʻia nāʻokoʻa o ka staining LacZ me ka hoʻohanaʻana i ka t-test unpaired.
Ua nānā ʻia nā MP ʻeono i wehewehe ʻia ma ka Papa 1 me ka hoʻohana ʻana i ka PCXI, a ua wehewehe ʻia ka ʻike ma ka Papa 2. ʻElua polystyrene MPs (MP1 a me MP2; 18 μm a me 0.25 μm, kēlā me kēia) ʻaʻole i ʻike ʻia ma lalo o PCXI, akā ʻike ʻia ke koena o nā mea hoʻohālike (hōʻike ʻia nā hiʻohiʻona ma Fe 1MP3 a me MP54%). ʻO 0.25 μm a me 0.9 μm, ʻo ia hoʻi) ʻike ʻia. neʻe aku nā mea magnet mai ka capillary, ua hoʻolōʻihi ʻia nā ʻāpana i nā kaula lōʻihi, akā i ka hoʻokokoke ʻana o nā mea magnet a me ka hoʻonui ʻana i ka ikaika o ka māla, ua pōkole nā ​​kaula i ka neʻe ʻana o nā ʻāpana i ka ʻili o luna o ka capillary (e nānā i ka Video S1: MP4), e hoʻonui ana i ka mānoanoa o ka ʻili. capillary (e nānā i ke wikiō S2: MP4). Ma hope o ka pau ʻana o ka neʻe ʻana o ka magnet, hoʻomau ka neʻe ʻana o nā ʻāpana no ka manawa pōkole ma hope o ka hiki ʻana i ke kūlana kaulike. I ka neʻe ʻana o ka MP i ka ʻaoʻao o luna o ka capillary, e huki pinepine nā ʻāpana magnetic i ka ʻōpala i loko o ka wai.
He ʻokoʻa ka ʻike ʻana o MP ma lalo o ka PCXI ma waena o nā laʻana. ʻO ka hoʻonui ʻana. E hoʻomaopopo ʻoe ʻaʻole i hoʻohālikelike ʻia nā anawaena o nā kiʻina magnet i nā kiʻi āpau a ʻoi aku ka nui o 100 mau manawa ma mua o ka mea i hōʻike ʻia.
I ka unuhi ʻana o ka magnet ma ka hema a me ka ʻākau ma ka piko o ka capillary, hoʻololi ke kihi o ke kaula MP e kūlike me ka magnet (e ʻike i ke kiʻi 6), no laila e wehewehe ana i nā laina kahua magnetic. S3:MP5) He mea maopopo loa keia i ke ki'i kokoke ana i ka hopena o ka capillary, ka mea e houluulu ai na MPs i ka wai-ea interface. O na particles i MP6, i oi aku ka paakiki o ka ike ana ma mua o MP3-5, aole i kauo i ka nee ana o ka magnet ma ka capillary, aka, ua wehe na kaula MP ma ke kahua o ka MP4. ʻO nā hihia, i ka wā i hoʻemi ʻia ai ke kahua magnetic i hoʻohana ʻia ma ka hoʻoneʻe ʻana i ka magnet i kahi mamao loa mai kahi kiʻi kiʻi, ua iho mālie nā MP i koe i ka ʻili o lalo o ka paipu ma o ka umekaumaha ʻoiai e waiho ana i ke kaula (e nānā i ke wikiō Hoʻohui S5: MP3).
Hoʻololi ke kihi o ke kaula MP i ka unuhi ʻia ʻana o ka magnet ma ka ʻākau ma luna o ka capillary.
Ua hōʻike kā mākou mau hoʻāʻo ʻana i ka neʻe ʻana o ka magnet i hope a i waho ma ka trachea e hoʻomaʻamaʻa i ka ʻike ʻana o MP i ka pōʻaiapili o ka neʻe paʻakikī i vivo.In vivo hoʻāʻo ʻaʻole i hana ʻia e like me ka polystyrene beads (MP1 a me MP2) ʻaʻole ʻike ʻia i loko o ka capillary. 3a), no ka mea, ua ʻoi aku ka lōʻihi o nā kaulahao MP a ʻoi aku ka maikaʻi ma mua o ka hoʻopau ʻana o ka hoʻonohonoho magnet. ʻAʻole i ʻike ʻia ka MP3, MP4 a me MP6 i ka trachea o nā holoholona ola. vivo hana o MP.
