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Pono e hoʻopaʻa ʻia nā gene vectors no ka mālama ʻana i ka pulmonary cystic fibrosis i nā ala ea conductive, no ka mea, ʻaʻohe hopena lapaʻau o ka transduction lung peripheral.ʻO ka maikaʻi o ka transduction viral pili pono i ka manawa noho o ka mea lawe.Eia nō naʻe, hoʻolaha maoli nā wai hoʻopuka e like me nā mea lawe gene i loko o ka alveoli i ka wā o ka inhalation, a hoʻoneʻe koke ʻia nā ʻāpana therapeutic o kēlā me kēia ʻano e ka lawe mucociliary.He mea koʻikoʻi ka hoʻonui ʻana i ka manawa noho o nā mea lawe gene ma ka ʻōpū hanu akā paʻakikī ke hoʻokō.Hiki i nā ʻāpana magnetic i hoʻohui ʻia i ka mea lawe i hiki ke kuhikuhi ʻia i ka ʻili o ka ʻōpū hanu hiki ke hoʻomaikaʻi i ka huli ʻāina.Ma muli o nā pilikia me ka in vivo imaging, ʻaʻole i hoʻomaopopo maikaʻi ʻia ke ʻano o ia mau mea liʻiliʻi magnetic ma ka ʻili o ke ea ma ke alo o kahi māla magnetic i noi ʻia.ʻO ka pahuhopu o kēia noiʻi ʻana ʻo ia ka hoʻohana ʻana i ka synchrotron imaging e nānā i loko o ka vivo i ka neʻe ʻana o kahi ʻano o nā ʻāpana magnetic i loko o ka trachea o nā ʻiole anesthetized i mea e aʻo ai i ka dynamics a me nā ʻano o ka ʻano o nā ʻāpana pākahi a me ka nui i loko o vivo.A laila noʻonoʻo pū mākou inā e hoʻonui ʻia ka lawe ʻana i nā ʻāpana magnetic lentiviral i mua o kahi māla magnetic i ka pono o ka transduction ma ka trachea iole.Hōʻike ke kiʻi X-ray Synchrotron i ka ʻano o nā ʻāpana magnetic i ka paʻa a me ka neʻe ʻana i nā māla magnetic in vitro a i vivo.ʻAʻole hiki ke kauo maʻalahi i nā ʻāpana ma luna o ka ʻili o nā ala ea ola me ka hoʻohana ʻana i nā magnet, akā i ka wā o ka lawe ʻana, hoʻopaʻa ʻia nā waihona i ke kahua o ka ʻike, kahi i ikaika loa ai ka māla magnetic.Ua hoʻonui ʻia ka maikaʻi o ka hoʻololi ʻana i ʻeono mau manawa i ka wā i hāʻawi ʻia ai nā ʻāpana magnetic lentiviral i mua o kahi kahua magnetic.I ka hui pū ʻia, ʻōlelo ʻia kēia mau hopena he mau ala kūpono e hoʻomaikaʻi ai i ka huli ʻana o nā gene vector a me nā pae transduction i nā ala ea conductive in vivo.
Hoʻokumu ʻia ka cystic fibrosis (CF) ma muli o nā ʻano like ʻole 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 cell epithelial he nui i loko o ke kino, me nā alaloa, kahi kahua nui i ka pathogenesis o ka cystic fibrosis.ʻO nā hemahema i loko o ka CFTR ke alakaʻi i ka lawe ʻana i ka wai ʻino, ka wai maloʻo o ka ʻili o ke ea, a me ka emi ʻana o ka hohonu o ka wai o ke ea (ASL).Hoʻopilikia pū ia i ka hiki ʻana o ka ʻōnaehana mucociliary transport (MCT) e hoʻomaʻemaʻe i nā alaloa o nā mea inhaled a me nā pathogens.ʻO kā mākou pahuhopu ka hoʻomohala ʻana i kahi lāʻau lapaʻau lentiviral (LV) e hāʻawi i ke kope pololei o ka gene CFTR a hoʻomaikaʻi i ka ASL, MCT, a me ke olakino māmā, a e hoʻomau i ka hoʻomohala ʻana i nā ʻenehana hou e hiki ke ana i kēia mau palena i vivo1.
ʻO nā vectors LV kekahi o nā moho alakaʻi no ka cystic fibrosis gene therapy, no ka mea hiki iā lākou ke hoʻohui mau i ka gene therapeutic i loko o nā sela basal ea (airway stem cell).He mea koʻikoʻi kēia no ka mea hiki iā lākou ke hoʻihoʻi i ka hydration maʻamau a me ka hoʻomaʻemaʻe mucus ma o ka hoʻokaʻawale ʻana i loko o nā pūnaewele i hoʻoponopono ʻia e nā gene-corrected ea i pili i ka cystic fibrosis, e hopena i nā pōmaikaʻi o ke ola.Pono e kuhikuhi ʻia nā vectors LV i nā ala ea conductive, ʻoiai ʻo ia kahi e hoʻomaka ai ke komo ʻana o ka māmā i CF.ʻO ka lawe ʻana o ka vector i loko o ka māmā hiki ke hopena i ka transduction alveolar, akā ʻaʻohe hopena therapeutic i ka cystic fibrosis.Eia nō naʻe, neʻe maoli nā wai e like me nā mea lawe gene i loko o ka alveoli i ka wā i hoʻomoʻa ʻia ma hope o ka hānau ʻana3,4 a hoʻokuʻu koke ʻia nā mea therapeutic i loko o ka lua waha e MCTs.ʻO ka maikaʻi o ka transduction LV pili pono i ka lōʻihi o ka manawa e noho kokoke ai ka vector i nā cell target e ʻae i ka lawe ʻana i ke kelepona - "manawa noho" 5 i hoʻopaʻa maʻalahi ʻia e ka ea ea kūloko a me ka hoʻopili ʻana o ka mucus a me nā ʻāpana MCT.No ka cystic fibrosis, ʻo ka hiki ke hoʻolōʻihi i ka manawa noho LV ma nā alaloa he mea nui ia e hoʻokō i nā kiʻekiʻe kiʻekiʻe o ka transduction ma kēia wahi, akā i kēia manawa ua paʻakikī.
No ka lanakila ʻana i kēia pilikia, manaʻo mākou e hiki ke kōkua ʻo LV magnetic particles (MPs) i ʻelua ala hoʻohui.ʻO ka mea mua, hiki iā lākou ke alakaʻi ʻia e kahi magnet i ka ʻili o ke ea e hoʻomaikaʻi i ka huli ʻana a kōkua i nā ʻāpana lawe gene i ka ʻāpana kūpono o ke alaloa;a me ASL) neʻe i loko o ka papa cell 6. Hoʻohana nui ʻia nā MP ma ke ʻano he mau kaʻa lawe lāʻau i manaʻo ʻia i ka wā e hoʻopaʻa ai lākou i nā antibodies, nā lāʻau lapaʻau chemotherapy, a i ʻole nā ​​molekole liʻiliʻi ʻē aʻe e pili ana i nā membrane cell a i ʻole e hoʻopaʻa ʻia i ko lākou mau cell surface receptors a hōʻiliʻili i nā wahi maʻi maʻi. hiki mai o ka uila paʻa.Nā kahua mākēneki no ka lāʻau maʻi maʻi maʻi 7. ʻO nā ʻano hana "hyperthermic" ʻē aʻe e kuhikuhi ana i ka pepehi ʻana i nā cell tumor ma o ka hoʻomehana ʻana i nā MP i ka wā e ʻike ʻia ai i nā māla magnetic oscillating.ʻO ke kumumanaʻo o ka hoʻololi magnetic, kahi e hoʻohana ʻia ai kahi māla magnetic ma ke ʻano he transfection agent e hoʻomaikaʻi ai i ka hoʻololi ʻana o ka DNA i loko o nā cell, hoʻohana mau ʻia i loko o ka vitro me ka hoʻohana ʻana i kahi ʻano o nā mea hoʻohana ʻole viral a viral no nā laina kelepona paʻakikī. ..Ua hoʻokumu ʻia ka pono o LV magnetotransfection me ka hāʻawi ʻana i ka LV MP in vitro i loko o kahi laina kelepona o ka epithelium bronchial kanaka i mua o kahi kahua magnetic static i hoʻokumu ʻia, e hoʻonui ana i ka pono o ka transduction e 186 mau manawa i hoʻohālikelike ʻia me ka vector LV wale nō.Ua hoʻohana pū ʻia ʻo LV MT i kahi kumu hoʻohālike in vitro o cystic fibrosis, kahi i hoʻonui ai ka transfection magnetic i ka transduction LV i nā moʻomeheu interface wai-wai ma kahi helu o 20 i mua o ka cystic fibrosis sputum10.Eia naʻe, i loko o vivo organ magnetotransfection ua loaʻa iki ka nānā ʻana a ua loiloi wale ʻia i loko o nā haʻawina holoholona liʻiliʻi11,12,13,14,15, ʻoi aku hoʻi i loko o nā māmā16,17.Eia nō naʻe, ʻike maopopo ʻia nā hiki o ka hoʻololi ʻana o ka magnetic i ka lung therapy i ka cystic fibrosis.Tan et al.(2020) i ʻōlelo ʻia "ʻo kahi haʻawina hōʻoia e pili ana i ka hāʻawi ʻana i ka pulmonary maikaʻi o nā nanoparticles magnetic e wehe i ke ala no nā hoʻolālā inhalation CFTR e hiki mai ana e hoʻomaikaʻi i nā hopena lapaʻau i nā maʻi me ka cystic fibrosis"6.
