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Pono nā vectors Gene no ka mālama ʻana i ka maʻi māmā cystic fibrosis e kuhikuhi i nā alaloa alakaʻi no ka mea ʻaʻole hāʻawi ka transduction māmā peripheral i ka pono therapeutic. Pili pono ka pono o ka transduction viral i ka manawa noho vector. Eia nō naʻe, ʻo nā wai lawe e like me nā mea lawe gene e hoʻolaha maoli i loko o ka alveoli i ka wā hoʻoulu, a ʻo nā ʻāpana therapeutic o kēlā me kēia ʻano e hoʻomaʻemaʻe koke ʻia e ka halihali mucociliary. He mea nui ka hoʻolōʻihi ʻana i ka manawa noho o nā mea lawe gene ma nā alaloa akā paʻakikī ke hoʻokō. Hiki i nā ʻāpana magnetic i hoʻohui ʻia e ka mea lawe gene i hiki ke kuhikuhi ʻia i ka ʻili o nā alaloa ke hoʻomaikaʻi i ka hoʻopaʻa ʻāpana. Ma muli o nā pilikia o ka nānā ʻana i loko o vivo, ʻaʻole maopopo loa ke ʻano o ia mau ʻāpana magnetic liʻiliʻi ma ka ʻili o ke alaloa i ke alo o kahi kahua magnetic i hoʻopili ʻia. ʻO ka pahuhopu o kēia haʻawina, ʻo ia ka hoʻohana ʻana i ke kiʻi synchrotron e ʻike i ka neʻe ʻana o loko o vivo o kahi moʻo o nā ʻāpana magnetic i loko o ka trachea o nā ʻiole anesthetized e nānā i nā dynamics a me nā ʻano o ke ʻano o kēlā me kēia a me ka nui o nā ʻāpana i loko o vivo. A laila ua loiloi mākou inā e hoʻonui ka lawe ʻana o nā ʻāpana magnetic lentiviral i ke alo o kahi kahua magnetic i ka pono transduction i loko o ka trachea ʻiole. Synchrotron X-ray Hōʻike ke kiʻi ʻana i ke ʻano o nā ʻāpana magnetic i loko o nā kahua magnetic paʻa a neʻe hoʻi i loko o vitro a i loko o vivo. ʻAʻole hiki ke kauō maʻalahi ʻia nā ʻāpana ma ka ʻili o ke ala ea ola me nā magnet, akā i ka wā o ka lawe ʻana, ua hoʻopili ʻia nā waihona ma ke kahua o ka ʻike kahi i ikaika loa ai ke kahua magnetic. Ua hoʻonui ʻia ka pono o ka transduction i ʻeono manawa i ka wā i hāʻawi ʻia ai nā ʻāpana magnetic lentiviral i ke alo o kahi kahua magnetic. I ka hui pū ʻana, hōʻike kēia mau hopena he mau ala waiwai paha nā ʻāpana magnetic lentiviral a me nā kahua magnetic e hoʻomaikaʻi i ka hoʻolālā vector gene a hoʻonui i nā pae transduction i ke alakaʻi ʻana i nā ala ea i loko o vivo.
Hoʻokumu ʻia ka Cystic fibrosis (CF) e ka loli ʻana o kahi gene hoʻokahi i kapa ʻia ʻo CF transmembrane conductance regulator (CFTR). ʻO ka protein CFTR kahi kahawai ion i loaʻa i loko o nā cell epithelial he nui ma ke kino, me nā alaloa ea, kahi kahua nui o ka pathogenesis CF. ʻO nā hemahema CFTR e alakaʻi i ka lawe ʻana o ka wai maʻamau, e hoʻomaloʻo ana i ka ʻili o ke alaloa a me ka hōʻemi ʻana i ka hohonu o ka papa wai o ka ʻili o ke alaloa (ASL). Hoʻopilikia pū kēia i ka hiki o ka ʻōnaehana lawe mucociliary (MCT) ke hoʻomaʻemaʻe i nā ʻāpana i hanu ʻia a me nā pathogens mai nā alaloa. ʻO kā mākou pahuhopu ka hoʻomohala ʻana i kahi lāʻau lapaʻau gene lentiviral (LV) e hāʻawi i ke kope pololei o ka gene CFTR a hoʻomaikaʻi i ke olakino ASL, MCT, a me ka māmā, a e hoʻomau i ka hoʻomohala ʻana i nā ʻenehana hou e hiki ke ana i kēia mau palena ma vivo1.
ʻO nā vectors LV kekahi o nā moho alakaʻi no ka hoʻōla ʻana i ka gene CF airway, ʻoiai no ka mea hiki iā lākou ke hoʻohui mau i ka gene therapeutic i loko o nā cell basal airway (nā cell stem airway). He mea nui kēia no ka mea hiki iā lākou ke hoʻihoʻi i ka hydration maʻamau a me ka hoʻomaʻemaʻe mucus ma o ka hoʻokaʻawale ʻana i nā cell ʻili airway i hoʻoponopono ʻia e ka gene functional CF, e hopena ana i nā pono ola holoʻokoʻa. Pono e kuhikuhi ʻia nā vectors LV e kūʻē i ke ala ea alakaʻi, ʻoiai ʻo ia kahi e hoʻomaka ai ka maʻi māmā CF. ʻO ka lawe ʻana o ka vector hohonu i loko o ka māmā e hopena i ka transduction alveolar, akā ʻaʻohe pono therapeutic i CF. Eia nō naʻe, neʻe maoli nā wai e like me nā mea lawe gene i ka alveoli ma hope o ka hoʻoulu ʻana ma hope o ka lawe ʻana3,4 a hoʻomaʻemaʻe koke ʻia nā ʻāpana therapeutic i loko o ka lua waha e MCT. Pili pono ka pono o ka transduction LV i ka lōʻihi o ka manawa e noho ai ka vector ma ka ʻaoʻao o nā cell target e ʻae i ka lawe ʻana o ka cellular - ka "manawa noho"5 - kahi i hoʻemi maʻalahi ʻia e ka airflow maʻamau a me ka hopu ʻana o ka mucus particle i hoʻonohonoho ʻia a me MCT. No CF, ʻo ka hiki ke hoʻolōʻihi i ka manawa noho o ka LV i loko o ke ala ea he mea nui ia e hoʻokō i nā pae kiʻekiʻe o ka transduction ma kēia ʻāina, akā ua paʻakikī a hiki i kēia manawa.
