本文選題：脫細(xì)胞豬角膜基質(zhì) + 載體支架��； 參考：《山東大學(xué)學(xué)報(bào)(理學(xué)版)》2017年05期

【摘要】：為了獲得基于異種角膜材料的理想組織工程角膜載體支架,首次建立了脫氧膽酸鈉(SD)和原釩酸鈉(SO)聯(lián)合處理的技術(shù)方法,利用新鮮豬角膜進(jìn)行了脫細(xì)胞角膜基質(zhì)(aPCS)的制備及其鑒定研究。用角膜板層刀從新鮮豬角膜中切下厚度450μm的角膜片,分別選用SD聯(lián)合SO、十二烷基磺酸鈉(SDS)和Triton X-100的去細(xì)胞處理方法制備出SD-aPCS、SDS-aPCS和Triton-aPCS共3種支架,利用外觀照相、分光光度計(jì)、石蠟切片蘇木紫-伊紅(HE)染色、冰凍切片DAPI和阿利新藍(lán)染色檢測了其理化性質(zhì)和組織結(jié)構(gòu);對SD-aPCS進(jìn)行掃描和透射電鏡鑒定后,進(jìn)而利用噻唑藍(lán)(MTT)、石蠟切片HE染色、冰凍切片DiI熒光觀察以及免疫熒光細(xì)胞化學(xué)染色評估了其對非轉(zhuǎn)染人角膜基質(zhì)(ntH CS)細(xì)胞的毒性與生物相容性。檢測結(jié)果發(fā)現(xiàn),3種aPCS的細(xì)胞脫除干凈且在干重和含水量上沒有顯著差異;其中,SD-aPCS的透明性與糖胺聚糖(GAG)含量最高、SDS-aPCS次之、Triton-aPCS最低;除Triton-aPCS的組織結(jié)構(gòu)出現(xiàn)了明顯的紊亂外,SD-aPCS和SDS-aPCS的組織結(jié)構(gòu)均排列規(guī)則。在電鏡下,SD-aPCS的前彈力層表面平整、無裂痕,板層結(jié)構(gòu)和膠原纖維超微結(jié)構(gòu)正常;此外,SD-aPCS浸提液對ntH CS細(xì)胞沒有毒性作用,注射接種到SD-aPCS支架內(nèi)的ntH CS細(xì)胞與支架嵌合緊密,隨體外培養(yǎng)時(shí)間的延長而逐漸伸展和遷移,且細(xì)胞仍保持有其固有標(biāo)志蛋白—波形蛋白,細(xì)胞連接蛋白—間隙連接蛋白-43和整聯(lián)蛋白,以及膜運(yùn)輸?shù)鞍住c鉀泵的陽性表達(dá)。由此可見,利用SD聯(lián)合SO的方法所制備SD-aPCS具有理想的理化性質(zhì)、組織結(jié)構(gòu)和生物相容性,可作為一種理想的載體支架用于組織工程角膜的體外構(gòu)建及其相關(guān)應(yīng)用研究。
[Abstract]:In order to obtain an ideal scaffold for corneal tissue engineering based on xenogeneic corneal materials, a new method of combined treatment of sodium deoxycholate and sodium orthovanadate (SOO) was established for the first time. The preparation and identification of acellular corneal stroma (APCS) from fresh porcine cornea were studied. Using lamellar knife to cut 450 渭 m corneal slices from fresh porcine cornea, SD combined with SO, SDS and Triton X-100 were used to prepare SD-aPCS SDS-aPCS and Triton-aPCS scaffolds, respectively. SD-aPCS and Triton-aPCS were used to produce SD-aPCS and Triton-aPCS respectively. The physicochemical properties and histological structure of paraffin sections were examined by DAPI and Alisin blue staining, and then the SD-aPCS were identified by scanning and transmission electron microscopy, and then the paraffin sections were stained with thiazolyl blue (MTT) and paraffin sections by HE staining. The toxicity and biocompatibility of frozen sections of human corneal stromal cells were evaluated by fluorescence observation and immunofluorescence cytochemical staining. The results showed that the cells of the three kinds of APCS were removed clean and had no significant difference in dry weight and water content, the transparency of SD-aPCS and the content of glycosaminoglycan (GAG) were the highest, and the content of SDS-aPCS was the lowest, and that of Triton-aPCS was the lowest. The organization structure of SD-aPCS and SDS-aPCS were arranged regularly except for the obvious disorder in the organizational structure of Triton-aPCS. Under electron microscope, the surface of the anterior elastic layer of SD-aPCS was flat, without cracks, the lamellar structure and the ultrastructure of collagen fibers were normal, in addition, the extract of SD-aPCS had no toxic effect on NTH CS cells, and the NTH CS cells injected into SD-aPCS scaffold were closely associated with the scaffold chimerism. With the extension of culture time in vitro, the cells gradually extended and migrated, and the cells still maintained the positive expression of vimentin, connexin 43 and integrin, as well as membrane transport protein-sodium potassium pump. It can be seen that SD-aPCS prepared by the method of SD combined with so has ideal physicochemical properties, tissue structure and biocompatibility, and can be used as an ideal carrier scaffold for in vitro construction of tissue engineered cornea and its related applications.
【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(diǎn)實(shí)驗(yàn)室;
【基金】：國家高技術(shù)研究發(fā)展計(jì)劃(863計(jì)劃)資助項(xiàng)目(2006AA02A132)
【分類號】：R318.08;R779.65

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