ʻO ke ʻano o nā ʻāpana liʻiliʻi liʻiliʻi ma ka ʻili o ka ʻōpū hanu i mua o kahi pā magnetic i hoʻopili ʻia he paʻakikī ke nānā a hoʻopaʻa ʻia, a no laila ʻaʻole maopopo ʻia lākou.Ma nā haʻawina ʻē aʻe, ua hoʻomohala mākou i kahi ʻano Synchrotron Propagation Based Phase Contrast X-Ray Imaging (PB-PCXI) no ke kiʻi non-invasive a me ka helu ʻana o nā minuke i loko o nā loli vivo i ka hohonu ASL18 a me ka ʻano MCT19, 20 e ana pololei i ka hydration o ke kahawai kinoea. a hoʻohana ʻia ma ke ʻano he hōʻailona hōʻailona maikaʻi o ka lāʻau lapaʻau.Eia hou, hoʻohana kā mākou ʻano helu helu MCT i nā ʻāpana 10-35 µm anawaena i haku ʻia me ka alumina a i ʻole ke aniani kuhikuhi refractive kiʻekiʻe e like me nā māka MCT i ʻike ʻia me PB-PCXI21.Ua kūpono nā ʻano ʻelua no ke kiʻi ʻana i nā ʻano ʻāpana, me nā MP.
Ma muli o ke kiʻekiʻe o ka spatial a me ka hoʻonā kino, ua kūpono kā mākou PB-PCXI-based ASL a me MCT assays e aʻo i ka dynamics a me nā ʻano ʻano o nā ʻāpana hoʻokahi a me ka nui i loko o vivo e kōkua iā mākou e hoʻomaopopo a hoʻopaʻa pono i nā kaʻina lawe gene MP.ʻO ke ala a mākou e hoʻohana ai ma ʻaneʻi e pili ana i kā mākou mau haʻawina e hoʻohana ana i ka SPring-8 BL20B2 beamline, kahi i ʻike ai mākou i ka neʻe ʻana o ka wai ma hope o ka lawe ʻana i kahi ʻano o kahi vector dummy i loko o nā ala ihu a me ka pulmonary o nā ʻiole e kōkua i ka wehewehe ʻana i kā mākou mau hiʻohiʻona hōʻike gen heterogeneous i nānā ʻia. i loko o kā mākou gene.nā haʻawina holoholona me ka nui o ka lawe lawe 3.4.
ʻO ka pahuhopu o kēia noiʻi ʻana ʻo ia ka hoʻohana ʻana i ka synchrotron PB-PCXI e ʻike i nā neʻe vivo o kahi ʻano o nā MP i ka trachea o nā ʻiole ola.Ua hoʻolālā ʻia kēia mau haʻawina kiʻi PB-PCXI no ka hoʻāʻo ʻana i ka pūʻulu MP, ka ikaika o ke kahua magnetic, a me kahi e hoʻoholo ai i ko lākou hopena i ka neʻe ʻana o MP.Ua manaʻo mākou e kōkua ke kahua magnetic waho i ka noho MF i hāʻawi ʻia a neʻe paha i ka wahi i kuhikuhi ʻia.Ua ʻae kēia mau haʻawina iā mākou e hoʻoholo i nā hoʻonohonoho magnet e hoʻonui i ka nui o nā ʻāpana i waiho ʻia i loko o ka trachea ma hope o ka waiho ʻana.Ma ka lua o nā haʻawina, ua manaʻo mākou e hoʻohana i kēia hoʻonohonoho maikaʻi loa e hōʻike i ke ʻano transduction i loaʻa mai ka lawe ʻana i loko o ka vivo o LV-MPs i nā ala ea ʻiole, ma ka manaʻo e hoʻopuka ʻia nā LV-MP ma ke ʻano o ka huli ʻana i ke ea. i ka hoʻonui ʻana i ka pono transduction LV..
Ua mālama ʻia nā haʻawina holoholona āpau e like me nā protocols i ʻae ʻia e ke Kulanui o Adelaide (M-2019-060 a me M-2020-022) a me ka SPring-8 Synchrotron Animal Ethics Committee.Ua hana ʻia nā hoʻokolohua e like me nā ʻōlelo aʻoaʻo a ARRIVE.
Ua lawe ʻia nā kiʻi x-ray a pau ma ka beamline BL20XU ma ka SPring-8 synchrotron ma Iapana me ka hoʻohana ʻana i kahi hoʻonohonoho e like me ka mea i wehewehe mua ʻia21,22.ʻO ka pōkole, aia ka pahu hoʻokolohua ma 245 m mai ke apo mālama mālama synchrotron.Hoʻohana ʻia kahi laʻana-a-detector mamao o 0.6 m no nā haʻawina kiʻi ʻāpana a me 0.3 m no nā haʻawina kiʻi in vivo e hana i nā hopena like ʻole.Ua hoʻohana ʻia kahi kukuna monochromatic me ka ikehu o 25 keV.Ua loaʻa nā kiʻi me ka hoʻohana ʻana i kahi transducer X-ray kiʻekiʻe (SPring-8 BM3) i hui pū ʻia me kahi mea ʻike sCMOS.Hoʻololi ka transducer i nā kukui X i ke kukui ʻike ʻia me ka scintillator mānoanoa 10 µm (Gd3Al2Ga3O12), a laila kuhikuhi ʻia i ka sensor sCMOS me ka hoʻohana ʻana i kahi pahuhopu microscope ×10 (NA 0.3).ʻO ka mea ʻike sCMOS he Orca-Flash4.0 (Hamamatsu Photonics, Iapana) me ka nui o 2048 × 2048 pixels a me ka nui pika maka o 6.5 × 6.5 µm.Hāʻawi kēia hoʻonohonoho i ka nui isotropic pixel kūpono o 0.51 µm a me kahi kahua o ka nānā ʻana ma kahi o 1.1 mm × 1.1 mm.Ua koho ʻia ka lōʻihi o ka hoʻolaha ʻana o 100 ms no ka hoʻonui ʻana i ka ratio signal-to-noise o nā ʻāpana magnetic i loko a i waho o nā alaloa ʻoiai e hōʻemi ana i nā mea hana i hana ʻia e ka hanu ʻana.No nā haʻawina in vivo, ua hoʻokomo ʻia kahi pani X-ray wikiwiki i ke ala X-ray e kaupalena i ka nui o ka radiation ma ke kāohi ʻana i ke kukui X-ray ma waena o nā hōʻike.