No ka lanakila ʻana i kēia pilikia, ke kuhikuhi nei mākou e hiki i nā ʻāpana magnetic LV (MPs) ke kōkua ma nā ʻano hoʻohui ʻelua. ʻO ka mea mua, hiki ke alakaʻi ʻia lākou me ka magnetic i ka ʻili o ke alaloa e hoʻomaikaʻi i ka hoʻolālā ʻana a kōkua i nā ʻāpana lawe gene e noho ma ka ʻāpana alaloa i makemake ʻia; a me ASL) e neʻe i ka papa cell 6. Ua hoʻohana nui ʻia nā MPs ma ke ʻano he mau kaʻa lawe lāʻau i kuhikuhi ʻia i ka wā e hoʻopaʻa ai lākou i nā antibodies, nā lāʻau chemotherapeutic, a i ʻole nā ​​​​mole liʻiliʻi ʻē aʻe e hoʻopili i nā membrane cell a i ʻole e hoʻopaʻa i nā mea loaʻa ʻili cell pili a hōʻiliʻili ma nā wahi tumor i ke alo o ka uila static. Nā Kahua Magnetic no ka Lapaʻau ʻana i ka Maʻi ʻAʻai 7. ʻO nā ʻano hana "hyperthermal" ʻē aʻe e hoʻolālā e hoʻomehana i nā MP ke hōʻike ʻia lākou i nā kahua magnetic oscillating, a laila e luku ana i nā cell tumor. ʻO ke kumumanaʻo o ka transfection magnetic, kahi e hoʻohana ʻia ai kahi kahua magnetic ma ke ʻano he transfection agent e hoʻonui i ka hoʻoili ʻana o ka DNA i nā cell, hoʻohana pinepine ʻia ma vitro me ka hoʻohana ʻana i kahi ʻano o nā vectors gene non-viral a me viral no nā laina cell paʻakikī e transduce. Ua hoʻokumu ʻia ka pono o ka LV magnetotransfection, me ka lawe ʻana i loko o vitro o LV-MPs i kahi laina cell epithelial bronchial kanaka i ke alo o kahi kahua magnetic static, e hoʻonui ana i ka pono transduction ma 186-fold i hoʻohālikelike ʻia me ka LV vector wale nō. Ua hoʻopili ʻia hoʻi ʻo LV-MP i kahi kumu hoʻohālike CF in vitro, kahi i hoʻonui ai ka transfection magnetic i ka transduction LV i nā moʻomeheu interface air-liquid ma 20-fold i ke alo o ka CF sputum10. Eia nō naʻe, ua loaʻa i ka magnetotransfection in vivo o nā ʻōpū ka nānā iki ʻole a ua loiloi wale ʻia i loko o kekahi mau haʻawina holoholona11,12,13,14,15, ʻoi aku hoʻi i ka nā māmā16,17.Eia naʻe, ua maopopo nā manawa kūpono no ka transfection magnetic i ka CF lung therapy.Ua ʻōlelo ʻo Tan et al.(2020) "ʻo kahi haʻawina hōʻoia-o-manaʻo o ka lawe ʻana o ka pulmonary nanoparticle magnetic kūpono e hoʻomākaukau i ke ala no nā hoʻolālā inhalation CFTR e hiki mai ana e hoʻomaikaʻi i nā hopena lapaʻau i nā poʻe maʻi CF"6.
He paʻakikī ke ʻike a me ke aʻo ʻana i ke ʻano o nā ʻāpana magnetic liʻiliʻi ma nā ʻili ea i ke alo o kahi kahua magnetic i hoʻopili ʻia, a no laila ua hoʻomaopopo maikaʻi ʻole ʻia. Ma nā haʻawina ʻē aʻe, ua hoʻomohala mākou i kahi ʻano hana synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) e ʻike maka ʻole a helu i nā loli in vivo minuke i ka hohonu ASL18 a me ke ʻano MCT19,20 e ana pololei i ka hydration o ka ʻili Canal kinoea a hoʻohana ʻia ma ke ʻano he hōʻailona mua o ka pono o ka lāʻau lapaʻau. Eia kekahi, hoʻohana kā mākou ʻano loiloi MCT i nā ʻāpana diameter 10-35 µm i haku ʻia me ka alumina a i ʻole ke aniani refractive index kiʻekiʻe e like me nā māka MCT i ʻike ʻia me ka hoʻohana ʻana iā PB-PCXI21. He kūpono nā ʻano hana ʻelua no ka nānā ʻana i nā ʻano ʻano ʻāpana, me MP.
Ma muli o kona hoʻonā kiʻekiʻe spatial a me temporal, kūpono loa kā mākou mau ʻano hana loiloi ASL a me MCT e pili ana iā PB-PCXI no ka nānā ʻana i nā dinamika a me nā ʻano o ke ʻano o nā ʻāpana hoʻokahi a me ka nui i loko o vivo e kōkua iā mākou e hoʻomaopopo a hoʻomaikaʻi i nā ʻano hana lawe ʻana o ka gene MP. Loaʻa ke ʻano a mākou e hoʻohana ai ma ʻaneʻi mai kā mākou mau haʻawina e hoʻohana ana i ka beamline SPring-8 BL20B2, kahi a mākou i ʻike ai i ka neʻe ʻana o ka wai ma hope o ka lawe ʻana o ka sham vector dose i loko o nā ala ea nasal a me ka pulmonary o nā ʻiole e kōkua i ka wehewehe ʻana i kā mākou mau ʻano hōʻike gene Non-uniform i ʻike ʻia i kā mākou mau haʻawina holoholona lawe gene dose 3,4.
ʻO ke kumu o kēia noiʻi, ʻo ia ka hoʻohana ʻana i ka synchrotron PB-PCXI e ʻike i nā neʻe ʻana o kahi moʻo o nā MP i loko o ka trachea o nā ʻiole ola. Ua hoʻolālā ʻia kēia mau haʻawina kiʻi PB-PCXI e hoʻāʻo i kahi ʻano o nā MP, nā ikaika o ke kahua magnetic, a me nā wahi e hoʻoholo ai i ko lākou hopena ma ka neʻe ʻana o MP. Ua kuhi mākou e kōkua kahi kahua magnetic i hoʻopili ʻia ma waho i ka MP i hāʻawi ʻia e noho a neʻe paha i kahi i manaʻo ʻia. Ua ʻae pū kēia mau haʻawina iā mākou e ʻike i nā hoʻonohonoho magnet e hoʻonui i ka nui o nā ʻāpana i mālama ʻia i loko o ka trachea ma hope o ka waiho ʻana. Ma kahi moʻo ʻelua o nā haʻawina, ua ʻimi mākou e hoʻohana i kēia hoʻonohonoho kūpono e hōʻike i ke ʻano transduction i loaʻa mai ka lawe ʻana o LV-MP i loko o ke alaloa o ka ʻiole, ma muli o ka manaʻo e hiki i ka lawe ʻana o LV-MP i loko o ke ʻano o ka hoʻolālā ʻana i ke alaloa e hoʻomaikaʻi i ka pono o ka transduction LV.
Ua hana ʻia nā haʻawina holoholona āpau e like me nā protocol i ʻāpono ʻia e ke Kulanui o Adelaide (M-2019-060 a me M-2020-022) a me ke Komite Hoʻoponopono Holoholona Synchrotron SPring-8. Ua hana ʻia nā hoʻokolohua e like me nā alakaʻi ARRIVE.
Ua hana ʻia nā kiʻi X-ray āpau ma ka BL20XU beamline ma ka SPring-8 synchrotron ma Iapana, me ka hoʻohana ʻana i kahi hoʻonohonoho like me ka mea i wehewehe mua ʻia21,22. I ka pōkole, aia ka pahu hoʻokolohua ma 245 m mai ke apo mālama synchrotron. Hoʻohana ʻia kahi mamao laʻana-a-detector 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 ʻokoʻa pae. Ua hoʻohana ʻia kahi ikehu kukuna monochromatic o 25 keV. Ua hopu ʻia nā kiʻi me ka hoʻohana ʻana i kahi mea hoʻololi X-ray kiʻekiʻe (SPring-8 BM3) i hoʻopili ʻia i kahi mea ʻike sCMOS. Hoʻololi ka mea hoʻololi i nā kukuna X i ka mālamalama ʻike ʻia me ka hoʻohana ʻana i kahi scintillator mānoanoa 10 µm (Gd3Al2Ga3O12), a laila kuhikuhi ʻia i kahi sensor sCMOS me ka hoʻohana ʻana i kahi pahuhopu microscope × 10 (NA 0.3). ʻO ka mea ʻike sCMOS ʻo Orca-Flash4.0 (Hamamatsu Photonics, Iapana) me ka nui o ka array o 2048 × 2048 mau pikela a me ka nui o ka pikela maka o 6.5 × 6.5 µm. Hoʻopuka kēia hoʻonohonoho i kahi nui pikela isotropic maikaʻi o 0.51 µm a me kahi kahua ʻike o kahi 1.1 mm × 1.1 mm. Ua koho ʻia kahi lōʻihi hōʻike o 100 ms e hoʻonui i ka lakio hōʻailona-i-walaʻau o nā ʻāpana magnetic i loko a ma waho o ke ala ea me ka hoʻemi ʻana i nā mea hana neʻe i hoʻoulu ʻia e ka hanu. No nā haʻawina in vivo, ua kau ʻia kahi pani X-ray wikiwiki ma ke ala X-ray e kaupalena i ka nui o ka radiation ma ke ālai ʻana i ke kukuna X-ray ma waena o nā hōʻike.
