肝细胞特异性杀伤载体的构建和应用

发布时间：2019-04-29 11:40

【摘要】：目的间充质干细胞(MSC)有重要的免疫调节和组织再生作用,但其治疗肝损伤的机制尚不完全清楚,既可能通过旁分泌因子调节炎症,也可直接转分化为肝细胞。为明确这两种机制,拟构建巨细胞病毒(CMV)启动子驱动的膜定位绿色荧光蛋白(GFP)与白蛋白启动子调控的PE40毒素的共表达质粒,以期杀伤转染MSC中向肝细胞转分化的细胞。方法用PCR获得GFP以及DLL1(Delta-like 1)跨膜区基因片段,融合成膜定位GFP基因并插入pFlag-CMV-1载体。将白蛋白基因启动子和PE40基因片段插入已构建好的膜定位GFP序列下游,构建真核表达载体,荧光显微镜检测GFP的表达。应用可表达白蛋白的人正常肝脏细胞检测该载体对肝细胞的杀伤。结果成功构建肝细胞特异性杀伤载体,该载体转染后对正常肝脏细胞具有杀伤作用,提示该载体可用于探索MSC治疗肝损伤的机制。结论成功构建能特异性杀伤肝细胞的载体。
[Abstract]:Aim Mesenchymal stem cells (MSC) play an important role in immunomodulation and tissue regeneration, but the mechanism of its treatment for liver injury is still unclear. It is possible to regulate inflammation through paracrine factors and to differentiate directly into hepatocytes. In order to clarify these two mechanisms, the co-expression plasmid of cytomegalovirus (CMV) promoter-driven green fluorescent protein (GFP) and albumin promoter-regulated PE40 toxin was constructed in order to kill and transfect the cells transformed into hepatocytes in MSC. Methods the transmembrane gene fragments of GFP and DLL1 (Delta-like-1) were obtained by PCR, and the GFP gene was fused into a membrane to locate the GFP gene and inserted into the pFlag-CMV-1 vector. The albumin gene promoter and PE40 gene fragment were inserted into the downstream of the constructed membrane-located GFP sequence to construct the eukaryotic expression vector. The expression of GFP was detected by fluorescence microscope. Human normal liver cells expressing albumin were used to detect the killing effect of the vector on hepatocytes. Results Hepatocyte-specific killing vector was successfully constructed. After transfection, the vector could kill normal liver cells, suggesting that the vector could be used to explore the mechanism of MSC in the treatment of liver injury. Conclusion the vector which can kill hepatocytes specifically was constructed successfully.
【作者单位】：第四军医大学基础部医学遗传与发育生物学教研室;
【基金】：国家自然科学基金重大研究计划培育项目(91029731);国家自然科学基金重点项目(81030010)
【分类号】：R575

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