高糖条件下p58促胰岛β细胞凋亡的机制

Molecular mechanism of β cell apoptosis induced by p58 in high glucose medium

2型糖尿病是一种具有明显遗传倾向的复杂的多基因疾病。CDC2L2是中国北方汉族人2型糖尿病的一个易感基因。目前,该基因与2型糖尿病发生发展的关系尚不清楚。本文针对CDC2L2编码的蛋白p58与胰岛β细胞凋亡的关系及其作用的分子机制进行了研究。INS-1(大鼠胰岛β细胞瘤细胞株)高糖浓度(20mmol/L)下培养,分为三个组:空白对照组、空载体对照组(转染载体pcDNA3.0)和实验组(转染质粒pcDNA3.0-HA-p58),Annexin V-FITC/PI双染法检测胰岛β细胞的凋亡状况。结果显示,在高糖培养条件下,p58高表达组INS-1细胞的凋亡率显著高于空白对照组和空载体对照组(P<0.01,P<0.05)。Western blot检测显示,与两对照组相比,p58高表达组的INS-1细胞中Caspase-3和Bax的表达水平明显上升(P<0.05,P<0.01),胞浆内CytoC的含量显著升高(P<0.01),Bcl-2的表达水平显著下降(P<0.05)。以上结果提示,p58在高糖条件下可能通过上调Bax和下调Bcl-2的表达,使线粒体外膜的通透性增加,从而引起CytoC由线粒体向胞浆的释放,最终活化Caspase-3,引起INS-1细胞凋亡。本实验为进一步研究CDC2L2与胰岛β细胞凋亡的关系及其分子作用机制奠定了重要的实验基础。

Type 2 diabetes is a complex disorder with a strong genetic background. CDC2L2 is one of the susceptibility genes of type 2 diabetes in Chinese Han population in northern area. The relationship between CDC2L2 and type 2 diabetes remains unknown. In this paper, the function and its molecular pathway of p58, a protein coded by CDC2L2, in β cell apoptosis were investigated. INS-1 cells cultured in high glucose （20 mmol/L） medium were divided into control, vector control （transfected with pcDNA3.0） and experimental （transfected with pcDNA3.0-HA-p58） groups. Beta cell apoptosis level was detected by Annexin V-FITC/PI double staining assay. The flow cytometry results showed that in high glucose medium （20 mmol/L）, high expression of p58 increased β cell apoptosis significantly compared with that in blank and vector controls （P〈0.01, P〈0.05）. Western blot revealed that the expressions of Caspase-3, Bax and cytochrome C in cytoplasm increased significantly （P〈0.05, P〈0.01, P〈0.01）, whereas the expression of Bcl-2 decreased significantly （P〈 0.05） in the INS-1 cells with high expression of p58, compared with those in both control groups. However, the Bad and Bik expression levels of INS-1 cells did not show obviously changes compared with those in both controls. The above results suggest that in high glucose condition, p58 may induce INS-1 cell apoptosis through up-regulating the expression of Bax and down-regulating the expression of Bcl-2, since both of them could promote the release of cytochrome C into cytoplasm, and finally activate Caspase-3. These results provide an important basis for the further exploration of the molecular mechanism of β cell apoptosis induced by CDC2L2.