ʻAʻole i hoʻohana ʻia ka mea lawe LV i kekahi mau haʻawina kiʻi SPring-8 PB-PCXI no ka mea ʻaʻole i hōʻoia ʻia ke keʻena kiʻi BL20XU e ka Biosafety Level 2. Akā, ua koho mākou i kahi ʻano o nā MP i ʻike maikaʻi ʻia mai ʻelua mau mea hoʻolako kalepa—e uhi ana i nā ʻano nui, nā mea hana, nā ʻano hao, a me nā noi—ʻo ka mea mua e hoʻomaopopo pehea e hoʻopilikia ai nā kahua magnetic i ka neʻe ʻana o MP i loko o nā capillaries aniani, a laila i loko o nā ala ea ola. ma luna o ka ʻili. Loaʻa ka nui o nā MP mai 0.25 a 18 μm a hana ʻia mai nā ʻano mea like ʻole (e nānā i ka Papa 1), akā ʻaʻole ʻike ʻia ka haku mele ʻana o kēlā me kēia laʻana, me ka nui o nā ʻāpana magnetic i loko o ka MP. Ma muli o kā mākou mau haʻawina MCT nui 19, 20, 21, 23, 24, manaʻo mākou e ʻike ʻia nā MP liʻiliʻi e like me 5 μm ma ka ʻili ea tracheal, no ka laʻana ma ka unuhi ʻana i nā kiʻi e pili ana e ʻike i ka ʻike maikaʻi ʻia o ka neʻe ʻana o MP. ʻOi aku ka liʻiliʻi o kahi MP hoʻokahi 0.25 μm ma mua o ka hoʻonā o ka hāmeʻa 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 kahi i waiho ʻia ai lākou ma hope o ka waiho ʻana.
Ua hoʻomākaukau ʻia nā laʻana no kēlā me kēia MP ma ka Papa 1 i loko o nā capillaries aniani 20 μl (Drummond Microcaps, PA, USA) me ke anawaena o loko o 0.63 mm. Loaʻa nā ʻāpana corpuscular i ka wai, ʻoiai loaʻa nā ʻāpana CombiMag i loko o ka wai ponoʻī o ka mea hana. Hoʻopiha ʻia kēlā me kēia paipu me ka hapalua o ka wai (ma kahi o 11 μl) a kau ʻia ma luna o ka mea paʻa laʻana (e nānā i ke Kiʻi 1). Ua kau ʻia nā capillaries aniani ma ke kahua laʻana i loko o ka pahu kiʻi, kēlā me kēia, a kau ʻia nā kihi o ka wai. Ua hoʻopili ʻia kahi magnet nickel shell 19 mm ke anawaena (28 mm ka lōʻihi) neodymium iron boron (NdFeB) rare earth (N35, cat. no. LM1652, Jaycar Electronics, Australia) me ke koena magnetization o 1.17 Tesla i kahi kahua unuhi kaʻawale e hoʻokō ai i ka hoʻololi ʻana i kona kūlana mamao i ka wā o ke kiʻi ʻana. Hoʻomaka ka loaʻa ʻana o ke kiʻi X-ray i ka wā e kau ʻia ai ka magnet ma kahi o 30 mm ma luna o ka laʻana, a loaʻa nā kiʻi ma ka wikiwiki o 4 mau kiʻi i kekona. I ka wā o ke kiʻi ʻana, ua lawe ʻia ka magnet kokoke i ka paipu capillary aniani (ma kahi o 1 mm ka mamao) a laila unuhi ʻia ma ke ʻano o ka paipu e loiloi i nā hopena o ka ikaika o ke kahua a me ke kūlana.
Hoʻonohonoho kiʻi in vitro e loaʻa ana nā laʻana MP i loko o nā capillaries aniani ma ke kahua unuhi xy laʻana. Hōʻailona ʻia ke ala o ke kukuna X-ray me kahi laina ʻulaʻula i kahakaha ʻia.
I ka manawa i hoʻokumu ʻia ai ka ʻike in vitro o nā MP, ua hoʻāʻo ʻia kahi hapa o lākou i loko o vivo i nā ʻiole wahine albino Wistar hihiu (~ 12 mau pule, ~ 200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Iapana), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Iapana) a me 4 mg/kg butorphanol (Vetorphale®, Meiji Seika) Ua hoʻomaʻamaʻa ʻia nā ʻiole me ka hui ʻana o Pharma), Iapana) ma o ka inikini intraperitoneal. Ma hope o ka anesthesia, ua hoʻomākaukau ʻia lākou no ke kiʻi ʻana ma ka wehe ʻana i ka huluhulu a puni ka trachea, e hoʻokomo ana i kahi paipu endotracheal (ET; 16 Ga iv cannula, Terumo BCT) a hoʻopaʻa iā lākou 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 ke kahua unuhi laʻana i loko o ka pahu kiʻi ma kahi kihi iki e hoʻohālikelike i ka trachea ma ke ʻano ākea ma ke kiʻi X-ray, e like me ka mea i hōʻike ʻia ma Kiʻi 2a.
(a) Hoʻonohonoho kiʻi in vivo i loko o ka pahu kiʻi SPring-8, ua hōʻailona ʻia ke ala o ke kukuna X-ray me kahi laina ʻulaʻula i kahakaha ʻia. (b,c) Ua hana ʻia ka localization Magnet ma ka trachea ma kahi mamao me ka hoʻohana ʻana i ʻelua mau kāmela IP i kau ʻia ma ke ʻano orthogonally. Ma ka ʻaoʻao hema o ke kiʻi pale, hiki ke ʻike ʻia ke kaula uea e paʻa ana i ke poʻo, a me ka cannula lawe ma kahi i loko o ka paipu ET.
Ua hoʻopili ʻia kahi ʻōnaehana pamu syringe mamao (UMP2, World Precision Instruments, Sarasota, FL) me ka hoʻohana ʻana i kahi syringe aniani 100 μl i ka paipu PE10 (OD 0.61 mm, ID 0.28 mm) ma o kahi nila 30 Ga. E kaha i ka paipu e hōʻoia i ka pae kūpono o ka piko i loko o ka trachea i ka wā e hoʻokomo ai i ka paipu ET. Me ka hoʻohana ʻana i ka micropump, ua huki ʻia ka plunger syringe ʻoiai ua hoʻokomo ʻia ka piko o ka paipu i loko o ka hāpana MP e hāʻawi ʻia. A laila ua hoʻokomo ʻia ka paipu hāʻawi i hoʻouka ʻia i loko o ka paipu endotracheal, e kau ana i ka piko i loko o ka ʻāpana ikaika loa o kā mākou kahua magnetic i manaʻo ʻia. Ua hoʻomalu ʻia ka loaʻa ʻana o ke kiʻi me ka hoʻohana ʻana i kahi mea ʻike hanu i hoʻopili ʻia i kā mākou pahu manawa Arduino, a ua hoʻopaʻa ʻia nā hōʻailona āpau (e laʻa me ka mahana, ka hanu ʻana, ka wehe ʻana/pani ʻana o ka pani a me ka loaʻa ʻana o ke kiʻi) me ka hoʻohana ʻana iā Powerlab a me LabChart (AD Instruments, Sydney, Australia) 22. I ke kiʻi ʻana I ka wā i hiki ʻole ai ke komo i ka pā, ua kau ʻia ʻelua mau kāmela 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 o ke kiʻi ʻana (Kiʻi 2b, c). No ka hōʻemi ʻana i nā mea hana neʻe, ua loaʻa hoʻokahi kiʻi no kēlā me kēia hanu i ka wā o ka pae kai hope.
