热休克蛋白70对心肌细胞内质网应激半胱氨酰天冬氨酸特异性蛋白酶-12、C／EBP同源蛋白和c-Jun氨基末端激酶信号通路的影响

Effects of transfected heat shock protein 70 on cysteinyl aspartate - specific protease - 12, C/EBP homologous protein and c - Jun amino - terminal kinase signal pathway in endoplasmic reticulumstress

目的观察热休克蛋白70（HSP70）表达对心肌细胞内质网应激半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-12、C／EBP同源蛋白（CHOP）和c-Jun氨基末端激酶（JNK）信号通路的影响。方法应用腺病毒基因表达技术诱导热休克蛋白70（HSP70）基因在SD乳鼠心肌细胞的表达和应用HSP70反义寡核苷酸导人技术抑制HSP70的表达，建立细胞培养和心肌细胞缺氧模型，测定细胞凋亡，提取内质网测定，采用免疫组织化学和Westernblot方法测定转染前后HSP70的表达水平及HSP70的表达对内质网应激导致的细胞凋亡、Caspase-12、CHOP、JNK通路引起的凋亡信息传递的改变。结果对照组细胞凋亡率（9．28±0．83）％、晚期（28．60±1．83）％，HSP70基因转染组早期细胞凋亡率（4．30±0．72）％、晚期（11．6±1．66）％，HSP70反义寡核苷酸处理组早期细胞凋亡率（29．3±2．54）％、晚期（34．4±2．43）％，免疫组织化学和Westernblot证实HSP70基因成功转染人心肌细胞内表达；HSP70表达抑制细胞凋亡，但HSPT0反义寡核苷酸则促进；HSPT0显著抑制Caspase-12和CHOP的表达，HSP70增加JNK的表达；HSP70反义寡核苷酸组明显增加CHOP的表达。结论转染HSP70表达通过调节内质网应激Caspase-12、CHOP和JNK信号通路调控心肌细胞凋亡。

Objective To study the effect of heat shock protein 70 （HSP70） on cysteinyl aspar- tare - specific protease （Caspase） - 12, C/EBP homologous protein （CHOP） and c - Jun N - terminal kinase （JNK） signal pathway in the endoplasmic reticulum stress. Methods HSP70 gene was transfected into SD neonatal rat cardiomyocytes by lipofectin. We used SD neonatal rat cardiomyocytes to establish a hypoxia cell model, and then we further detected the role of HSP70 in cell apoptosis, in expression of Casepase - 12, CHOP, JNK pathway in ER caused by ER stress, by overexpression or downexpression of HSP70. Results The early apoptosis rate of the blank group （9. 28 ± 0. 83） %, late （28.60 ± 1.83） %, HSP70 gene transfection group early apoptosis rate （4. 30 ± 0. 72） %, late （ 11.6 ± 1.66） %, HSP70 treated with antisense oligonucleotides early apoptosis rate （29. 3 ± 2. 54 ）%, late （34. 4 ± 2. 43 ）%. Hypoxia-induced cell apoptosis was inhibited by HSP70, but was promoted by HSP70 anti -sense oligonucleotide. Hypoxia - induced ROS production was also inhibited by HSP70, but was not inhibited by HSP70 anti - sense oligonucleotide. HSP70 signifiantly inhibited the expression of Caspase - 12 and CHOP. HSP70 increased the expression of JNK. HSP70 anti - sense oligonucleotide significantly increased the expression of CHOP,but did not signifiantly inflent other tested molecules. Conclusion This study demonstrated that HSPT0 reduce myocardial cell apoptosis by regulating the Caspase - 12, C/EBP homologous protein and c -Jun aminoterminal kinase signal pathway of endoplasmic reticulum.