【摘要】：為了評價深海魚類膠原蛋白用于制備組織工程角膜載體支架的可行性,首次利用魚皮膠原蛋白進行了三維膠原支架的制備及其生物相容性研究。用源于太平洋鱈魚(Gadus macrocephalus)的純化魚皮膠原蛋白,經(jīng)冷凍干燥后選用1-乙基-3-(3-二甲基氨基丙基)碳二亞胺(EDC)/N-羥基丁二酰亞胺(NHS)交聯(lián)劑制備出三維膠原支架,利用外觀照相、光度計透光率、冰凍切片蘇木紫-伊紅(HE)染色及掃描電鏡檢測了其透明度和組織結(jié)構(gòu);在接種Di I(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)熒光標(biāo)記的非轉(zhuǎn)染人角膜基質(zhì)(HCS)細(xì)胞后,利用冰凍切片HE染色和熒光觀察鑒定了細(xì)胞的遷入及在支架內(nèi)的分布情況,生物相容性評價采用MTT(噻唑藍(lán))和免疫細(xì)胞熒光技術(shù)檢測了支架對HCS細(xì)胞活力和標(biāo)志蛋白及功能蛋白表達(dá)的影響。結(jié)果顯示,所制備的三維膠原支架透明度極高,呈現(xiàn)出均勻的網(wǎng)格狀結(jié)構(gòu),孔徑大小為50~130μm;接種HCS細(xì)胞并體外培養(yǎng)3 d后,發(fā)現(xiàn)有大量細(xì)胞遷入支架內(nèi)且分布較為均勻;支架抽提物對HCS細(xì)胞活力沒有任何不良影響;且遷入支架內(nèi)的細(xì)胞仍保持有其標(biāo)志蛋白—波形蛋白,細(xì)胞連接形成蛋白—整聯(lián)蛋白β1、E-鈣黏蛋白和間隙連接蛋白-43,膜運輸?shù)鞍住c鉀泵,以及抗UV蛋白—乙醛脫氫酶的陽性表達(dá)。綜上所述,利用太平洋鱈魚皮膠原蛋白制備的三維膠原支架具有良好的透明度與組織結(jié)構(gòu),不僅適合HCS細(xì)胞的順利遷入,而且對HCS細(xì)胞具有良好的生物相容性,有望作為載體支架用于組織工程角膜的體外構(gòu)建研究。
[Abstract]:In order to evaluate the feasibility of deep-sea fish collagen used to prepare tissue engineering corneal carrier scaffold, the three-dimensional collagen scaffold was prepared by using fish skin collagen for the first time and its biocompatibility was studied. Purified fish skin collagen derived from Pacific cod (Gadus macrocephalus), Three-dimensional collagen scaffolds were prepared by using 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) crosslinking agent after freeze-drying. The transparency and structure of frozen section were examined by (HE) staining and scanning electron microscopy. After inoculated with Di I (1 ~ (-1) -dioctadecyl-3 ~ (3 +) -tetramethylindocarbocyanine perchlorate, the migration and distribution of human corneal stromal (HCS) cells were identified by HE staining and fluorescence observation in frozen sections. Biocompatibility was evaluated by MTT (thiazolyl blue) and immunofluorescence technique. The effects of stents on the viability and expression of marker proteins and functional proteins in HCS cells were investigated. The results showed that the three-dimensional collagen scaffold was highly transparent, with a uniform reticular structure with a pore size of 50 ~ 130 渭 m. After inoculating HCS cells and cultured in vitro for 3 days, a large number of cells were found to migrate into the scaffold and distributed evenly. The scaffold extract had no adverse effect on the viability of HCS cells. The cells that migrated into the scaffold still had their marker protein vimentin, cell junction protein-integrin 尾 1-E-cadherin and gap junction protein-43, membrane transport protein-sodium potassium pump. And the positive expression of anti UV protein-acetaldehyde dehydrogenase. In conclusion, the three-dimensional collagen scaffold prepared from Pacific cod skin collagen has good transparency and tissue structure, which is not only suitable for the smooth migration of HCS cells, but also has good biocompatibility for HCS cells. It is expected to be used as carrier scaffold for in vitro construction of tissue engineered cornea.
【作者單位】： 中國海洋大學(xué)海洋生命學(xué)院角膜組織工程重點實驗室;
【基金】：國家高技術(shù)研究發(fā)展計劃(863計劃)資助項目(2006AA02A132)
【分類號】：R318.08

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