Hoʻopili ʻia kahi magnet i kahi pae ʻelua e hiki ke loaʻa mamao mai waho o ka hale kiʻi. Ua hoʻāʻo ʻia nā kūlana magnet like ʻole a me nā hoʻonohonoho ʻana, me: Kau ʻia ma kahi kihi o kahi 30° ma luna o ka trachea (nā hoʻonohonoho i hōʻike ʻia 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 e huki (Kiʻi 3b); hoʻokahi magnet ma luna o ka holoholona a ʻo kekahi ma lalo, me nā pou i hoʻonohonoho ʻia e kipaku (Kiʻi 3c); a hoʻokahi magnet ma luna a kū pololei i ka trachea (Kiʻi 3d). Ke hoʻonohonoho ʻia ka holoholona a me ka magnet a hoʻouka ʻia ka MP e hoʻāʻo ʻia i loko o ka pamu syringe, e hāʻawi i kahi mahele 50 μl ma ka wikiwiki o 4 μl/sec i ka wā e loaʻa ai nā kiʻi. A laila hoʻoneʻe ʻia ka magnet i mua a i mua ma ke ala a i ʻole ma ka ʻaoʻao o ka trachea me ka hoʻomau ʻana i ka loaʻa ʻana o nā kiʻi.
ʻO ke ʻano o ka Magnet no ke kiʻi ʻana i loko o ke kino (a) kahi magnet hoʻokahi ma luna o ka trachea ma kahi kihi o 30°, (b) ʻelua mau magnet i hoʻonohonoho ʻia e huki, (c) ʻelua mau magnet i hoʻonohonoho ʻia e pale aku, (d) hoʻokahi magnet ma luna a kū pololei i loko o ka trachea. Nānā ka mea nānā i lalo mai ka waha a i nā māmā ma o ka trachea, a hele ka kukuna X-ray ma ka ʻaoʻao hema o ka ʻiole a puka i waho o ka ʻaoʻao ʻākau. Neʻe ʻia ka magnet ma ka lōʻihi o ke ala ea a i ʻole hema a ʻākau ma luna o ka trachea ma ke kuhikuhi o ke kukuna X-ray.
Ua ʻimi pū mākou e hoʻoholo i ka ʻike a me ke ʻano o nā ʻāpana i loko o nā alaloa i ka wā ʻaʻohe hanu huikau a me ka neʻe ʻana o ka puʻuwai. No laila, i ka hopena o ka manawa kiʻi, ua pepehi kanaka ʻia nā holoholona no ka overdose pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). Ua waiho ʻia kekahi mau holoholona ma ke kahua kiʻi, a i ka pau ʻana o ka hanu ʻana a me ka puʻuwai o ka naʻau, ua hana hou ʻia ke kaʻina hana kiʻi, me ka hoʻohui ʻana i kahi mahele hou o MP inā ʻaʻohe MP i ʻike ʻia ma ka ʻili o ke alaloa.
Ua hoʻoponopono ʻia nā kiʻi i loaʻa ma ke kahua pālahalaha a me ke kahua pouli a laila ua hōʻuluʻulu ʻia i loko o kahi kiʻiʻoniʻoni (20 mau mōlina i ke kekona; 15-25 × ka wikiwiki maʻamau ma muli o ka nui o ka hanu ʻana) me ka hoʻohana ʻana i kahi palapala maʻamau i kākau ʻia ma MATLAB (R2020a, The Mathworks).
Ua mālama ʻia nā haʻawina lawe vector gene LV āpau ma ka Laboratory Animal Research Facility ma ke Kulanui o Adelaide a ua manaʻo ʻia e hoʻohana i nā hopena o ka hoʻokolohua SPring-8 e loiloi inā hiki i ka lawe ʻana o LV-MP i ke alo o kahi kahua magnetic ke hoʻonui i ka hoʻoili gene in vivo. No ka loiloi ʻana i nā hopena o MP a me ke kahua magnetic, ua mālama ʻia ʻelua mau hui holoholona: ua hāʻawi ʻia kekahi hui iā LV-MP me kahi magnet i kau ʻia, a ua loaʻa i kekahi hui kahi hui kaohi me LV-MP me ka ʻole o kahi magnet.
Ua hana ʻia nā vectors gene LV me ka hoʻohana ʻana i nā ʻano hana i wehewehe mua ʻia 25, 26. Hōʻike ka vector LacZ i ka gene beta-galactosidase nuclear-localized i hoʻokele ʻia e ka mea hoʻolaha MPSV constitutive (LV-LacZ), kahi e hana ai i kahi huahana hopena polū i loko o nā cell transduced, ʻike ʻia ma nā alo o ka ʻiʻo māmā a me nā ʻāpana ʻiʻo. Ua hana ʻia ka titration i loko o nā moʻomeheu cell ma ka helu lima ʻana i ka helu o nā cell LacZ maikaʻi me kahi hemocytometer e helu i ka titer ma TU/ml. Hoʻopaʻa ʻia nā mea lawe ma -80 °C, hoʻoheheʻe ʻia ma mua o ka hoʻohana ʻana, a hoʻopaʻa ʻia i CombiMag ma ke kāwili ʻana ma kahi ratio 1: 1 a me ka hoʻomoʻa ʻana ma luna o ka hau no ka liʻiliʻi he 30 mau minuke ma mua o ka lawe ʻana.
Ua hoʻomaʻamaʻa ʻia nā ʻiole Sprague Dawley maʻamau (n = 3/hui, ~2-3 i loko o ka ʻōpū me ka hui ʻana o 0.4 mg/kg medetomidine (Domitor, Ilium, Australia) a me 60 mg/kg ketamine (Ilium, Australia) mahina-makahiki) ip) injection a me ka cannulation waha ʻole me ka 16 Ga iv cannula. No ka hōʻoia ʻana e loaʻa i ka ʻiʻo ea tracheal ka transduction LV, ua hoʻomaʻamaʻa ʻia me ka hoʻohana ʻana i kā mākou protocol perturbation mechanical i wehewehe mua ʻia, kahi i hamo ʻia ai ka ʻili ea tracheal me kahi hīnaʻi uea (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. A laila ua hana ʻia ka hoʻokele ʻana o LV-MP ma Tracheal i loko o kahi keʻena palekana biological ma kahi o 10 mau minuke ma hope o ka perturbation.
Ua hoʻonohonoho ʻia ke kahua magnetic i hoʻohana ʻia ma kēia hoʻokolohua ma ke ʻano like me ke aʻo ʻana i ka kiʻi X-ray in vivo, me nā magnet like i paʻa ma luna o ka trachea me ka hoʻohana ʻana i nā clip stent distillation (Kiʻi 4). Ua hāʻawi ʻia kahi nui 50 μl (2 × 25 μl aliquots) o LV-MP i loko o ka trachea (n = 3 mau holoholona) me ka hoʻohana ʻana i kahi pipette e loaʻa ana kahi tip gel e like me ka mea i wehewehe mua ʻia. Ua loaʻa i kahi hui hoʻomalu (n = 3 mau holoholona) nā LV-MP like me ka hoʻohana ʻole ʻana i kahi magnet. Ma hope o ka pau ʻana o ka infusion, ua wehe ʻia ka cannula mai ka ʻōmole ET a ua extubated ka holoholona. Noho ka magnet ma kahi no 10 mau minuke, a laila ua wehe ʻia. Ua loaʻa i nā ʻiole kahi mahele subcutaneous o meloxicam (1 ml/kg) (Ilium, Australia) a ukali ʻia e ka hoʻohuli ʻana o ka anesthesia ma ka ip injection o 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia). Ua mālama ʻia nā ʻiole i ka mahana a nānā ʻia a hiki i ka hoʻōla piha ʻana mai ka anesthesia.
