论文部分内容阅读
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。
OBJECTIVE: Mesenchymal stem cells (MSCs) have important immunomodulatory and tissue regeneration effects. However, their mechanisms of treating liver injury are not fully understood. They may mediate inflammation through paracrine factors and direct transdifferentiation into hepatocytes. In order to clarify these two mechanisms, it is proposed to construct a co-expression plasmid of cytomegalovirus (CMV) promoter-driven membrane localization green fluorescent protein (GFP) and albumin promoter-regulated PE40 toxin in order to kill the transfected MSC into hepatocytes Transdifferentiated cells. Methods GFP and DLL1 (Delta-like 1) transmembrane region gene fragments were obtained by PCR. The GFP gene was fused and inserted into pFlag-CMV-1 vector. The albumin gene promoter and PE40 gene fragment were inserted into the downstream of the constructed membrane localization GFP sequence to construct the eukaryotic expression vector, and the expression of GFP was detected by fluorescence microscopy. Normal human liver cells, which express albumin, were tested for their ability to kill hepatocytes. Results The hepatocyte-specific killing vector was successfully constructed. The vector has a killing effect on normal liver cells after transfection, suggesting that the vector can be used to explore the mechanism of liver injury treated by MSCs. Conclusion The successful construction of a carrier that can specifically kill hepatocytes.