组织蛋白酶B促进肿瘤坏死因子α诱导的血管平滑肌细胞凋亡  

Cathepsin B promoted the apoptosis of vascular smooth muscle cells as induced by tumor necrosis factor-α

作　　者：张涛[1] 羊镇宇[1] 薄小萍[1] 吴小庆[1] 李宗斌[1] 郭素侠[1] 

机构地区：[1]南京医科大学附属无锡市人民医院心脏中心,无锡214320

出　　处：《中华医学杂志》2011年第12期845-849,共5页National Medical Journal of China

摘　　要：目的 以肿瘤坏死因子α/放线菌素（TNF-α/act D）诱导细胞凋亡作为一种血管平滑肌细胞损伤模型,探讨组织蛋白酶B在TNF-α/act D诱导血管平滑肌细胞凋亡中的作用.方法 通过脂质体转染人主动脉平滑肌细胞株pcDNA3.1-组织蛋白酶B和pSilencer 2.1-组织蛋白酶B质粒,经过筛选鉴定建立组织蛋白酶B过表达组与基因沉默组,以TNF-α/act D诱导人主动脉平滑肌细胞凋亡,分别从细胞活性分析,形态学观察及凋亡蛋白检测细胞凋亡情况,并在组织蛋白酶B过表达细胞中加入组织蛋白酶抑制剂（E64d）和组织蛋白酶B特异性抑制剂（CA-074ME）,观察细胞活性改变.结果 噻唑蓝分析正常培养条件下组织蛋白酶B过表达组、基因沉默组细胞活性和对照组无明显差别;经TNF-α/act D诱导后细胞活性明显降低,与对照组（14.60%±1.34%）比较,过表达组（9.98%±1.04%）明显下降,加入E64d、CA-074ME组（18.23%±1.05%,17.40%±1.03%）较过表达组活性增加;基因沉默组（21.30%±2.37%）活性增加;吖啶橙/溴化乙啶染色分析得到相似结果;Western印迹Bcl-2蛋白表达,基因沉默组显著高于对照组,而过表达组水平明显低于对照组,E64d、CA-074ME组的水平均明显高于过表达组.Bax蛋白表达在各组间差异无统计学意义.结论 过表达组织蛋白酶B能够促进TNF-α诱导的血管平滑肌细胞凋亡,而组织蛋白酶B基因沉默以及加入组织蛋白酶抑制剂可以抑制TNF-α诱导的血管平滑肌细胞凋亡.Objective To establish the cathepsin B over-expression group and cathepsin B genesilencing group so as to investigate whether cathepsin B was capable of promoting the apoptosis of VSMC （vascular smooth muscle cell） induced by TNF-α/act D.Methods Human aortic smooth muscle cell （HA-VSMC） was transfected with pcDNA3.1-cathepsin B and pSilencer2.1-cathepsin B plasmids by lipofection to establish the over-expression and gene-silencing groups.Through TNF-α induced apoptosis,the cell viability was observed by MTT assay,morphological observation and assays of apoptotic proteins as indicators of apoptosis.Results The cathepsin B over-expression and gene-silencing group were successfully established.MTT assay showed no significant difference between the transfected cell and blank control.After the intervention of TNF-α,the HA-VSMC viability decreased significantly.As compared with control group,the over-expression group significantly decreased （9.98% ± 1.04 % vs 14.60% ± 1.34% ）.As compared with the over-expression group,the E64d and CA-074ME groups （18.23% ± 1.05%,17.40% ± 1.03% ） increased significantly while the silent group （ 21.30% ± 2.37% ） significantly increased.The analysis of acridine orange/ethidium bromide staining showed similar results.Western blot showed the Bcl-2 protein were significantly higher in the silent group than that in the control group.And the over-expression group was significantly lower than the control group.The E64d and CA-074ME groups were significantly higher than that in the over-expression group.But the Bax protein level had no significant difference among all groups.Conclusion The over-expression of cathepsin B promotes TNF-α-induced VSMC apoptosis while Cathepsin B gene silencing and the addition of cathepsin inhibitor in over-expression group inhibit the TNF-α induced VSMC apoptosis.

关 键 词：组织蛋白酶B 血管平滑肌细胞 细胞凋亡 肿瘤坏死因子Α 

分 类 号：R543.5[医药卫生—心血管疾病]