ʻO ka mea lawe LV-MP i loko o kahi keʻena palekana olaola. Hiki ke ʻike ʻia ke kikowaena Luer hina māmā o ka paipu ET e puka mai ana mai ka waha a ua 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 100% CO2 inhalation a ua loiloi ʻia ka hōʻike ʻana o LacZ me ka hoʻohana ʻana i kā mākou lāʻau X-gal maʻamau. Ua wehe ʻia nā apo cartilaginous ʻekolu caudal loa e hōʻoia i ka hōʻino ʻana o kekahi pōʻino mechanical a i ʻole ka paʻa ʻana o ka wai mai ke kau ʻana o ka paipu endotracheal i ka nānā ʻana. Ua ʻoki ʻia kēlā me kēia trachea ma ke ʻano lōʻihi e hana i ʻelua hapalua no ka nānā ʻana, a ua kau ʻia lākou i loko o kahi kīʻaha i loaʻa ka silicone rubber (Sylgard, Dow Inc) me ka hoʻohana ʻana i kahi nila Minutien (Fine Science Tools) e nānā i ka ʻili luminal. Ua hōʻoia ʻia ka hoʻolaha a me ke ʻano o nā cell transduced e ke kiʻi mua e hoʻohana ana i kahi microscope Nikon (SMZ1500) me kahi kāmela DigiLite a me ka polokalamu TCapture (Tucsen Photonics, Kina). Ua loaʻa nā kiʻi ma ka hoʻonui 20x (me ka hoʻonohonoho kiʻekiʻe loa no ka laulā piha o ka trachea), me ka lōʻihi holoʻokoʻa o ka trachea i kiʻi ʻia i kēlā me kēia ʻanuʻu, e hōʻoia ana i ka lawa ʻana o ka overlap ma waena o kēlā me kēia kiʻi e ʻae ai i ke kiʻi "humuhumu". ʻO nā kiʻi mai kēlā me kēia trachea a laila ua hōʻuluʻulu ʻia i loko o hoʻokahi kiʻi hui pū ʻia me ka hoʻohana ʻana i ka Image Composite Editor v2.0.3 (Microsoft Research) me ka hoʻohana ʻana i kahi algorithm neʻe planar. Ua helu ʻia nā wahi hōʻike LacZ i nā kiʻi hui o ka trachea mai kēlā me kēia holoholona me ka hoʻohana ʻana i kahi 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 Value < 0.7. Ma ka hahai ʻana i nā contours o ka ʻiʻo, ua hana lima ʻia kahi mask ma GIMP v2.10.24 no kēlā me kēia kiʻi hui pū ʻia i mea e ʻike ai i ka ʻāpana ʻiʻo a pale i nā ʻike wahaheʻe mai waho o ka ʻiʻo tracheal. Ua hōʻuluʻulu ʻia nā wahi i hoʻoluʻu ʻia mai nā kiʻi hui pū ʻia mai kēlā me kēia holoholona e hana i ka huina o ka wahi i hoʻoluʻu ʻia no kēlā holoholona. A laila ua puʻunaue ʻia ka wahi i hoʻoluʻu ʻia e ka huina o ka wahi mask e hana i ka wahi i hoʻonohonoho ʻia.
Ua hoʻokomo ʻia kēlā me kēia trachea i loko o ka paraffin a ua ʻoki ʻia nā ʻāpana 5 μm. Ua hoʻoluʻu ʻia nā ʻāpana me ka ʻulaʻula wikiwiki kūlike no 5 min a ua loaʻa nā kiʻi me ka hoʻohana ʻana i kahi microscope Nikon Eclipse E400, kāmela DS-Fi3 a me ka polokalamu hopu element NIS (mana 5.20.00).
Ua hana ʻia nā loiloi helu āpau ma GraphPad Prism v9 (GraphPad Software, Inc.). Ua hoʻonohonoho ʻia ke koʻikoʻi helu ma p ≤ 0.05. Ua hōʻoia ʻia ka maʻamau me ka hoʻohana ʻana i ka hoʻāʻo Shapiro-Wilk, a ua loiloi ʻia nā ʻokoʻa ma ka hoʻoluʻu LacZ me ka hoʻohana ʻana i ka hoʻāʻo t-paired ʻole.
Ua nānā ʻia nā ʻeono MP i wehewehe ʻia ma ka Papa 1 me ka hoʻohana ʻana iā PCXI, a ua wehewehe ʻia ka ʻike ʻana ma ka Papa 2. ʻAʻole ʻike ʻia ʻelua mau polystyrene MP (MP1 a me MP2; 18 μm a me 0.25 μm, kēlā me kēia) ma lalo o PCXI, akā ua ʻike ʻia ke koena o nā laʻana (hōʻike ʻia nā laʻana ma ke Kiʻi 5). ʻIke iki ʻia ʻo MP3 a me MP4 (10-15% Fe3O4; 0.25 μm a me 0.9 μm, kēlā me kēia). ʻOiai aia kekahi o nā ʻāpana liʻiliʻi loa i hoʻāʻo ʻia, ʻo MP5 (98% Fe3O4; 0.25 μm) ka mea i ʻike nui ʻia. He paʻakikī ke ʻike i ka huahana CombiMag MP6. I nā hihia āpau, ua hoʻonui nui ʻia ko mākou hiki ke ʻike iā MP ma ka unuhi ʻana i ka magnet i mua a i hope e like me ke capillary. I ka neʻe ʻana o nā magnet mai ke capillary, ua hoʻolōʻihi ʻia nā ʻāpana i nā kaula lōʻihi, akā i ka wā e kokoke mai ai nā magnet a ua piʻi ka ikaika o ke kahua magnetic, ua pōkole nā ​​kaula ʻāpana i ka neʻe ʻana o nā ʻāpana i ka ʻili luna o ke capillary (e ʻike i ke wikiō hoʻohui S1: MP4), e hoʻonui ana i ka ka nui o nā ʻāpana o ka ʻili. I ka ʻaoʻao ʻē aʻe, ke wehe ʻia ka magnet mai ke capillary, emi ka ikaika o ke kahua a hoʻonohonoho hou nā MP i nā kaula lōʻihi e hoʻolōʻihi ana mai ka ʻili o luna o ke capillary (e ʻike i ke wikiō hoʻohui S2:MP4). Ma hope o ka pau ʻana o ka neʻe ʻana o ka magnet, hoʻomau nā ʻāpana i ka neʻe ʻana no kekahi manawa pōkole ma hope o ka hiki ʻana i ke kūlana kaulike. Ke neʻe nei ka MP i kahi a mamao aku mai ka ʻili o luna o ke capillary, huki pinepine nā ʻāpana magnetic i nā ʻōpala ma o ke wai.
ʻOkoʻa nui ka ʻike ʻia 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 a pau i hōʻike ʻia ma ʻaneʻi me kahi magnet i loaʻa ma kahi o 10 mm ma luna pono o ke capillary. ʻO nā pōʻai nui i ʻike ʻia he mau pehu ea i paʻa i loko o nā capillaries, e hōʻike maopopo ana i nā hiʻohiʻona ʻeleʻele a me keʻokeʻo o ke kiʻi ʻokoʻa pae. Aia ka pahu ʻulaʻula i ka hoʻonui ʻana i ka hoʻonui ʻana i ka hoʻohālikelike. E hoʻomaopopo ʻaʻole i kūlike nā diameters o nā schematics magnet i nā kiʻi a pau a ma kahi o 100 mau manawa ʻoi aku ka nui ma mua o ka mea i hōʻike ʻia.