ʻAʻole i hoʻohana ʻia ka media 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 ka laulā o ka maikaʻi-characterized MPs mai elua kalepa kalepa e uhi ana i ka laulā o ka nui, mea, hao hoʻopalekana, a me nā noi , - mua i mea e hoʻomaopopo i ka hana magnetic pā i ka neʻeʻana o nā MPs i ke aniani capillaries, a laila ma ea ola.ʻilikai.ʻOkoʻa ka nui o ka MP mai ka 0.25 a hiki i ka 18 µm a ua hana ʻia mai nā mea like ʻole (e ʻike i ka Papa 1), akā ʻaʻole ʻike ʻia ke ʻano o kēlā me kēia laʻana, me ka nui o nā ʻāpana magnetic i ka MP.Ma muli o kā mākou noiʻi nui MCT 19, 20, 21, 23, 24, manaʻo mākou e ʻike ʻia nā MP a hiki i ka 5 µm ma ka ʻaoʻao o ka ea tracheal, no ka laʻana, ma ka unuhi ʻana i nā papa kuhikuhi e ʻike i ka ʻike maikaʻi ʻana o ka neʻe ʻana o MP.ʻOi aku ka liʻiliʻi o kahi MP hoʻokahi o 0.25 µm ma mua o ka hoʻonā ʻana o ka hāmeʻa kiʻi kiʻi, akā manaʻo ʻia ʻo PB-PCXI e ʻike i kā lākou volumetric contrast a me ka neʻe ʻana o ka wai wai i waiho ʻia ma hope o ka waiho ʻia ʻana.
Nā laʻana no kēlā me kēia MP i ka pākaukau.Ua hoʻomākaukau ʻia ʻo 1 i nā capillaries aniani 20 μl (Drummond Microcaps, PA, USA) me ke anawaena o loko o 0.63 mm.Loaʻa nā ʻāpana kino i ka wai, aʻo nā ʻāpana CombiMag i loaʻa i ka wai waiwai o ka mea hana.Hoʻopiha ʻia kēlā me kēia paipu me ka wai (ma kahi o 11 µl) a kau ʻia ma luna o ka mea paʻa (e nānā i ke Kiʻi 1).Hoʻonoho ʻia nā capillaries aniani ma ke kahua ma ke keʻena kiʻi, kēlā me kēia, a hoʻonoho ʻia ma nā kihi o ka wai.He 19 mm anawaena (28 mm ka lōʻihi) nickel-shell magnet i hana ʻia me ka honua laha ʻole, neodymium, hao a boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Australia) me ke koena o 1.17 T i hoʻopili ʻia i kahi ʻO ka papa hoʻololi hoʻokaʻawale e hoʻokō E hoʻololi mamao i kou kūlana i ka wā hoʻoili.Hoʻomaka ke kiʻi kiʻi X-ray ke hoʻonoho ʻia ka magnet ma kahi o 30 mm ma luna o ka hāpana a loaʻa nā kiʻi ma 4 mau kiʻi i kekona.I ka wā o ke kiʻi kiʻi, ua hoʻokokoke ʻia ka magnet i ke aniani capillary tube (ma kahi mamao o 1 mm) a laila neʻe ʻia ma ka paipu e nānā i ka hopena o ka ikaika a me ke kūlana.
ʻO kahi hoʻonohonoho kiʻi in vitro i loaʻa nā laʻana MP i nā capillaries aniani i ka pae o ka unuhi ʻana o ka laʻana xy.Hōʻailona ʻia ke ala o ke kukui X-ray me kahi laina kiko ʻulaʻula.
I ka manawa i hoʻokumu ʻia ai ka ʻike in vitro o nā MP, ua hoʻāʻo ʻia kahi ʻāpana o lākou i vivo ma nā ʻiole Wistar albino wahine ʻano hihiu (~ 12 mau pule, ~ 200 g).Medetomidine 0.24 mg / kg (Domitor®, Zenoaq, Iapana), midazolam 3.2 mg / kg (Dormicum®, Astellas Pharma, Iapana) a me butorphanol 4 mg / kg (Vetorphale®, Meiji 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 ana i kahi paipu endotracheal (ET; 16 Ga intravenous cannula, Terumo BCT), a hoʻopaʻa ʻole iā lākou ma kahi kūlana ma luna o kahi pā kiʻi i hana ʻia me kahi ʻeke wela. e mālama i ka mahana o ke kino.22. A laila ua hoʻopili ʻia ka pā kiʻi i ka pae hoʻohālike i loko o ka pahu kiʻi ma kahi kihi liʻiliʻi e hoʻohālikelike i ka trachea ma ke kiʻi x-ray e like me ka hōʻike ʻana ma ke Kiʻi 2a.
(a) In vivo imaging setup in the SPring-8 imaging unit, X-ray beam alahele i kaha ʻia me ka laina kiko ʻulaʻula.(b,c) Ua hana mamao ʻia ka localization magnet tracheal me ka hoʻohana ʻana i ʻelua mau kiʻi kiʻi IP orthogonally.Ma ka ʻaoʻao hema o ke kiʻi ma ka pale, hiki iā ʻoe ke ʻike i ka loop loop e paʻa ana i ke poʻo a me ka cannula lawe i hoʻokomo ʻia i loko o ka paipu ET.
Ua hoʻopili ʻia kahi ʻōnaehana pamu syringe mamao (UMP2, World Precision Instruments, Sarasota, FL) me ka 100 µl aniani syringe i kahi paipu PE10 (0.61 mm OD, 0.28 mm ID) me ka hoʻohana ʻana i kahi nila 30 Ga.E kaha i ka paipu e hōʻoia i ke kūlana kūpono o ka piko i loko o ka trachea i ka wā e hoʻokomo ai i ka paipu endotracheal.Ma ka hoʻohana ʻana i kahi micropump, ua wehe ʻia ka plunger syringe a ua hoʻokomo ʻia ka piko o ka paipu i loko o ka hāpana MP e hāʻawi ʻia.A laila hoʻokomo ʻia ka paipu hāʻawi i hoʻouka ʻia i loko o ka paipu endotracheal, e kau ana i ka piko ma ka ʻaoʻao ikaika loa o kā mākou māla magnetic i manaʻo ʻia.Ua mālama ʻia ka loaʻa ʻana o nā kiʻi me ka hoʻohana ʻana i ka mea ʻike hanu i hoʻopili ʻia i kā mākou pahu manawa i hoʻokumu ʻia ʻo Arduino, a ua hoʻopaʻa ʻia nā hōʻailona āpau (e laʻa, ka wela, ka hanu ʻana, ka wehe ʻana/pani ʻana, a me ka loaʻa kiʻi) ua hoʻopaʻa ʻia me Powerlab a me LabChart (AD Instruments, Sydney, Australia) 22 Ke Kiʻi I ka wā i loaʻa ʻole ai ka hale, ua hoʻonoho ʻia ʻelua mau pahu kiʻi IP (Panasonic BB-SC382) ma kahi o 90 ° kekahi i kekahi a hoʻohana ʻia e hoʻomalu i ke kūlana o ka magnet e pili ana i ka trachea i ka wā o ke kiʻi (Figure 2b, c).No ka hōʻemi ʻana i nā kiʻi ʻoniʻoni, ua loaʻa hoʻokahi kiʻi i kēlā me kēia hanu i ka wā o ka pae hoʻokele hanu.
Hoʻopili ʻia ka magnet i ka pae ʻelua, aia paha ma kahi mamao ma waho o ke kino kiʻi.Ua hoʻāʻo ʻia nā kūlana like ʻole a me nā hoʻonohonoho o ka magnet, e like me: kau ʻia ma kahi kihi o 30 ° ma luna o ka trachea (hōʻike ʻia nā hoʻonohonoho ma nā Kiʻi 2a a me 3a);hoʻokahi magnet ma luna o ka holoholona a ʻo kekahi ma lalo, me nā pou i hoʻonohonoho ʻia no ka huki ʻana (Figure 3b)., hoʻokahi magnet ma luna o ka holoholona a me kekahi ma lalo, me nā pou i hoʻonohonoho ʻia no ka hoʻokuke ʻana (Figure 3c), a hoʻokahi magnet ma luna a kū pololei i ka trachea (Figure 3d).Ma hope o ka hoʻonohonoho ʻana i ka holoholona a me ka magnet a me ka hoʻouka ʻana i ka MP ma lalo o ka hoʻāʻo ʻana i loko o ka pamu syringe, e hāʻawi i kahi ʻano o 50 µl ma ka wikiwiki o 4 µl/sec ma ka loaʻa ʻana o nā kiʻi.A laila hoʻoneʻe ʻia ka magnet i hope a i ʻole ma ka trachea me ka hoʻomau ʻana i ka loaʻa ʻana o nā kiʻi.
