Tcf7l2基因DNA甲基化对胰岛β细胞功能的影响及其机制的研究

The reason why there is a progressive deterioration in pancreatic beta-cell (β-cell) function induced by glucolipotoxicity in type 2 diabetes remains poorly understood. In our previous study, RIN-m5F cells were cultured in high-glucose and high-fat medium, an abnormal DNA methylation in the promoter region (CpG island) of Tcf7l2 gene has been found, and there was a contradictory between transcription and translation in Tcf7l2 gene. The results suggested that the interaction between environment and gene might play an important role in deterioration of β-cell function. We plan to construct the reporter gene plasmids with site-specific DNA methylation in Tcf7l2 gene promoter (CpG island), these plasmids will be transfected into NIT-1 cells. We will study the effects of the specific DNA methylation in Tcf7l2 gene on β-cell function and TCF7L2 splice variants expression in this transfected cells. In order to understand the mechanism of the deterioration of β-cell function induced by glucolipotoxicity, we plan to find the concentration of glucose and free acid which is needed to cause DNA methlyation of Tcf7l2 gene and study the effects of the DNA methlyation inhibitor on DNA methlyation of Tcf7l2 gene and Tcf7l2 splice variants expression induced by high-glucose and -fat. The effects of DNA methlyation in Tcf7l2 gene on the β-cells function will also be studied in experiment animal models. It might be a original study, and helpful to provide a new method in prevention or treatment of type 2 diabetes.

糖脂毒性引起2型糖尿病胰岛β细胞功能进行性减退的机制目前仍不清楚。我们前期研究发现，高糖+高脂会引起胰岛β细胞Tcf7l2基因启动子区域CpG岛异常甲基化，Tcf7l2基因转录和翻译出现分离，提示环境与遗传相互作用可能在胰岛β细胞功能减退中起重要作用。本研究拟构建Tcf7l2基因启动子特异性甲基化重组质粒，转染NIT-1胰岛细胞，研究Tcf7l2基因特异性甲基化对胰岛β细胞功能、Tcf7l2基因转录产物剪切的影响；确定引起Tcf7l2基因DNA甲基化的高糖、高脂条件，研究甲基化抑制剂对Tcf7l2基因DNA甲基化、Tcf7l2基因转录产物剪切的作用，探讨糖脂毒性的分子机制；在实验动物水平进一步研究Tcf7l2基因DNA甲基化对胰岛β细胞功能的影响及其机制。对该基因甲基化的研究具有源头创新性，有助于阐明糖脂毒性对胰岛β细胞影响的分子机制，有可能为2型糖尿病的防治提供新的方法。

TCF7L2 (transcription factor 7 like 2) 与2型糖尿病关联度最高的基因之一，但许多没有携带TCF7L2突变位点的人仍然会得2型糖尿病。我们主要研究了高脂饮食导致的Tcf7l2基因DNA甲基化和基因表达水平改变。我们用高脂饮食喂养C57BL/6小鼠8周后进行腹腔葡萄糖耐量试验，并分离小鼠胰岛进行亚硫酸盐PCR测序检测Tcf7l2基因DNA甲基化水平。我们还将克隆的Tcf7l2基因DNA片段用甲基转移酶预处理，使其异常甲基化，然后通过基因重组技术转染到MIN-6细胞当中，来验证Tcf7l2的异常甲基化对基因表达及胰岛β细胞功能的影响。我们发现高脂喂养的小鼠胰岛Tcf7l2基因启动子转录起始位点上游165bp处的CpG岛出现异常甲基化，同时Tcf7l2基因mRNA和蛋白表达均下降。我们还发现在MIN-6细胞中Tcf7l2基因甲基化抑制了基因表达和MIN-6细胞生存和分泌胰岛素的能力下降。我们的研究发现了告知饮食能导致小鼠胰岛Tcf7l2基因异常甲基化并导致其基因表达异常。这一发现为营养摄入和基因表达之间的相互关系提供了理论依据，对不良饮食导致2型糖尿病发生的发病机制进行了深入探讨，为2型糖尿病的防治提供了新的思路。