I ka unuhi ʻia ʻana o ka magnet ma ka hema a me ka ʻākau ma ke poʻo o ke capillary, loli ke kihi o ke kaula MP e kūlike me ka magnet (e nānā i ke Kiʻi 6), no laila e wehewehe ana i nā laina kahua magnetic. No MP3-5, ma hope o ka hiki ʻana o ke kaula i kahi kihi paepae, ua kauō ʻia nā ʻāpana ma ka ʻili luna o ke capillary. ʻO ka hopena pinepine kēia i ka hui ʻana o nā MP i nā hui nui kokoke i kahi i ʻoi aku ka ikaika o ke kahua magnetic (e nānā i ke wikiō hoʻohui S3:MP5). He mea maopopo loa kēia i ka wā e kiʻi ai i kahi kokoke i ka hopena capillary, kahi e hōʻuluʻulu ai nā MP a hoʻopaʻa i ka interface fluid-air. ʻO nā ʻāpana ma MP6, i ʻoi aku ka paʻakikī ke ʻike ma mua o MP3-5, ʻaʻole i kauō ʻia i ka neʻe ʻana o ka magnet ma ke capillary, akā ua hoʻokaʻawale ʻia nā kaula MP, e waiho ana i nā ʻāpana ma ke kahua o ka ʻike (e nānā i ke wikiō hoʻohui S4:MP6). I kekahi mau hihia, i ka wā i hoʻemi ʻia ai ke kahua magnetic i hoʻopili ʻia ma ka hoʻoneʻe ʻana i ka magnet i kahi mamao loa mai kahi o ke kiʻi ʻana, ua iho mālie nā MP i koe i ka ʻili lalo o ka paipu ma o ka umekaumaha ʻoiai e noho ana i loko o 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 i ka ʻākau ma luna o ke capillary. (a) MP3, (b) MP4, (c) MP5 a me (d) MP6. Aia i loko o ka pahu ʻulaʻula ka hoʻonui ʻokoʻa. E hoʻomaopopo he ʻike nā wikiō hoʻohui no ka mea e hōʻike ana lākou i ke ʻano koʻikoʻi o nā ʻāpana a me ka ʻike dynamic i hiki ʻole ke ʻike ʻia ma kēia mau kiʻi static.
Ua hōʻike kā mākou mau hoʻāʻo ʻana e hoʻoneʻe mālie ana ka magnet i mua a i hope ma ke trachea i ka ʻike ʻana o MP i loko o ke ʻano o ka neʻe paʻakikī i vivo. ʻAʻole i hana ʻia ka hoʻāʻo ʻana i loko o vivo no ka mea ʻaʻole ʻike ʻia nā beads polystyrene (MP1 a me MP2) i loko o ke capillary. Ua hoʻāʻo ʻia kēlā me kēia o nā MP ʻehā i koe i loko o vivo me ke axis lōʻihi magnet i hoʻonohonoho ʻia ma luna o ka trachea ma kahi kihi o kahi 30° a i luna (e nānā i nā Kiʻi 2b a me 3a), ʻoiai ua hopena kēia i nā kaulahao MP lōʻihi a ʻoi aku ka maikaʻi ma mua o ka hoʻopau ʻana o ka hoʻonohonoho magnet. ʻAʻole i ʻike ʻia ʻo MP3, MP4 a me MP6 i loko o ka trachea o nā holoholona ola. I ka wā i kiʻi ʻia ai nā ala ea o ka ʻiole ma hope o ka pepehi kanaka ʻia ʻana o nā holoholona, ​​ua noho ʻike ʻole ʻia nā ʻāpana ʻoiai ua hoʻohui ʻia ka nui hou me ka hoʻohana ʻana i ka pamu syringe. ʻO MP5 ka mea kiʻekiʻe loa o ka hao oxide a ʻo ia wale nō ka ʻāpana i ʻike ʻia, a no laila ua hoʻohana ʻia e loiloi a wehewehe i ke ʻano in vivo o MP.
ʻO ke kau ʻana i ka magnet ma luna o ka trachea i ka wā o ka lawe ʻana o ka MP ua hopena i ka nui o nā MP, akā ʻaʻole nā ​​​​mea āpau, i hoʻopaʻa ʻia ma ke kahua o ka ʻike. ʻIke maikaʻi ʻia nā ʻāpana e komo ana i ka trachea i nā holoholona i mōhai ʻia e ke kanaka. Kiʻi 7 a me ke wikiō hoʻohui S6: Hōʻike ka MP5 i ka hopu wikiwiki ʻana o ka magnetic a me ke kaulike ʻana o nā ʻāpana ma ka ʻili o ka trachea ventral, e hōʻike ana hiki ke kuhikuhi ʻia nā MP i nā wahi i makemake ʻia o ka trachea. I ka wā e ʻimi ana ma kahi mamao loa ma ka trachea ma hope o ka lawe ʻana o ka MP, ua loaʻa kekahi mau MP kokoke i ka carina, e hōʻike ana ʻaʻole lawa ka ikaika o ke kahua magnetic e hōʻiliʻili a mālama i nā MP āpau, ʻoiai ua hāʻawi ʻia lākou ma o ka ʻāpana o ka ikaika o ke kahua magnetic kiʻekiʻe loa i ka wā o ke kaʻina wai. Eia naʻe, ʻoi aku ka kiʻekiʻe o nā kikowaena MP postpartum a puni ka wahi i kiʻi ʻia, e hōʻike ana he nui nā MP i noho ma nā wahi ea kahi i kiʻekiʻe loa ai ka ikaika o ke kahua magnetic i hoʻopili ʻia.
Nā kiʻi mai (a) ma mua a me (b) ma hope o ka hāʻawi ʻana o MP5 i loko o ka trachea o kahi ʻiole i hoʻopau ʻia me ka magnet i kau pololei ʻia ma luna o ka wahi kiʻi. Aia ka wahi i kiʻi ʻia ma waena o nā apo cartilage ʻelua. Ma mua o ka hāʻawi ʻana o MP, aia kekahi wai i loko o ke ala ea. Aia ka pahu ʻulaʻula i ka hoʻonui hoʻonui ʻokoʻa. ʻO kēia mau kiʻi mai ke wikiō i hōʻike ʻia ma ke wikiō Hoʻohui S6:MP5.
ʻO ka unuhi ʻana i ka magnet ma ke trachea in vivo i hoʻololi ai ke kaulahao MP i ke kihi i loko o ka ʻili o ke alaloa ma ke ʻano like me ka mea i ʻike ʻia ma nā capillaries (e ʻike i ke Kiʻi 8 a me ke wikiō hoʻohui S7:MP5). Eia naʻe, i kā mākou noiʻi ʻana, ʻaʻole hiki ke kauō ʻia nā MP ma ke ʻili o ke alaloa ola e like me kā lākou hiki me nā capillaries. I kekahi mau hihia, e lōʻihi ke kaulahao MP i ka neʻe ʻana o ka magnet i ka hema a me ka ʻākau. He mea hoihoi, ua ʻike pū mākou e hoʻololi ke kaula ʻāpana i ka hohonu o ka papa wai o ka ʻili ke neʻe ʻia ka magnet ma ke ʻano lōʻihi ma ke trachea, a hoʻonui ʻia ke neʻe pololei ʻia ka magnet ma luna a hoʻohuli ʻia ke kaula ʻāpana i kahi kūlana kū (e ʻike i ke wikiō hoʻohui S7). : MP5 ma 0:09, lalo ʻākau). Ua loli ke ʻano o ka neʻe ʻana i ka wā i unuhi ʻia ai ka magnet ma luna o ka trachea ma ka ʻaoʻao (ʻo ia hoʻi, ma ka hema a i ʻole ka ʻākau o ka holoholona ma mua o ka lōʻihi o ka trachea). Ua ʻike maopopo ʻia nā ʻāpana i ko lākou neʻe ʻana, akā i ka wā i wehe ʻia ai ka magnet mai ka trachea, ua ʻike ʻia nā wēlau o nā kaula ʻāpana (e ʻike i ke wikiō hoʻohui S8:MP5, e hoʻomaka ana ma 0:08). He kūlike kēia me ke ʻano MP a mākou i ʻike ai ma lalo o kahi kahua magnetic i hoʻopili ʻia i loko o kahi capillary aniani.