ʻO ka waiho ʻana i ka magnet ma luna o ka trachea i ka wā o ka hāʻawi ʻana i ka MP i hopena i nā mea he nui, akā ʻaʻole nā ​​​​mea a pau, ʻo nā MP i hoʻopaʻa ʻia i ke kahua o ka ʻike. I ka huli ʻana ma kahi mamao loa ma ka trachea ma hope o ka hāʻawi ʻana o MP, ua ʻike ʻia kekahi mau MP ma kahi kokoke i ka carina, e hōʻike ana ʻaʻole lawa ka ikaika o ke kahua magnetic e hōʻiliʻili a mālama i nā MP a pau, ʻoiai ua hāʻawi ʻia lākou ma ka ʻāina o ka ikaika loa o ka mākēneki i ka wā o ke kaʻina wai.
Nā kiʻi mai (a) ma mua a (b) ma hope o ka lawe ʻana o MP5 i loko o ka trachea o kahi ʻiole euthanized hou me ka magnet i hoʻonoho pololei ʻia ma luna o ka wahi kiʻi. Aia ka wahi kiʻi ma waena o nā apo cartilage ʻelua.
ʻO ka unuhi ʻana i ka magnet ma ka trachea in vivo ua hoʻololi ke kaulahao MP i ke kihi o ka ʻili ea e like me ka mea i ʻike ʻia i nā capillaries (e ʻike i ke kiʻi 8 a me ke wikiō hou S7: MP5). ʻIke ʻia e hoʻololi i ka hohonu o ka ʻili wai o ke kaula i ka wā e hoʻoneʻe ʻia ai ka magnet i ka lōʻihi ma ka trachea, a e hoʻonui i ka wā e hoʻoneʻe pololei ʻia ai ka mākēneki ma luna o ke poʻo a hoʻohuli ʻia ke kaula ʻāpana i kahi kūlana kū (e nānā i ke wikiō Hoʻohui S7). : MP5 ma 0:09, lalo akau). Ua loli ke ano o ka oni i ka unuhiia ana o ka magnet ma ka piko o ka trachea ma ka aoao (oia hoi, ma ka hema a akau paha o ka holoholona ma mua o ka loa o ka trachea). e kūlike me ka ʻano MP a mākou i ʻike ai ma lalo o kahi kahua magnetic i hoʻopili ʻia i loko o kahi capillary aniani.
Nā kiʻi laʻana e hōʻike ana i ka MP5 i loko o ka trachea o kahi ʻiole hoʻomaʻamaʻa ola.
I ka hoʻonohonoho ʻia ʻana o nā pou ʻelua ma ka ʻaoʻao ʻākau-hema ma luna a ma lalo o ka trachea (ʻo ia hoʻi ka huki ʻana; Fig. 3b), ua ʻoi aku ka lōʻihi o ka MP chords a aia ma ka ʻaoʻao ʻaoʻao o ka trachea ma mua o ka dorsal tracheal surface (e nānā i ke wikiō Hoʻohui S9: MP5). Ua hoʻohana ʻia kahi mea manaʻo pālua, ʻo ia ka mea maʻamau i ka wā e hoʻohana ʻia ai ka mea hoʻohana hoʻokahi. i ke ala ea akā e hele ana ma ke ala ea ma 90 degere i hiki ai i nā laina kahua ke ʻaʻa i ka paia tracheal orthogonally (Fig. 3d), kahi orientation i hoʻolālā ʻia e hoʻoholo inā hiki ke nānā ʻia ka ʻāpana particle ma ka paia ʻaoʻao. 3a) i koho ʻia no nā haʻawina ʻo vivo gene carrier.
Ke kiʻi pinepine ʻia ka holoholona ma hope koke o ka pepehi kanaka ʻana, ʻo ka nele o ka neʻe ʻana o ka ʻiʻo huikau, ʻo ia ka mea hiki ke ʻike ʻia nā laina ʻāpana liʻiliʻi a ʻoi aku ka maikaʻi ma ke kahua interchondral akaka, "wobbly" i ka laina me ka neʻe unuhi o ka magnet. Eia nō naʻe, ʻaʻole hiki ke ʻike maopopo i ke alo a me ka neʻe ʻana o nā ʻāpana MP6.