ʻO ka hoʻonohonoho hoʻonohonoho ʻana no ka kiʻi vivo (a) hoʻokahi magnet ma luna o ka trachea ma kahi kihi o 30°, (b) ʻelua mau magnet i hoʻonohonoho ʻia no ka huki ʻana, (c) ʻelua mau magnet i hoʻonohonoho ʻia no ka repulsion, (d) hoʻokahi magnet ma luna a kū pololei i ka ʻūhā.Ua nānā ka mea nānā mai ka waha a i ka māmā ma o ka trachea a ua hele ka kukui X-ray ma ka ʻaoʻao hema o ka ʻiole a puka i ka ʻaoʻao ʻākau.Hoʻoneʻe ʻia ka magnet ma ka lōʻihi o ke ala ea a i ʻole hema a ʻākau paha ma luna o ka trachea ma ke kuhikuhi o ke kukui X-ray.
Ua ʻimi pū mākou e hoʻoholo i ka ʻike a me ke ʻano o nā ʻāpana i nā alaloa me ka ʻole o ka hui ʻana o ka hanu a me ka puʻuwai puʻuwai.No laila, i ka pau ʻana o ka manawa kiʻi, ua hoʻopau ʻia nā holoholona ma muli o ka overdose pentobarbital (Somnopentyl, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg ip).Ua waiho ʻia kekahi mau holoholona ma luna o ke kahua kiʻi, a ma hope o ka pau ʻana o ka hanu a me ka puʻuwai, ua hana hou ʻia ke kaʻina hana kiʻi, me ka hoʻohui ʻana i kahi maʻi MP inā ʻaʻohe MP i ʻike ʻia ma ka ʻili o ke ea.
Ua hoʻoponopono ʻia nā kiʻi i loaʻa no ke kahua pālahalaha a me ka ʻeleʻele a laila hui ʻia i kahi kiʻiʻoniʻoni (20 mau kiʻi i kēlā me kēia kekona; 15–25 × ka wikiwiki maʻamau e pili ana i ka nui o ka hanu) me ka hoʻohana ʻana i kahi palapala maʻamau i kākau ʻia ma MATLAB (R2020a, The Mathworks).
Ua alakaʻi ʻia nā haʻawina āpau e pili ana i ka lawe ʻana o LV gene vector ma ke Kulanui o Adelaide Laboratory Animal Research Center a ua manaʻo e hoʻohana i nā hopena o ka hoʻokolohua SPring-8 e loiloi inā hiki i ka lawe ʻana o LV-MP ma ke alo o kahi māla magnetic ke hoʻonui i ka hoʻololi ʻana i loko o vivo .No ka loiloi i nā hopena o ka MF a me ke kahua magnetic, ua mālama ʻia nā pūʻulu holoholona ʻelua: hoʻokahi hui i hoʻopaʻa ʻia me LV MF me ka hoʻokomo magnet, a ʻo kekahi hui i hoʻopaʻa ʻia me kahi pūʻulu mana me LV MF me ka ʻole o ka magnet.
Ua hoʻokumu ʻia nā vector gene LV me ka hoʻohana ʻana i nā ala i wehewehe mua ʻia 25, 26.Hōʻike ka lacZ vector i kahi ʻano beta-galactosidase nuklea localized i alakaʻi ʻia e ka MPSV constitutive promoter (LV-LacZ), e hoʻopuka ana i kahi huahana pane ʻulaʻula i loko o nā cell transduced, ʻike ʻia ma nā alo a me nā ʻāpana o ka ʻiʻo māmā.Hana ʻia ka titration ma nā moʻomeheu cell ma ka helu lima lima i ka helu o nā cell LacZ-positive me ka hemocytometer e helu i ka titer ma TU/ml.Mālama ʻia nā mea lawe ma -80°C, hoʻoheheʻe ʻia ma mua o ka hoʻohana ʻana, a hoʻopaʻa ʻia iā CombiMag ma ka hui ʻana i ka 1:1 a me ka incubating ma ka hau no ka liʻiliʻi he 30 mau minuke ma mua o ka lawe ʻana.
Nāʻiole Sprague Dawley maʻamau (n = 3 / hui, ~ 2-3 ip anesthetized me kahi hui o 0.4mg / kg medetomidine (Domitor, Ilium, Australia) a me 60mg / kg ketamine (Ilium, Australia) ma 1 mahina o ka makahiki) ip ) ka hoʻokolo ʻana a me ka hoʻoheheʻe waha ʻole me ka cannula intravenous 16 Ga.No ka hōʻoia i ka loaʻa ʻana o ka LV transduction, ua hoʻokaʻawale ʻia ia me ka hoʻohana ʻana i kā mākou protocol perturbation mechanical i wehewehe mua ʻia kahi i hoʻopili ʻia ai ka ʻili o ke ea tracheal me kahi hīnaʻi uea (N-Circle, nitinol stone extractor me ka ʻole NTSE-022115) -UDH, Cook Medical, USA) 30 p28.A laila, ma kahi o 10 mau minuke ma hope o ka hoʻopilikia ʻana i ka keʻena biosafety, ua hana ʻia ka hoʻokele tracheal o LV-MP.
Ua hoʻonohonoho like ʻia ke kahua mākēneki i hoʻohana ʻia i kēia hoʻokolohua me kahi noiʻi x-ray in vivo, me nā mea magnet like i hoʻopaʻa ʻia ma luna o ka trachea me nā stent clamps distillation (Figure 4).Hāʻawi ʻia kahi 50 µl volume (2 x 25 µl aliquots) o LV-MP i ka trachea (n = 3 holoholona) me ka hoʻohana ʻana i ka pipette gel-tipped e like me ka mea i wehewehe mua ʻia.Ua loaʻa i ka pūʻulu mana (n = 3 holoholona) ka LV-MP like me ka hoʻohana ʻole ʻana i kahi magnet.Ma hope o ka pau ʻana o ka infusion, wehe ʻia ka cannula mai ka paipu endotracheal a hoʻopau ʻia ka holoholona.Noho ka magnet ma kahi o 10 mau minuke ma mua o ka wehe ʻana.Hoʻopili ʻia nā ʻiole me ka meloxicam (1 ml / kg) (Ilium, Australia) ma hope o ka hemo ʻana o ka anesthesia e ka intraperitoneal injection o 1 mg / kg atipamazole hydrochloride (Antisedan, Zoetis, Australia).Mālama ʻia nā ʻiole me ka mahana a nānā ʻia a hiki i ka hoʻōla piha ʻana mai ka anesthesia.