Nā kiʻi hoʻohālike e hōʻike ana i ka MP5 i loko o ka trachea o kahi ʻiole ola i hoʻomaʻamaʻa ʻia. (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 o ka magnet i ka ʻākau. Aia i loko o ka pahu ʻulaʻula ka hoʻonui hoʻonui ʻokoʻa. Mai ke wikiō i hōʻike ʻia ma ke wikiō hoʻohui S7:MP5 kēia mau kiʻi.
I ka wā i hoʻonohonoho ʻia ai nā pou ʻelua ma kahi kuhikuhi ʻākau-hema ma luna a ma lalo o ka trachea (ʻo ia hoʻi ka huki ʻana; Kiʻi 3b), ua ʻike ʻia nā MP chords lōʻihi a aia ma ka ʻaoʻao o ka trachea ma mua o ka ʻili o ka tracheal dorsal (e ʻike i ke wikiō hoʻohui S9:MP5). Eia naʻe, ʻaʻole i ʻike ʻia nā kiʻekiʻe o nā ʻāpana ma kahi hoʻokahi (ʻo ia hoʻi, ka ʻili dorsal o ka trachea) ma hope o ka lawe ʻana i ka wai i ka wā i hoʻohana ʻia ai kahi mea dual-magnet, kahi mea maʻamau i ka wā e hoʻohana ʻia ai kahi mea hoʻokahi-magnet. A laila i ka wā i hoʻonohonoho ʻia ai kekahi magnet e pale aku i nā pou i hoʻohuli ʻia (Kiʻi 3c), ʻaʻole i ʻike ʻia ka nui o nā ʻāpana i ʻike ʻia ma ke kahua o ka ʻike e piʻi ma hope o ka lawe ʻana. He paʻakikī ka hoʻonohonoho ʻana o nā hoʻonohonoho dual-magnet ʻelua ma muli o nā ikaika kahua magnetic kiʻekiʻe e huki a paʻi paha i nā magnet, kēlā me kēia. A laila ua hoʻololi ʻia ka hoʻonohonoho ʻana i hoʻokahi magnet e like me ke alaloa akā e hele ana ma ke alaloa ma 90 degere i hiki ai i nā laina kahua ke hele i ka paia tracheal orthogonally (Kiʻi 3d), kahi kuhikuhi i hoʻolālā ʻia e hoʻoholo ai inā hiki ke ʻike ʻia ka hōʻuluʻulu ʻana o nā ʻāpana ma ka paia ʻaoʻao. Eia naʻe, i loko Ma kēia hoʻonohonoho ʻana, ʻaʻohe neʻe ʻana o ka hōʻiliʻili MP a i ʻole ka neʻe ʻana o ka magnet. Ma muli o kēia mau hopena āpau, ua koho ʻia kahi hoʻonohonoho hoʻonohonoho hoʻokahi-magnet, 30-degere (Kiʻi 3a) no nā haʻawina lawe gene in vivo.
I ka wā i kiʻi pinepine ʻia ai ka holoholona ma hope koke iho o ka pepehi kanaka ʻana, ʻo ka nele o ka neʻe ʻana o nā ʻiʻo e hōʻike ana ua hiki ke ʻike ʻia nā laina ʻāpana maikaʻi a pōkole i loko o ke kahua interchondral maopopo, "wiliwili" e like me ka neʻe ʻana o ka magnet. Eia naʻe, ʻaʻole hiki ke ʻike maopopo i ke alo a me ka neʻe ʻana o nā ʻāpana MP6.
ʻO ka titer LV-LacZ 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 kahi mahele tracheal 50 μl o 9 × 107 TU/ml LV vehicle (ʻo ia hoʻi 4.5 × 106 TU/iole). ). Ma kēia mau haʻawina, ma kahi o ka unuhi ʻana i ka magnet i ka wā hānau, ua hoʻopaʻa mākou i ka magnet ma kahi hoʻokahi e hoʻoholo ai inā hiki ke hoʻomaikaʻi ʻia ka transduction LV (a) i hoʻohālikelike ʻia me ka lawe ʻana o ka vector i ka loaʻa ʻole o kahi kahua magnetic, a me (b) hiki ke kālele ʻia. Hoʻololi ʻia nā pūnaewele airway i nā wahi i manaʻo ʻia he magnetic o ke alaloa o luna.
ʻAʻole i ʻike ʻia ka loaʻa ʻana o nā magnet a me ka hoʻohana ʻana o CombiMag i hui pū ʻia me nā vectors LV i nā hopena maikaʻi ʻole i ke olakino holoholona, ​​​​e like me kā mākou protocol lawe vector LV maʻamau. Ua hōʻike nā kiʻi mua o ka ʻāpana tracheal i hoʻopilikia ʻia e ka mechanical perturbation (Supplementary Fig. 1) he nui nā pae transduction kiʻekiʻe i ka hui o nā holoholona i mālama ʻia me LV-MP i ka wā i loaʻa ai ka magnet (Fig. 9a). He liʻiliʻi wale nō o ka pena polū LacZ i loaʻa i ka hui kaohi (Fig. 9b). Ua hōʻike ka helu ʻana o nā wahi i hoʻoluʻu ʻia ʻo X-Gal i hoʻonohonoho ʻia ua hoʻopuka ka hoʻokele ʻana o LV-MP i ke alo o kahi kahua magnetic i kahi hoʻomaikaʻi 6-fold (Fig. 9c).
Nā kiʻi hoʻohālikelike e hōʻike ana i ka hoʻoili ʻana o ka tracheal e LV-MP (a) i ke alo o kahi kahua magnetic a me (b) i ka loaʻa ʻole o kahi magnet. (c) Hoʻomaikaʻi koʻikoʻi ma ka helu helu ma ka wahi hoʻoili LacZ i hoʻonohonoho ʻia 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).
Ua hōʻike nā ʻāpana ʻulaʻula wikiwiki kūlike ʻole (ka laʻana i hōʻike ʻia ma ke Kiʻi Hoʻohui 2) i nā pūnaewele i hoʻoluʻu ʻia e LacZ i loko o kahi ʻano like a me kahi e like me ka mea i hōʻike mua ʻia.
ʻO kahi pilikia koʻikoʻi no ka hoʻōla ʻana i nā gene airway ke kau nei i ka localization pololei o nā ʻāpana lawe i nā wahi hoihoi a me ka hoʻokō ʻana i nā pae kiʻekiʻe o ka transduction efficiency i loko o ka māmā e neʻe ana i ke alo o ke kahe ʻana o ka ea a me ka hoʻomaʻemaʻe mucus ikaika. No nā mea lawe LV i hoʻolālā ʻia e mālama i ka maʻi CF airway, ʻo ka hoʻonui ʻana i ka manawa noho o nā ʻāpana lawe i loko o nā alaloa alakaʻi he pahuhopu paʻakikī ia a hiki i kēia manawa. E like me ka mea i kuhikuhi ʻia e Castellani et al., ʻo ka hoʻohana ʻana i nā kahua magnetic e hoʻomaikaʻi i ka transduction he mau pono i hoʻohālikelike ʻia me nā ʻano lawe gene ʻē aʻe e like me ka electroporation, no ka mea hiki ke hoʻohui i ka maʻalahi, ka uku-effectiveness, ka localization lawe, ka hoʻonui ʻana i ka pono, a me nā manawa incubation pōkole, a me kahi liʻiliʻi o ka dose lawe10. Eia nō naʻe, ʻaʻole i wehewehe ʻia ka waiho ʻana o ka in vivo a me ke ʻano o nā ʻāpana magnetic i loko o nā alaloa ma lalo o ka mana o nā mana magnetic waho, ʻaʻole hoʻi i hōʻike ʻia ka hiki ke hana i kēia ʻano hana i loko o vivo e hoʻonui i nā pae hōʻike gene i nā alaloa ola.
