高效Pdx1/insulin双报告基因载体的构建及其初步应用

Construction of a high efficient pancreatic duodenal homeobox 1/insulin dual-reporter vector and its preliminary application

背景:胰岛细胞移植是治疗1型糖尿病最有效的方法之一,然而移植细胞来源短缺限制了其临床应用。多能干细胞有望成为胰岛细胞移植的理想种子细胞,但其向胰岛β细胞分化是一个复杂的过程。目的:构建高效Pdx1/insulin双报告基因载体,探讨其实时监测诱导多能干细胞向胰岛β细胞分化中关键性基因表达的可行性。方法:首先在pT iger载体上引入嘌呤霉素抗性,然后以646 bp的小鼠Ins1启动子替换了原来载体上410 bp的小鼠Ins1启动子,获得高效Pdx1/insulin双报告基因载体。最后,将载体导入INS-1细胞和诱导多能干细胞,验证其功能。结果与结论:(1)实验成功构建了高效Pdx1/insulin双报告基因载体;(2)在INS-1细胞中验证了载体获得嘌呤霉素抗性,提高了insulin表达效率,并在INS-1细胞中检测到Pdx1和insulin的表达;(3)实验初步证明了Pdx1/insulin双报告基因能够有效监测细胞分化过程中Pdx1和insulin基因的表达。

BACKGROUND：Islet beta cell replacement therapy is one of the most promising approaches for treating type 1 diabetes mellitus. However, its large scale application is hampered by a shortage of islet beta cells for transplantation. Pluripotent stem cells are one of ideal seed cells for islet beta cell replacement therapy, but pancreatic beta-cell differentiation is time-consuming and labor-intensive. OBJECTIVE：To construct a high efficient pancreatic and duodenal homeobox 1 （Pdx1）/insulin dual-reporter vector and to monitor the key genes expression during pancreatic beta-cell differentiation from pluripotent stem cells. METHODS：In order to construct a high efficient Pdx1/insulin dual-reporter vector, puromycin resistance gene was firstly introduced into pTiger vector, and then the original 410 bp mouse Ins1 promoter of the vector was replaced by 646 bp mouse Ins1 promoter. Finally, the dual-reporter vector was transduced into INS-1 and human induced pluripotent stem cells to testify its function. RESULTS AND CONCLUSION：The high efficient Pdx1/insulin dual-reporter vector was constructed successfully. The vector successfully acquired puromycin resistance gene and high gene expression efficacy of insulin in INS-1 cells. The specific gene expression pattern of Pdx1/insulin was first found in INS-1 cells. To conclude, the real-time monitoring function of Pdx1/insulin expression is preliminarily confirmed during pancreatic beta-cell differentiation.