LV-MP mea lawe i loko o kahi keʻena palekana olaola.Hiki iā ʻoe ke ʻike i ka puka ʻana o ka lima Luer-lock hina hina o ka paipu ET mai ka waha, a ua hoʻokomo ʻia ka piko gel 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 hoʻokele LV-MP, ua kaumaha ʻia nā holoholona me ka inhalation o 100% CO2 a ua loiloi ʻia ka ʻōlelo LacZ me ka hoʻohana ʻana i kā mākou hana X-gal maʻamau.Ua wehe ʻia nā apo cartilage ʻekolu o ka caudal no ka hōʻoia ʻana ʻaʻole e hoʻokomo ʻia kekahi pōʻino mechanical a i ʻole ka paʻa ʻana o ka wai ma muli o ka hoʻokomo ʻana o ka paipu endotracheal i ka loiloi.Ua ʻoki ʻia kēlā me kēia trachea i ka lōʻihi no ka loaʻa ʻana o ʻelua ʻāpana no ka nānā ʻana a waiho ʻia i loko o ke kīʻaha i loko o ke kīʻaha silicone (Sylgard, Dow Inc) me ka hoʻohana ʻana i kahi nila Minutien (Fine Science Tools) e ʻike i ka ʻili o ka luminal.Ua hōʻoia ʻia ka hoʻolaha ʻana a me ke ʻano o nā cell transduced e ke kiʻi mua me ka microscope Nikon (SMZ1500) me kahi kamera DigiLite a me ka polokalamu TCapture (Tucsen Photonics, Kina).Ua kiʻi ʻia nā kiʻi ma 20x hoʻonui (me ka hoʻonohonoho kiʻekiʻe no ka laulā piha o ka trachea), me ka lōʻihi holoʻokoʻa o ka trachea i hōʻike ʻia i kēlā me kēia pae, e hāʻawi ana i ka nui o ka overlap ma waena o kēlā me kēia kiʻi e hiki ai i nā kiʻi ke "huʻi".Hoʻohui ʻia nā kiʻi mai kēlā me kēia trachea i hoʻokahi kiʻi hui pū me ka hoʻohana ʻana i ka Composite Image Editor version 2.0.3 (Microsoft Research) me ka hoʻohana ʻana i ka algorithm planar motion. Ua helu ʻia ka ʻāpana o ka ʻōlelo LacZ i loko o nā kiʻi hui puʻupuʻu mai kēlā me kēia holoholona me ka palapala MATLAB automated (R2020a, MathWorks) e like me ka mea i wehewehe mua ʻia28, me ka hoʻohana ʻana i nā hoʻonohonoho o 0.35 <Hue <0.58, Saturation> 0.15, a me ka waiwai <0.7. Ua helu ʻia ka ʻāpana o ka hōʻike LacZ i loko o nā kiʻi hui tracheal mai kēlā me kēia holoholona me ka palapala MATLAB automated (R2020a, MathWorks) e like me ka mea i wehewehe mua ʻia28, me ka hoʻohana ʻana i nā hoʻonohonoho o 0.35 <Hue <0.58, Saturation> 0.15, a me ka waiwai <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. Ua helu ʻia ka ʻāpana o ka ʻōlelo LacZ i nā kiʻi tracheal composite mai kēlā me kēia holoholona me ka hoʻohana ʻana i kahi palapala MATLAB automated (R2020a, MathWorks) e like me ka mea i wehewehe mua ʻia28 me ka hoʻohana ʻana i nā hoʻonohonoho o 0.350.15 a me ka waiwai <0 .7.如前所述，使用自动MATLAB 脚本（R2020a，MathWorks）对来自每只动物的气管复合图像中的LacZ 表达区域进行量化，使用0.35 < 色调< 0.58、饱和度> 0.15 和值< 0.7 的设置。如 前所前所前所 自动自动 malolab 脚本 ((R2020, MA mat 的 气管 I像 IMA像 IMA像 的 I.3 0 5 <0.7 0.5 0.8 0.5 0.8 0.5 0. .. . . . . . . . .. HIP 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 . Ua helu ʻia nā ʻāpana o ka ʻōlelo LacZ ma nā kiʻi hui pū o ka trachea o kēlā me kēia holoholona me ka palapala MATLAB automated (R2020a, MathWorks) e like me ka mea i wehewehe mua ʻia me ka hoʻohana ʻana i nā hoʻonohonoho o 0.35 <hue <0.58, saturation> 0.15 a me ka waiwai <0.7.Ma ka hahai ʻana i nā kiko kiko ma GIMP v2.10.24, ua hana lima ʻia kahi mask no kēlā me kēia kiʻi composite e ʻike i ka ʻāpana kiko a pale i nā ʻike hoʻopunipuni ma waho o ka ʻiʻo tracheal.Ua hōʻuluʻulu ʻia nā wahi ʻeleʻele mai nā kiʻi i hui ʻia mai kēlā me kēia holoholona e hāʻawi i ka nui o ka ʻāpana ʻeleʻele no kēlā holoholona.Ua mahele ʻia ka wahi i pena ʻia e ka huina o ka mask e loaʻa ai kahi wahi maʻamau.
Hoʻokomo ʻia kēlā me kēia trachea i loko o ka paraffin a hoʻokaʻawale ʻia he 5 µm mānoanoa.Ua hoʻopaʻa ʻia nā ʻāpana me ka ʻulaʻula wikiwiki kūʻokoʻa no 5 mau minuke 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 hana ʻia nā ʻikepili helu āpau ma GraphPad Prism v9 (GraphPad Software, Inc.).Ua hoʻonoho ʻia ke koʻikoʻi helu ma ka p ≤ 0.05.Ua hoʻāʻo ʻia ke ʻano maʻamau me ka hoʻohana ʻana i ka hoʻāʻo ʻo Shapiro-Wilk a ua loiloi ʻia nā ʻokoʻa o ka staining LacZ me ka hoʻohana ʻana i kahi hoʻāʻo t-paired.
ʻO nā MP ʻeono i hōʻike ʻia ma ka Papa 1 ua nānā ʻia e PCXI, a ua wehewehe ʻia ka ʻike ma ka Papa 2. ʻElua polystyrene MP (MP1 a me MP2; 18 µm a me 0.25 µm, kēlā me kēia) ʻaʻole i ʻike ʻia e PCXI, akā hiki ke ʻike ʻia nā mea i koe. (hōʻike ʻia nā laʻana ma ke Kiʻi 5).ʻIke nāwaliwali ʻia ka MP3 a me MP4 (10-15% Fe3O4; 0.25 µm a me 0.9 µm, kēlā me kēia).ʻOiai ʻo MP5 (98% Fe3O4; 0.25 µm) i loaʻa kekahi o nā mea liʻiliʻi loa i hoʻāʻo ʻia, ʻo ia ka mea i ʻōlelo ʻia.ʻO ka huahana CombiMag MP6 paʻakikī ke ʻike.I nā hihia a pau, ua hoʻomaikaʻi maikaʻi ʻia ko mākou hiki ke ʻike i nā MF ma ka neʻe ʻana i ka magnet i hope a i waho e like me ka capillary.I ka neʻe ʻana aku o nā mākēneki mai ka capillary, ua huki ʻia nā ʻāpana ma nā kaulahao lōʻihi, akā i ka hoʻokokoke ʻana mai o nā mea magnet a me ka hoʻonui ʻana i ka ikaika o ke kahua magnetic, ua pōkole nā ​​kaulahao i ka neʻe ʻana o nā ʻāpana i ka ʻili o luna o ka capillary (e nānā i ke wikiō Hoʻohui S1. : MP4), e hoʻonui ana i ka mānoanoa o ka ʻili.ʻO ka mea ʻē aʻe, ke hoʻoneʻe ʻia ka magnet mai ka capillary, e emi ana ka ikaika o ke kahua a hoʻonohonoho hou nā MP i nā kaulahao lōʻihi e hoʻonui ana mai ka ʻili o luna o ka capillary (e nānā i ke wikiō Hoʻohui S2: MP4).Ma hope o ka pau ʻana o ka neʻe ʻana o ka magnet, hoʻomau ka neʻe ʻana o nā ʻāpana no kekahi manawa 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 nā ʻāpana magnetic i ka ʻōpala ma o ka wai.
ʻOkoʻa ka ʻike ʻana o MP ma lalo o PCXI ma waena o nā laʻana.(a) MP3, (b) MP4, (c) MP5 a me (d) MP6.Ua lawe ʻia nā kiʻi āpau i hōʻike ʻia ma ʻaneʻi me kahi magnet i hoʻonoho ʻia ma kahi o 10 mm ma luna pono o ka capillary.ʻO nā pōʻai nui i ʻike ʻia he mau ea i hoʻopaʻa ʻia i loko o nā capillaries, e hōʻike pono ana i nā hiʻohiʻona ʻeleʻele a keʻokeʻo o ke kiʻi ʻokoʻa.Hōʻike ka pahu ʻulaʻula i ka hoʻonui e hoʻonui ai i ka ʻokoʻa.E hoʻomanaʻo, ʻaʻole like ke anawaena o nā kaapuni magnet i nā kiʻi āpau a ʻoi aku ka nui o 100 mau manawa ma mua o ka mea i hōʻike ʻia.