Ua hōʻike ʻia kā mākou mau hoʻokolohua synchrotron PCXI in vitro ua ʻike ʻia nā ʻāpana āpau a mākou i hoʻāʻo ai, koe wale nō ka polystyrene MP, i ka hoʻonohonoho kiʻi a mākou i hoʻohana ai. I ke alo o kahi kahua magnetic, hana nā MP i nā kaula nona ka lōʻihi e pili ana i ke ʻano o ka ʻāpana a me ka ikaika o ke kahua magnetic (ʻo ia hoʻi ka 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 nānā ai ma muli o ka magnetized ʻia o kēlā me kēia ʻāpana a me ka hoʻoulu ʻana i kāna kahua magnetic kūloko ponoʻī. Hoʻoulu kēia mau kahua kaʻawale i nā ʻāpana like ʻē aʻe e hōʻuluʻulu a hoʻopili, me nā neʻe like me ke kaula hui ma muli o nā ikaika kūloko mai nā ikaika hoihoi a me nā mana repulsive kūloko o nā ʻāpana ʻē aʻe.
Hōʻike kiʻi (a,b) nā kaʻaahi ʻāpana i hana ʻia i loko o nā capillaries i piha i ka wai a me (c,d) trachea i piha i ka ea. E hoʻomaopopo ʻaʻole i kaha ʻia nā capillaries a me ka trachea i ka unahi. Loaʻa pū ka Panel (a) i kahi wehewehe o ka MP, nona nā ʻāpana Fe3O4 i hoʻonohonoho ʻia i loko o nā kaula.
I ka wā i neʻe ʻia ai ka magnet ma luna o ke capillary, ua hōʻea ke kihi o ke kaula ʻāpana i kahi paepae koʻikoʻi no MP3-5 e loaʻa ana iā Fe3O4, ma hope iho ʻaʻole i noho hou ke kaula ʻāpana i ke kūlana mua, akā ua neʻe ma ka ʻili i kahi kūlana hou. magnet. Hiki ke hana ʻia kēia hopena no ka mea ua lawa ka laumania o ka ʻili capillary aniani e ʻae i kēia neʻe ʻana. ʻO ka mea hoihoi, ʻaʻole i hana ʻo MP6 (CombiMag) i kēia ʻano, malia paha no ka mea ua liʻiliʻi nā ʻāpana, he ʻokoʻa nā uhi a i ʻole nā ​​​​uku ʻili, a i ʻole kahi wai lawe ponoʻī i hoʻopilikia i ko lākou hiki ke neʻe. ʻOi aku ka nāwaliwali o ke kiʻi o nā ʻāpana CombiMag, e hōʻike ana he like nā densities o ka wai a me nā ʻāpana a no laila ʻaʻole maʻalahi ka neʻe ʻana i kekahi i kekahi. Hiki ke paʻa nā ʻāpana inā neʻe wikiwiki ka magnet, e hōʻike ana ʻaʻole hiki i ka ikaika o ke kahua magnetic ke lanakila mau i ka friction ma waena o nā ʻāpana i loko o ka wai, e hōʻike ana ʻaʻole paha he mea kupanaha ka ikaika o ke kahua magnetic a me ka mamao ma waena o ka magnet a me ka wahi i manaʻo ʻia. He mea nui loa. I ka hui pū ʻana, hōʻike pū kēia mau hopena, ʻoiai hiki i nā magnet ke hopu i nā MP he nui e kahe ana ma o ka wahi i manaʻo ʻia, ʻaʻole paha e hilinaʻi ʻia nā magnet. e hoʻoneʻe i nā ʻāpana CombiMag ma ka ʻili o ka trachea. No laila, ke hoʻoholo nei mākou e hoʻohana nā haʻawina LV-MP in vivo i nā kahua magnetic static e hoʻopaʻa kino i nā wahi kikoʻī o ka lāʻau ea.
Ke hoʻouna ʻia nā ʻāpana i loko o ke kino, he paʻakikī ke ʻike i loko o ke ʻano o nā ʻiʻo kino e neʻe paʻakikī ana, akā ua hoʻonui ʻia ka hiki ke ʻike iā lākou ma ka unuhi ʻana i ka magnet ma luna o ka trachea e "wili" i nā kaula MP. ʻOiai hiki ke kiʻi ola, ʻoi aku ka maʻalahi o ka ʻike ʻana i ka neʻe ʻana o nā ʻāpana ke pepehi kanaka ʻia ka holoholona. ʻOi aku ka nui o nā kikowaena MP ma kēia wahi i ka wā i kau ʻia ai ka magnet ma luna o ka wahi kiʻi, ʻoiai ua loaʻa pinepine kekahi mau ʻāpana ma kahi o ka trachea. I ka hoʻohālikelike ʻana i nā haʻawina in vitro, ʻaʻole hiki ke kauō ʻia nā ʻāpana ma ka trachea ma ka unuhi ʻana i ka magnet. Kūlike kēia ʻike me ke ʻano o ka mucus e uhi ana i ka ʻili o ka trachea e hana maʻamau i nā ʻāpana i hanu ʻia, e hoʻopaʻa ana iā lākou i loko o ka mucus a ma hope e hoʻomaʻemaʻe ʻia e ka mīkini hoʻomaʻemaʻe mucociliary.
Ua kuhi mākou ʻo ka hoʻohana ʻana i nā magnet no ka huki ʻana ma luna a ma lalo o ka trachea (Kiʻi 3b) e hopena paha i kahi kahua magnetic like ʻole, ma mua o kahi kahua magnetic i hoʻopaʻa nui ʻia ma kahi kiko hoʻokahi, e alakaʻi ana paha i kahi hoʻolaha like o nā ʻāpana. Eia naʻe, ʻaʻole i loaʻa i kā mākou noiʻi mua nā hōʻike maopopo e kākoʻo i kēia kuhiakau. Pēlā nō, ʻo ka hoʻonohonoho ʻana i kahi pālua o nā magnet e pale aku (Kiʻi 3c) ʻaʻole i hopena i ka waiho ʻana o nā ʻāpana i loko o ka wahi i kiʻi ʻia. Hōʻike kēia mau ʻike ʻelua ʻaʻole i hoʻomaikaʻi nui ka hoʻonohonoho dual-magnet i ka mana kūloko o ka MP targeting, a he paʻakikī ke hoʻonohonoho ʻana i nā mana magnetic ikaika i hopena ʻia, e hoʻolilo ana i kēia ʻano hana i mea kūpono ʻole. Pēlā nō, ʻo ka hoʻonohonoho ʻana i ka magnet ma luna a ma o ka trachea (Kiʻi 3d) ʻaʻole hoʻi i hoʻonui i ka helu o nā ʻāpana i mālama ʻia i loko o ka wahi i kiʻi ʻia. ʻAʻole paha e holomua kekahi o kēia mau hoʻonohonoho ʻē aʻe no ka mea e hopena lākou i nā ikaika kahua magnetic haʻahaʻa i loko o ka wahi waiho. No laila, ʻo ka hoʻonohonoho magnet kihi 30-degere hoʻokahi (Kiʻi 3a) i manaʻo ʻia ʻo ia ke ʻano maʻalahi a me ka maikaʻi loa no ka hoʻāʻo ʻana i loko o vivo.
Ua hōʻike ʻia ke aʻo ʻana o 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 hoʻonui nui ʻia nā pae transduction i loko o ka trachea i hoʻohālikelike ʻia me nā kaohi. Ma muli o nā haʻawina kiʻi synchrotron a me nā hopena LacZ, ua hiki i ke kahua magnetic ke mālama i ka LV i loko o ka trachea a hōʻemi i ka helu o nā ʻāpana vector i komo koke i loko o ka māmā. Hiki i kēlā mau hoʻomaikaʻi ʻana i ka pahuhopu ke alakaʻi i ka pono kiʻekiʻe aʻe me ka hōʻemi ʻana i nā titers i hāʻawi ʻia, ka transduction off-target, nā hopena ʻaoʻao mumū a me ka pale ʻana, 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 hoʻoili gene ʻē aʻe, me nā vectors viral ʻē aʻe (e like me AAV) a me nā waikawa nucleic.