本文選題：腺病毒　切入點(diǎn)：骨形態(tài)發(fā)生蛋白　出處：《中國骨質(zhì)疏松雜志》2014年08期 　論文類型：期刊論文

【摘要】：目的用腺病毒載體介導(dǎo)人骨形態(tài)發(fā)生蛋白-2及EGFP基因轉(zhuǎn)染兔骨髓基質(zhì)干細(xì)胞(rBMSC),種植DBM(脫鈣骨)支架體外構(gòu)建組織工程骨。方法兔髓基質(zhì)干細(xì)胞(rBMSC)的分離、培養(yǎng);流式細(xì)胞儀檢測細(xì)胞表面標(biāo)記;轉(zhuǎn)染后,熒光顯微鏡觀察細(xì)胞最適感染復(fù)數(shù)(MOI)及蛋白印跡檢測外源基因的表達(dá)情況;采用Urist提供的方法制備脫鈣骨(DBM),用細(xì)胞計(jì)量法測定BMSCs與DBM復(fù)合培養(yǎng)的黏附率。然后將轉(zhuǎn)染后細(xì)胞接種到DBM支架上,用熒光顯微鏡觀察細(xì)胞是否成功粘附于支架材料上,掃描電鏡觀察細(xì)胞貼附、生長狀況。結(jié)果 BMSCs細(xì)胞表型鑒定:CD44表達(dá)陽性,CD45表達(dá)陰性;轉(zhuǎn)染后,蛋白印跡試驗(yàn)檢測到BMP-2表達(dá)增高;骨髓間充質(zhì)干細(xì)胞與脫鈣骨基質(zhì)的平均粘附率為(72.74±1.99)%;掃描電鏡見轉(zhuǎn)染細(xì)胞生長良好,伸出絲狀突起,相互連接。結(jié)論成功培養(yǎng)及鑒定兔BMSCs,Ad-BMP-2/EGFP可高效轉(zhuǎn)染兔BMSCs,轉(zhuǎn)染后的細(xì)胞種植于DBM支架材料后生長狀況良好,組織工程骨構(gòu)建成功。
[Abstract]:Objective to transfect human bone morphogenetic protein-2 and EGFP gene into rabbit bone marrow stromal stem cells (BMSCs) by adenovirus vector, and to construct tissue engineered bone with DBM (decalcified bone) scaffold in vitro. Methods Rabbits bone marrow stromal cells were isolated and cultured. After transfection, the expression of exogenous genes was detected by fluorescence microscopy and Western blotting. The method provided by Urist was used to prepare decalcified DBM. The adhesion rate of BMSCs and DBM co-cultured was measured by cell metrology. Then the transfected cells were inoculated into DBM scaffold, and fluorescence microscope was used to observe whether the cells adhered to the scaffold material successfully. Results BMSCs cell phenotypic identification showed that the positive expression of CD44 was negative, and the expression of BMP-2 was increased by Western blotting after transfection. The average adhesion rate of bone marrow mesenchymal stem cells to decalcified bone matrix was 72.74 鹵1.99%. Conclusion BMSCs can be transfected with Ad-BMP-2 / EGFP successfully. The transfected cells grow well after implantation on DBM scaffold, and the tissue engineering bone is successfully constructed.
【作者單位】： 桂林醫(yī)學(xué)院;桂林醫(yī)學(xué)院附屬醫(yī)院;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(31160199)
【分類號】：R318

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