I ka neʻe ʻana o ka magnet i ka hema a me ka ʻākau ma ka piko o ka capillary, e hoʻololi ke kihi o ke kaula MP e kūlike me ka magnet (e nānā i ke Kiʻi 6), pēlā e wehewehe ai i nā laina kahua magnetic.No ka MP3-5, ma hope o ka hōʻea ʻana o ka chord i ka huina paepae, e kauo nā ʻāpana ma ka ʻili o luna o ka capillary.ʻO kēia ka hopena pinepine i ka hui ʻana o nā MP i loko o nā pūʻulu nui ma kahi kokoke i kahi e ikaika loa ai ke kahua magnetic (e ʻike i ke wikiō Hoʻohui S3: MP5).ʻIke ʻia kēia i ka wā e kiʻi kokoke ana i ka hope o ka capillary, ka mea e hōʻuluʻulu ai ka MP a hoʻopaʻa ʻia i ke kikowaena wai-ea.ʻO nā ʻāpana o ka MP6, i ʻoi aku ka paʻakikī o ka ʻike ʻana ma mua o ka MP3-5, ʻaʻole i kauo i ka wā i neʻe ai ka magnet ma ka capillary, akā ua hoʻokaʻawale ʻia nā kaula MP, a waiho i nā ʻāpana i ka nānā ʻana (e nānā i ke wikiō Hoʻohui S4: MP6).I kekahi mau hihia, i ka wā i hoʻemi ʻia ai ka mākēneki i hoʻohana ʻia ma ka hoʻoneʻe ʻana i ka magnet ma kahi lōʻihi mai ke kahua kiʻi, ua iho mālie nā MP i koe i ka ʻili o lalo o ka paipu e ka umekaumaha, e koe ana i ke kaula (e nānā i ke wikiō Hoʻohui S5: MP3) .
Hoʻololi ke kihi o ke kaula MP i ka neʻe ʻana o ka magnet i ka ʻākau ma luna o ka capillary.(a) MP3, (b) MP4, (c) MP5 a me (d) MP6.Hōʻike ka pahu ʻulaʻula i ka hoʻonui e hoʻonui ai i ka ʻokoʻa.E ʻoluʻolu e hoʻomaopopo ʻia nā wikiō hou aʻe no nā kumu ʻike no ka mea e hōʻike ana lākou i ke ʻano mea nui a me ka ʻike ikaika i hiki ʻole ke ʻike ʻia i kēia mau kiʻi paʻa.
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 ka MF ma ke ʻano o ka neʻe paʻakikī i loko o vivo.ʻAʻole i hana ʻia nā hoʻokolohua in vivo no ka mea ʻaʻole ʻike ʻia nā peʻa polystyrene (MP1 a me MP2) i loko o ka capillary.Ua hoʻāʻo ʻia kēlā me kēia o nā MF ʻehā i koe i loko o ka vivo me ke koʻi lōʻihi o ka magnet i hoʻonoho ʻia ma luna o ka trachea ma kahi kihi o kahi 30° a i ke kūpaʻa (e nānā i nā Kiʻi 2b a me 3a), no ka mea, ua loaʻa i nā kaulahao MF lōʻihi a ʻoi aku ka maikaʻi. ma mua o ka magnet..hoʻopau ʻia ka hoʻonohonoho.ʻAʻole i loaʻa ka MP3, MP4 a me MP6 i ka trachea o nā holoholona ola.I ka nānā ʻana i ka ʻōpū hanu o nā ʻiole ma hope o ka pepehi kanaka ʻana i nā holoholona, ​​ʻaʻole ʻike ʻia nā ʻāpana ʻoiai ke hoʻohui ʻia ka leo hou me ka hoʻohana ʻana i ka pamu syringe.Loaʻa i ka MP5 ka mea kiʻekiʻe loa o ka hao oxide a ʻo ia wale nō ka mea i ʻike ʻia, no laila ua hoʻohana ʻia e loiloi a hōʻike i ka ʻano MP i vivo.
ʻO ka hoʻokomo ʻana o ka magnet ma luna o ka trachea i ka wā o ka hoʻokomo ʻana i ka MF i hopena i ka nui, akā ʻaʻole nā ​​​​mea a pau, ʻo nā MF i hoʻopaʻa ʻia i ke kahua o ka ʻike.ʻIke maikaʻi ʻia ke komo ʻana o nā ʻāpana i loko o nā holoholona euthanized kanaka.Kiʻi 7 a me ka wikiō hou S6: Hōʻike ka MP5 i ka hopu wikiwiki a me ka hoʻolikelike ʻana o nā ʻāpana ma ka ʻili o ka trachea ventral, e hōʻike ana e hiki ke kuhikuhi ʻia nā MP i nā wahi i makemake ʻia o ka trachea.I ka huli ʻana ma kahi mamao loa ma ka trachea ma hope o ka hoʻopuka ʻana o MF, ua ʻike ʻia kekahi mau MF ma kahi kokoke i ka carina, e hōʻike ana i ka lawa ʻole o ka ikaika o ke kahua magnetic e hōʻiliʻili a paʻa i nā MF āpau, no ka mea, ua hāʻawi ʻia lākou ma ka ʻāpana o ka ikaika mākēneki kiʻekiʻe i ka wā o ka hoʻokele wai.kaʻina hana.Eia naʻe, ʻoi aku ka kiʻekiʻe o nā manaʻo MP ma hope o ka hānau ʻana a puni ka wahi kiʻi, e hōʻike ana he nui nā MP i noho ma nā wahi ala ea kahi i kiʻekiʻe ai ka ikaika o ke kahua magnetic.
Nā kiʻi o (a) ma mua a me (b) ma hope o ka hoʻopuka ʻana o MP5 i loko o ka trachea o kahi ʻiole i hoʻopau hou ʻia me kahi magnet i kau ʻia ma luna pono o ka wahi kiʻi.Aia ka wahi i hōʻike ʻia ma waena o nā apo cartilaginous ʻelua.Aia kekahi wai ma nā ala ea ma mua o ka hāʻawi ʻia ʻana o ka MP.Hōʻike ka pahu ʻulaʻula i ka hoʻonui e hoʻonui ai i ka ʻokoʻa.Lawe ʻia kēia mau kiʻi mai ke wikiō i hōʻike ʻia ma S6: MP5 Pākuʻi Video.
ʻO ka hoʻoneʻe ʻana i ka magnet ma ka trachea in vivo i hoʻololi ʻia i ke kihi o ke kaulahao MP ma ka ʻili o ke ea, e like me ka mea i ʻike ʻia ma nā capillaries (e nānā i ke Kiʻi 8 a me ke wikiō Hoʻohui S7: MP5).Eia nō naʻe, i kā mākou noiʻi ʻana, ʻaʻole hiki ke kauo ʻia nā MP ma ka ʻili o nā ʻōpū hanu ola, e like me ka hana ʻana o nā capillaries.I kekahi mau hihia, hoʻolōʻihi ʻia ke kaulahao MP i ka neʻe ʻana o ka magnet i ka hema a me ka ʻākau.ʻO ka mea e mahalo ai, ua ʻike pū mākou ua hoʻololi ke kaulahao particle i ka hohonu o ka ʻili o ka wai i ka wā e hoʻoneʻe ʻia ai ka magnet i ka lōʻihi ma ka trachea, a hoʻonui i ka wā e hoʻoneʻe pololei ʻia ai ka magnet ma luna o ke poʻo a ua hoʻohuli ʻia ke kaulahao particle i kahi kūlana kūpaʻa. Pākuʻi wikiō S7).: MP5 ma 0:09, lalo ʻākau).Ua hoʻololi ʻia ke ʻano o ka neʻe ʻana i ka wā i hoʻoneʻe ʻia ka magnet ma ka ʻaoʻao o ka piko o ka trachea (ʻo ia hoʻi, ma ka hema a ʻākau paha o ka holoholona, ​​​​ma mua o ka lōʻihi o ka trachea).ʻIke maopopo ʻia nā ʻāpana i ka wā o kā lākou neʻe ʻana, akā i ka wā i wehe ʻia ai ka magnet mai ka trachea, ua ʻike ʻia nā kihi o nā kaula ʻāpana (e nānā i ke wikiō Hoʻohui S8: MP5, e hoʻomaka ana ma 0:08).Ua ʻae kēia me ke ʻano i ʻike ʻia o ke kahua magnetic ma lalo o ka hana a kahi māla i hoʻopili ʻia i loko o kahi capillary aniani.