ʻO ka LV-LacZ titer he 1.8 × 108 TU / ml, a ma hope o ka hui ʻana o 1: 1 me CombiMag MP (MP6), ua loaʻa i nā holoholona he 50 μl tracheal dose o 9 × 107 TU / ml LV kaʻa (ie 4.5 × 106 TU / ʻiole). ).Ma kēia mau haʻawina, ma kahi o ka unuhi ʻana i ka magnet i ka wā hana, ua hoʻopaʻa mākou i ka magnet ma kahi kūlana e hoʻoholo ai inā hiki ke hoʻomaikaʻi ʻia ka transduction LV (a) i ka hoʻohālikelike ʻana i ka lawe ʻana i ka vector i ka loaʻa ʻole o kahi kahua magnetic, a (b) hiki ke hoʻohuli ʻia ʻo Airway cell i transduced i nā ʻāpana i hoʻopaʻa ʻia o ka ea luna.
ʻO ka loaʻa ʻana o nā magnet a me ka hoʻohana ʻana o CombiMag i hui pū ʻia me nā vectors LV ʻaʻole i ʻike ʻia he hopena maikaʻi ʻole i ke olakino holoholona, ​​​​e like me kā mākou maʻamau LV vector delivery protocol. ʻO ka hoʻopaʻa ʻana i loko o ka pūʻulu mana (Fig. 9b). ʻO ka helu ʻana o nā wahi i hoʻopaʻa ʻia ʻo X-Gal maʻamau i hōʻike ʻia i ka hoʻokele ʻana o LV-MP ma ke alo o kahi kahua magnetic i hana i kahi hoʻomaikaʻi 6-fold (Fig. 9c).
He laʻana kiʻi hoʻohuihui e hōʻike ana i ka hoʻololi ʻana o ka tracheal e LV-MP (a) ma ke alo o kahi mākēneki a me (b) i ka loaʻa ʻole o kahi magnet. (c) ʻO ka hoʻomaikaʻi koʻikoʻi o ka helu ʻana i ka wahi transduction LacZ maʻamau i loko o ka trachea i ka wā e hoʻohana ai i ka magnet (*p = 0.029, t-test, n = 3 no kēlā me kēia hui, mean ± SEM).
ʻO nā ʻāpana ʻulaʻula wikiwiki kūʻokoʻa (laʻana i hōʻike ʻia ma ka Fig.
ʻO kahi paʻakikī koʻikoʻi no ke ea gene therapy ʻo ka hoʻonohonoho pono ʻana o nā mea lawe i nā wahi hoihoi a loaʻa i nā kiʻekiʻe kiʻekiʻe o ka transduction maikaʻi i ka māmā neʻe i ke alo o ka ea a me ka hoʻomaʻemaʻe ʻana o ka mucus. Loaʻa ka maikaʻi o ka transduction i ka hoʻohālikelike ʻana i nā ʻano hana hoʻoili ʻē aʻe e like me ka electroporation, no ka mea hiki iā ia ke hoʻohui i ka maʻalahi, ka maikaʻi o ke kumukūʻai, ka hoʻopuka ʻana i ka localization, ka hoʻonui ʻana i ka pono, a me ka pōkole o ka manawa incubation, a ʻoi aku ka liʻiliʻi o ka lawe lawe dose10. ma na ala ea ola paa.
ʻO kā mākou mau hoʻokolohua in vitro synchrotron PCXI ua ʻike ʻia nā ʻāpana āpau a mākou i hoʻāʻo ai, koe naʻe ka polystyrene MP, i ka hoʻonohonoho kiʻi i hoʻohana ʻia e mākou. kahua. ʻO kēia mau kahua kaʻawale ke kumu e hōʻuluʻulu ai a hoʻohui ʻia nā ʻāpana like ʻole, me nā neʻe e like me ke kaula o ka hui ma muli o nā ikaika kūloko mai nā mana hoihoi a hoʻopailua o nā ʻāpana ʻē aʻe.
