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为了构建猪胰岛素启动子(IP)-绿色荧光蛋白(GFP)报告系统,评价其在胰岛间充质干细胞(PIMSCs)诱导分化过程中的应用价值,试验以胎猪胰腺组织基因组DNA为模板,PCR扩增获得IP基因片段,T载体连接测序后,构建p EGFP-IP重组质粒,经PCR和双酶切鉴定后,分别转染原代胎猪胰腺细胞和PIMSCs,48 h后观察荧光表达情况,转染重组质粒的PIMSCs体外诱导2周后,观察绿色荧光表达情况,并进行GFP和胰岛素的Western-blot检测。结果表明:PCR扩增得到1条659 bp大小的基因片段,其测序结果与Gen Bank公布的IP基因序列一致。经PCR和双酶切鉴定,p EGFPIP重组质粒构建成功。重组质粒转染48 h后,部分原代胎猪胰腺细胞强表达绿色荧光,而PIMSCs则不表达。诱导2周后,部分PIMSCs表达绿色荧光,同时随着绿色荧光的表达增强,其胰岛素表达也相应增强。说明成功构建出了猪胰岛素启动子-绿色荧光蛋白报告系统,该报告系统具有表达特异性,可用于检测由PIMSCs诱导分化得到的胰岛素分泌细胞。
In order to construct a porcine insulin (IP) -green fluorescent protein (GFP) reporter system and evaluate its application in the induction and differentiation of islet mesenchymal stem cells (PIMSCs), the genomic DNA of the pancreas was used as a template and PCR After the T vector was ligated and sequenced, the recombinant plasmid of p EGFP-IP was constructed. After PCR and double enzyme digestion, the primary cultured fetal porcine pancreatic cells and PIMSCs were transfected with pEGFP-IP, and the expression of pEGFP-IP was observed 48 h later. After two weeks of in vitro induction of PIMSCs transfected with the recombinant plasmids, the expression of green fluorescence was observed, and the detection of GFP and insulin by Western-blot. The results showed that a 659 bp gene fragment was amplified by PCR and its sequencing result was consistent with that of GenBank. The recombinant plasmid p EGFPIP was successfully constructed by PCR and double enzyme digestion. 48 h after transfection, some primary porcine pancreatic cells strongly expressed green fluorescence, but not PIMSCs. After two weeks of induction, some of the PIMSCs expressed green fluorescence, and at the same time, the expression of green fluorescence increased, and the expression of insulin increased correspondingly. This indicated that the porcine insulin promoter-green fluorescent protein reporter system was successfully constructed. The reporter system has the specificity of expression and can be used to detect insulin-secreting cells induced by PIMSCs.