Nā kiʻi laʻana e hōʻike ana i ka MP5 i ka trachea o kahi ʻiole anesthetic ola.(a) Hoʻohana ʻia ka magnet e kiʻi i nā kiʻi ma luna a ma ka hema o ka trachea, a laila (b) ma hope o ka neʻe ʻana i ka magnet i ka ʻākau.Hōʻike ka pahu ʻulaʻula i ka hoʻonui e hoʻonui ai i ka ʻokoʻa.ʻO kēia mau kiʻi mai ke wikiō i hōʻike ʻia ma S7's Supplementary Video: MP5.
I ka hoʻokani ʻia ʻana o nā pou ʻelua ma ka ʻākau-hema ma luna a ma lalo o ka trachea (ʻo ia hoʻi, huki; Fig. 3b), ua ʻoi aku ka lōʻihi o nā mele MP a aia ma ka paia ʻaoʻao o ka trachea ma mua o ka ʻili hope o ka pahu. trachea (e nānā i ka Appendix).Video S9:MP5).Eia nō naʻe, ʻaʻole i ʻike ʻia ka nui o nā ʻāpana kiʻekiʻe ma kahi kahua hoʻokahi (ʻo ia hoʻi, ka ʻili hope o ka trachea) ma hope o ka hoʻohana ʻana i ka wai me ka hoʻohana ʻana i kahi mea mana magnet ʻelua, ka mea maʻamau me kahi mea magnet hoʻokahi.A laila, i ka wā i hoʻonohonoho ʻia ai kahi magnet e hoʻihoʻi i nā pou kūʻē (Figure 3c), ʻaʻole i hoʻonui ʻia ka helu o nā ʻāpana i ʻike ʻia ma ke kahua ʻike ma hope o ka lawe ʻana.He mea paʻakikī ka hoʻonohonoho ʻana i nā hoʻonohonoho magnet ʻelua ma muli o ke kiʻekiʻe kiʻekiʻe o ke kahua magnetic e huki a hoʻoneʻe paha i nā magnet.A laila ua hoʻololi ʻia ka hoʻonohonoho ʻana i hoʻokahi magnet e like me nā ala ea akā e hele ana ma nā ala ea ma kahi kihi 90 degere i hiki ai i nā laina o ka ikaika ke ʻaʻa i ka paia tracheal orthogonally (Figure 3d), kahi kuhikuhi i manaʻo ʻia e hoʻoholo i ka hiki ke hoʻohui ʻia ma luna. ka paia aoao.e malamaia.Eia nō naʻe, i kēia hoʻonohonoho, ʻaʻohe ʻike ʻia ka neʻe ʻana o ka MF accumulation a i ʻole ka neʻe magnet.Ma muli o kēia mau hopena a pau, ua koho ʻia kahi hoʻonohonoho me kahi magnet hoʻokahi a me kahi orientation 30-degree no nā haʻawina in vivo o nā mea lawe gene (Fig. 3a).
Ke kiʻi ʻia ka holoholona i nā manawa he nui ma hope koke o ka mōhai ʻia ʻana o ke kanaka, ʻo ka nele o ka neʻe ʻana o ke kino, ʻo ia ka mea hiki ke ʻike ʻia nā laina liʻiliʻi liʻiliʻi ma ke kahua intercartilaginous akaka, 'swaying' e like me ka neʻe unuhi o ka magnet.ʻike maopopo i ke alo a me ka neʻe ʻana o nā ʻāpana MP6.
ʻO ka titer o LV-LacZ he 1.8 x 108 IU/mL, a ma hope o ka hui ʻana i ka 1: 1 me CombiMag MP (MP6), ua hoʻopaʻa ʻia nā holoholona me 50 µl o kahi ʻano tracheal o 9 x 107 IU/ml o ka kaʻa LV (ie 4.5 x 106 TU/iole).)).Ma kēia mau haʻawina, ma kahi o ka hoʻoneʻe ʻana i ka magnet i ka wā o ka 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 me ka loaʻa ʻole o kahi māla magnetic, a (b) inā hiki i ke ea. e kālele ana.Ke lawe ʻia nei nā pūnaewele ma nā wahi i manaʻo ʻia o ka ʻōpū hanu luna.
ʻO ka hele ʻana o nā magnets a me ka hoʻohana ʻana o CombiMag i hui pū ʻia me nā vectors LV ʻaʻole i ʻike ʻia e hoʻopilikia i ke olakino holoholona, ​​​​e like me kā mākou LV vector delivery protocol.ʻO nā kiʻi mua o ka'āpana tracheal i hoʻopiliʻia i ka mechanical perturbation (Supplementary Fig. 1) hōʻike i ka hui LV-MP i mālamaʻia he kiʻekiʻe loa o ka transduction i mua o kahi magnet (Fig. 9a).ʻO kahi liʻiliʻi liʻiliʻi o ka polū LacZ i loaʻa i ka pūʻulu mana (Figure 9b).ʻO ka helu ʻana o nā ʻāpana maʻamau i hoʻopaʻa ʻia e X-Gal i hōʻike ʻia i ka hoʻokele ʻana o LV-MP ma ke alo o kahi māla magnetic i hoʻonui ʻia ma kahi o 6-fold (Fig. 9c).
He laʻana o nā kiʻi i hui pū ʻia e hōʻike ana i ka hoʻololi ʻana o ka tracheal me LV-MP (a) i ke alo o kahi mākēneki a (b) i ka loaʻa ʻole o kahi magnet.(c) Ka hoʻomaikaʻi nui ʻana o ka helu helu ma ka wahi maʻamau o LacZ transduction i loko o ka trachea me ka hoʻohana ʻana i kahi magnet (*p = 0.029, t-test, n = 3 no kēlā me kēia hui, mean ± hewa maʻamau o ka mean).
ʻO nā ʻāpana ʻulaʻula wikiwiki kūʻokoʻa (laʻana i hōʻike ʻia ma ka Fig.
ʻO ka luʻi nui i ka ea gene therapy e noho mau i ka localization pololei o nā mea lawe i nā wahi hoihoi a me ka loaʻa ʻana o kahi kiʻekiʻe o ka transduction pono i loko o ka māmā kelepona i mua o ka lele ʻana o ka ea a me ka hoʻomaʻemaʻe ʻana i ka mucus.No nā mea lawe LV i manaʻo ʻia no ka mālama ʻana i nā maʻi hanu i ka cystic fibrosis, ʻo ka hoʻonui ʻana i ka manawa noho o nā mea lawe i nā ala ea conductive a hiki i kēia manawa he pahuhopu hiki ʻole ke loaʻa.E like me ka mea i kuhikuhi ʻia e Castellani et al., ʻo ka hoʻohana ʻana i nā māla magnetic e hoʻomaikaʻi i ka transduction ʻoi aku ka maikaʻi ma mua o nā ʻano hana hāʻawi ʻē aʻe e like me ka electroporation no ka mea hiki ke hoʻohui i ka maʻalahi, ka hoʻokele waiwai, ka hoʻopuka kūloko, hoʻonui i ka pono, a me ka manawa incubation pōkole.a he haʻahaʻa haʻahaʻa paha o ke kaʻa10.Eia naʻe, ʻaʻole i hōʻike ʻia i loko o ka vivo deposition a me ke ʻano o nā ʻāpana magnetic i nā ala ea ma lalo o ka mana o nā mana magnetic o waho, a ʻo ka mea hiki i kēia ʻano hana ke hoʻonui i nā pae hōʻike gene i nā ala ea ola ʻaʻole i hōʻike ʻia ma vivo.
