热休克蛋白70在缺糖损伤的HeLa细胞中对Bax和Bcl-2以及Bax构象改变的影响

Effect of heat shock protein 70 on the expression of Bax and Bcl-2 and the conformational change of Bax in glucose deprived HeLa cells

目的观察热休克蛋白70（Hsp70）对缺糖引起的HeLa细胞凋亡的影响,探讨其与凋亡相关的Bcl-2家族成员的相互作用关系。方法用人正义Hsp70重组质粒稳定转染HeLa细胞获得Hsp70过表达细胞。采用Hsp70过表达和正常HeLa细胞无糖培养建立两组细胞损伤模型,并均取3个平行样本。四甲基偶氮唑盐（MTT）法测细胞活率;Hoechst染色法和Giemsa染色法检测细胞凋亡率;RT-PCR法检测Bax和Bcl-2的表达变化,并计算其灰度比值;细胞免疫化学和荧光免疫法检测Bax构象改变情况。结果缺糖48h内,Hsp70明显抑制了HeLa细胞凋亡并增强其活力,同时也抑制了凋亡细胞中Bax/Bcl-2的增高以及Bax的构象改变。结论缺糖诱导下,热休克蛋白70在HeLa细胞中能够通过与Bcl-2家族成员作用而抑制凋亡的发生。

Objective To observe the effect of heat shock protein 70 （Hsp70） on apoptosis of HeLa cells induced by glucose deprivation and investigate the relationship between Hsp70 and the key proteins of apoptosis： Bcl-2 family members. Methods HeLa cells were stably transfected with the plasmid of pcDNA3.1 （ ＋ ）-Hsp70 to establish the Hsp70 over-expressed cell model; Hsp70 normal-expression and over-expression ceils cultured with glucose free medium to create stress models. Every detection had three parallel samples. MTT assay was applied to evaluate the cell viability; Hoechst 33258 and Giemsa stain were used to examine the rate of cell apoptosis. RT-PCR was used to determine the expression level of Bax and Bel-2 and to calculate the ratio of Bax/Bcl-2; Immunofluorescence and immunocytochemistry were applied to examine the conformational change of Bax. Results Cell viability was increased and the rate of cell apoptosis was decreased in Hsp70 over-expression cells cultured with glucose free medium. At the same time, the expression of Bax and Bcl-2 and the conformational change of Bax were also inhibited by Hsp70. Conclusion Hsp70 blocks glucose- deprivation-induced apoptosis primarily by inhibiting the up-regulation of Bax/Bcl- 2 and the conformational change of Bax.