2型糖尿病患者Caveolin-3基因K15N突变蛋白分析及功能研究

发布时间：2018-03-02 09:33

本文关键词： Caveolin-3 2型糖尿病基因突变糖代谢 GLUT-4　出处：《广西医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：[目的]2型糖尿病(Type 2 diabetes mellitus, T2DM)的遗传背景与基因多态性密切相关。Caveolin-3 (Cav-3)是肌肉特异性蛋白,它可以调节胰岛素信号通路。课题组前期研究发现T2DM病人Cav-3基因外显子存在多处变异位点。为了观察Cav-3基因突变是否影响胰岛素受体信号途径,改变骨骼肌细胞糖代谢,从而参与T2DM的发病机制,我们将Cav-3 K15N突变基因导入C2C12细胞,对其表达的突变蛋白进行相关功能研究。[方法]利用生物信息学预测Cav-3第一外显子K15N (KU971296)、 D16H和N33N三个突变点蛋白质的二级结构,然后选择二级结构改变明显的K15N突变进行研究。分别构建阴性对照组、野生型组以及Cav-3 K15N突变组表达载体转染C2C12细胞。G418筛选后,胰岛刺激30min,运用Western Blot技术和IF技术鉴定Cav-3、AKT、pAKT以及GLUT-4等蛋白的表达情况；在12h、24h测定C2C12肌细胞对葡萄糖的摄取量和36h细胞内糖原合成量。[结果]1.分析发现K15N、D16H属于错义突变,N33N属于同义突变。生物信息学预测Cav-3蛋白质二级无规则卷曲结构变为螺旋结构的有K15N和D16H两个位点。2.经400μg/ml的G418筛选后,得到稳定表达的细胞系。Westernblot鉴定证实筛选成功,组间转染效率没有统计学差异。3.与野生型组相比,K15N突变使骨骼肌细胞Cav-3蛋白总体表达降低,而且富集在细胞核周围,细胞膜分布减少。其下游信号分子AKT磷酸化减少,但总AKT蛋白表达水平无明显变化。细胞GLUT-4蛋白不仅总体表达水平下降,而且在细胞膜上聚集成团。4.Cav-3 K15N突变蛋白导致C2C12细胞对葡萄糖摄取减少。在12h,K15N突变组与阴性对照组和野生型组相比,p值分别等于0.042和0.000；在24h,K15N突变组与野生型组比较p=0.010,有显著的统计学差异,但是与阴性对照组相比p=0.366,没有统计学意义。在36小时K15N突变组细胞糖原合成减少(p0.050)。[结论]Cav-3 K15N突变导致C2C12细胞膜Cav-3表达减少,影响胰岛素信号通路上AKT磷酸化和GLUT-4蛋白的转位,导致骨骼肌细胞葡萄糖摄取障碍,糖原合成减少。我们推测骨骼肌细胞利用葡萄糖减少可能会引起高血糖,是促使T2DM发生发展的机制之一。
[Abstract]:[objective] the genetic background of type 2 diabetes mellitus type 2 diabetes mellitus (T2DM) is closely related to gene polymorphism. Caveolin-3 Cav-3) is a muscle-specific protein. In order to observe whether the mutation of Cav-3 gene affects insulin receptor signaling pathway and changes glucose metabolism in skeletal muscle cells, we found that there are many mutation sites in the exon of Cav-3 gene in T2DM patients. In order to participate in the pathogenesis of T2DM, we introduced the Cav-3 K15N mutation gene into C2C12 cells. [methods] Bioinformatics was used to predict the secondary structures of three mutant proteins, K15N, KU971296, D16H and N33N, in the first exon of Cav-3. Then the K15N mutation with obvious changes in secondary structure was selected to construct negative control group, wild-type group and Cav-3 K15N mutant expression vector transfected into C2C12 cells. G418 was used to screen the expression vector. After 30 minutes of islet stimulation, Western Blot and if techniques were used to identify the expression of Cav-3AK Blot and GLUT-4 protein. The glucose uptake of C2C12 myocytes and the intracellular glycogen synthesis in C2C12 cells were determined at 12h and 24h. [results] 1. It was found that K15N D16H belongs to synonymous mutation. Bioinformatics predicts the secondary irregular convolution structural change of Cav-3 protein. They were K15N and D16H loci with helical structure. After screening with 400 渭 g / ml G418, The stable expressed cell line. Western blot confirmed that the selection was successful, and the transfection efficiency was not significantly different between the two groups. Compared with the wild type group, K15N mutation decreased the overall expression of Cav-3 protein in skeletal muscle cells and enriched around the nucleus. The distribution of cell membrane decreased, the phosphorylation of downstream signal molecule AKT decreased, but the expression level of total AKT protein did not change significantly. The expression level of GLUT-4 protein not only decreased, but also decreased. Cav-3 K15N mutant protein decreased glucose uptake of C2C12 cells at 12h, which was equal to 0.042 and 0.000 in 12h K15N mutation group compared with negative control group and wild type group, respectively, and compared with wild type group at 24 h after Cav-3 K15N mutation. There was a significant statistical difference between the two groups. However, compared with the negative control group, there was no statistical significance. The glycogen synthesis in the K15N mutant group decreased p0.050 at 36 hours. [conclusion] the Cav-3 K15N mutation resulted in the decrease of Cav-3 expression on the C2C12 cell membrane, which affected the phosphorylation of AKT and the translocation of GLUT-4 protein in the insulin signaling pathway. We speculate that the decrease in glucose utilization by skeletal muscle cells may lead to hyperglycemia, which is one of the mechanisms that promote the development of T2DM.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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