ʻO kā mākou mau hoʻokolohua in vitro ma ka PCXI synchrotron ua hōʻike ʻia nā ʻāpana āpau a mākou i hoʻāʻo ai, me ka ʻole o ka MP polystyrene, ʻike ʻia i ka hoʻonohonoho kiʻi i hoʻohana ai mākou.Ma ke alo o kahi mākia, hana nā mākena magnetic i nā kaula, ʻo ka lōʻihi o ia mea e pili ana i ke ʻano o nā mea ʻāpana a me ka ikaika o ka māla magnetic (ʻo ia hoʻi, ke kokoke a me ka neʻe ʻana o ka magnet).E like me ka mea i hōʻike ʻia ma ke Kiʻi 10, ua hoʻokumu ʻia nā kaula a mākou e ʻike nei i ka lilo ʻana o kēlā me kēia ʻāpana i mea magnetized a hoʻoulu i kona māla magnetic kūloko.ʻO kēia mau kahua kaʻawale ke kumu o nā ʻāpana like ʻole e hōʻiliʻili a hoʻohui pū me nā neʻe kaula hui ma muli o nā ikaika kūloko mai nā ikaika kūloko a me ka hoʻohuli ʻana i nā mea ʻē aʻe.
Kiʻikuhi e hōʻike ana (a, b) nā kaulahao o nā ʻāpana i hana ʻia i loko o nā capillaries i piha i ka wai a me (c, d) kahi trachea piha i ka ea.E hoʻomaopopo ʻaʻole huki ʻia nā capillaries a me ka trachea i ka unahi.Aia i loko o ka Panel (a) ka wehewehe ʻana o ka MF i loaʻa nā ʻāpana Fe3O4 i hoʻonohonoho ʻia i nā kaulahao.
I ka neʻe ʻana o ka magnet ma luna o ka capillary, ua hōʻea ke kihi o ka string particle i ka paepae koʻikoʻi no MP3-5 i loaʻa iā Fe3O4, a laila ʻaʻole i noho hou ke kaula ʻāpana i kona kūlana mua, akā neʻe ma ka ʻili i kahi kūlana hou.magnet.Loaʻa paha kēia hopena no ka maʻalahi o ka ʻili o ka capillary aniani e ʻae i kēia neʻe ʻana.ʻO ka mea e mahalo ai, ʻaʻole i hana like ʻo MP6 (CombiMag) ma muli paha o ka liʻiliʻi o nā ʻāpana, he ʻokoʻa ka uhi ʻana a i ʻole ka hoʻouka ʻana i luna, a i ʻole ka wai lawe waiwai i hoʻopilikia i ko lākou hiki ke neʻe.ʻOi aku ka nāwaliwali o ka ʻokoʻa o ke kiʻi ʻāpana CombiMag, e hōʻike ana he like ka nui o ka wai a me nā ʻāpana a no laila ʻaʻole hiki ke neʻe maʻalahi kekahi i kekahi.Hiki ke hoʻopaʻa ʻia nā ʻāpana inā holo wikiwiki ka magnet, e hōʻike ana ʻaʻole hiki i ka ikaika o ke kahua magnet ke lanakila mau i ka friction ma waena o nā ʻāpana i loko o ka wai, e hōʻike ana ʻaʻole e hele mai ka ikaika o ke kahua magnetic a me ka mamao ma waena o ka magnet a me ka wahi i hoʻopaʻa ʻia. kāhāhā.mea nui.Hōʻike pū ʻia kēia mau hopena ʻoiai hiki i nā mea magnet ke hopu i nā microparticles he nui e kahe ana ma ka wahi i hoʻopaʻa ʻia, ʻaʻole hiki ke hilinaʻi ʻia nā magnet e hoʻoneʻe i nā ʻāpana CombiMag ma ka ʻili o ka trachea.No laila, ua hoʻoholo mākou e hoʻohana nā haʻawina vivo LV MF i nā māla magnetic static e hoʻopaʻa kino i nā wahi kikoʻī o ka lāʻau ea.
Ke hāʻawi ʻia nā ʻāpana i loko o ke kino, paʻakikī lākou e ʻike i ka pōʻaiapili o ka ʻiʻo neʻe paʻakikī o ke kino, akā ua hoʻomaikaʻi ʻia ko lākou hiki ke ʻike ma ka neʻe ʻana i ka magnet ma luna o ka trachea e "wiggle" i nā kaula MP.ʻOiai hiki ke kiʻi i ka manawa maoli, ʻoi aku ka maʻalahi o ka ʻike ʻana i ka neʻe ʻana o nā ʻāpana ma hope o ka pepehi ʻia ʻana o ka holoholona.ʻOi aku ka kiʻekiʻe o nā manaʻo MP ma kēia wahi i ka wā i hoʻonoho ʻia ai ka magnet ma luna o ka wahi kiʻi, ʻoiai ʻike pinepine ʻia kekahi mau ʻāpana ma lalo o ka trachea.ʻAʻole like me nā haʻawina in vitro, ʻaʻole hiki ke huki ʻia nā ʻāpana i lalo o ka trachea e ka neʻe ʻana o kahi magnet.Ua kūlike kēia ʻike me ke ʻano o ka hana ʻana 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 ma hope o ka hoʻomaʻemaʻe ʻana iā lākou ma o ka mīkini hoʻomaʻemaʻe muco-ciliary.
Manaʻo mākou ʻo ka hoʻohana ʻana i nā mea magnet ma luna a ma lalo o ka trachea no ka huki ʻana (Fig. 3b) hiki ke hopena i kahi kahua mākēneki like ʻole, ma mua o ke kahua hoʻokalakupua i hoʻopaʻa nui ʻia i ka manawa hoʻokahi, hiki ke hopena i ka hāʻawi like ʻana o nā ʻāpana..Akā naʻe, ʻaʻole i loaʻa i kā mākou noiʻi mua nā hōʻike maopopo e kākoʻo i kēia kuhiakau.Pēlā hoʻi, ʻo ka hoʻonohonoho ʻana i ʻelua mau magnet e hoʻokuʻu (Fig. 3c) ʻaʻole ia i hopena i ka hoʻopaʻa ʻana o nā ʻāpana i ka wahi kiʻi.Hōʻike kēia mau ʻike ʻelua ʻaʻole i hoʻomaikaʻi maikaʻi ka hoʻonohonoho ʻelua-magnet i ka mana kūloko o ke kuhikuhi ʻana o MP, a he paʻakikī ke hoʻolohe ʻana i nā mana magnetic ikaika, e hoʻemi i kēia ala.Pēlā nō, ʻaʻole i hoʻonui ʻia ka heluna o nā ʻāpana i koe ma ka wahi i kiʻi ʻia.ʻAʻole paha e kūleʻa kekahi o kēia mau hoʻonohonoho ʻokoʻa no ka mea e hoʻemi ʻia ka ikaika o ke kahua magnetic ma ka ʻāpana deposition.No laila, ʻo ka hoʻonohonoho hoʻonohonoho magnet hoʻokahi ma 30 degere (Fig. 3a) i manaʻo ʻia ʻo ka maʻalahi a maikaʻi loa i ke ʻano hoʻokolohua 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 kahua magnetic, ua piʻi nui nā pae transduction ma ka trachea i hoʻohālikelike ʻia me nā mana.Ma muli o nā haʻawina kiʻi synchrotron a me nā hopena LacZ, ua ʻike ʻia e hiki i ke kahua magnetic ke mālama i ka LV i loko o ka trachea a hoʻemi i ka nui o nā ʻāpana vector i komo koke i loko o ka māmā.Hiki i kēia mau hoʻomaikaʻi ʻana ke alakaʻi i ka hana ʻoi aku ka maikaʻi ʻoiai e hōʻemi ana i nā titers i hāʻawi ʻia, ka transduction non-targeted, inflammatory and immune side effects, a me nā kumukūʻai hoʻoili gene.ʻO ka mea nui, e like me ka mea hana, hiki ke hoʻohana ʻia ʻo CombiMag me ka hui pū ʻana me nā ʻano hoʻololi ʻē aʻe, me nā vectors viral ʻē aʻe (e like me AAV) a me nā waikawa nucleic.