Hōʻike ʻia nā kaʻaahi ʻāpana (a,b) i hana ʻia i loko o nā capillaries piha wai a me (c,d) trachea piha i ka ea.
Ke hoʻoneʻe ʻia ka magnet ma luna o ka capillary, ua hiki ke kihi o ke kaula ʻāpana i kahi paepae koʻikoʻi no MP3-5 i loaʻa Fe3O4, a laila ʻaʻole i noho hou ke kaula ʻāpana i ke kūlana kumu, akā neʻe ma ka ʻili i kahi kūlana hou. ʻoi aku ka liʻiliʻi, he ʻokoʻa nā uhi a i ʻole nā ​​​​uku o ka ʻili, a i ʻole kahi wai lawe waiwai i hoʻopilikia i ko lākou hiki ke neʻe. ʻOi aku ka nāwaliwali o ke kiʻi ʻokoʻa o nā ʻāpana CombiMag, e hōʻike ana he like like ka wai a me nā ʻāpana a no laila ʻaʻole hiki ke neʻe maʻalahi kekahi i kekahi. ʻO ka ikaika o ka mākēneki a me ka mamao ma waena o ka magnet a me ka wahi i hoʻopaʻa ʻia.
Ke hāʻawi ʻia nā ʻāpana i loko o ke kino, paʻakikī lākou e ʻike ma ke ʻano o ka ʻiʻo o ke kino paʻakikī, akā ua hoʻonui ʻia ka hiki ke ʻike iā lākou ma ka unuhi ʻana i ka magnet ma luna o ka trachea e "wiwili" i nā kaula MP. trachea. He ʻokoʻa me nā haʻawina in vitro, ʻaʻole hiki ke kauo ʻia nā ʻāpana ma ka trachea ma ka unuhi ʻana i ka magnet. Ua kūlike kēia ʻike me ka hana maʻamau o ka mucus e uhi ana i ka ʻili o ka trachea i nā ʻāpana inhaled, e hoʻopaʻa iā lākou i loko o ka mucus a laila hoʻomaʻemaʻe ʻia e ka mīkini hoʻomaʻemaʻe mucociliary.
Manaʻo mākou ʻo ka hoʻohana ʻana i nā mea hoʻokalakupua no ka huki ʻana ma luna a ma lalo o ka trachea (Fig. 3b) hiki ke loaʻa i kahi kahua mākēneki like ʻole, ma mua o kahi kahua magnetic i hoʻopaʻa nui ʻia i kahi manawa, hiki ke alakaʻi i kahi mahele like ʻole o nā particles. Ke hōʻike nei kēia mau ʻike ʻelua ʻaʻole i hoʻomaikaʻi maikaʻi ka hoʻonohonoho ʻelua-magnet i ka mana kūloko o ka huli ʻana o MP, a he mea paʻakikī ke hoʻonohonoho ʻana i nā mana magnetic ikaika, no ka mea, ʻaʻole i hoʻonui ʻia ka nui o nā mea i hoʻopaʻa ʻia ma kēia kahua kiʻi. i loko o ka waiho ʻana. No laila, ʻo ka 30-degree angle magnet configuration (Figure 3a) i manaʻo ʻia ʻo ke ala maʻalahi a maikaʻi loa no ka hoʻāʻo in vivo.
Ua hōʻike ʻia ka haʻawina LV-MP i ka wā i hui pū ʻia ai nā vectors LV me CombiMag a hāʻawi ʻia ma hope o ka hoʻopilikia kino ʻana i ke alo o kahi māla magnetic, ua hoʻonui nui ʻia nā pae transduction ma ka trachea i hoʻohālikelike ʻia me nā mana. alakaʻi i ka efficacy kiʻekiʻe i ka wā e hōʻemi ana i nā titers i hāʻawi ʻia, transduction off-target, inflammatory a me nā hopena ʻaoʻao immune, a me nā kumukūʻai lawe gene. ʻO ka mea nui, e like me ka mea hana, hiki ke hoʻohana ʻia ʻo CombiMag me nā ʻano hana hoʻololi ʻē aʻe, me nā vectors viral ʻē aʻe (e like me AAV) a me nā waikawa nucleic.