南京大学学报(自然科学版) ›› 2012, Vol. 48 ›› Issue (6): 809–816.
• • 上一篇
张涛，羊镇宇**，薄小萍，王强，陈茂华，李坚，郭素峡，冯健，尤华彦
Zhang Tao，Yang Zhen-Yu，Bo Xiao-Ping，Wang Qiang，Chen Mao-Hua，Li Jian，
Guo Su-Xia，Feng Jian，You Hua-Yan
摘要： 摘要:构建并转染pcDNA3. 1-cathcpsin B和pSilcncer2.l-cathcpsin B质粒，建立组织蛋白酶B过表
达细胞株与基因沉默细胞株，初步探讨组织蛋白酶B对血管平滑肌细胞凋亡的作用.通过RT-PCR法扩
增获得组织蛋白酶Y全基因序列并构建pcDNA3. 1-cathcpsin B表达质粒，同时设计针对组织蛋白酶B
靶序列的siRNA，按shRNA表达质粒的克降策略，设计针对该序列的双链DNA序列并构建pSilcncer
2. 1-cathcpsin B质粒，通过脂质体转染人主动脉平滑肌细胞(HA-VSMC)，经过筛选鉴定建立组织蛋白
酶B过表达株与基因沉默株.通过TNF-α/actD(放线菌素D)诱导细胞凋亡，从细胞活性分析、流式细胞
术观察组织蛋白酶H对血管平滑肌细胞的作用，并加入组织蛋白酶抑制剂(E64d)和组织蛋白酶H特异
性抑制剂(CA-074ME)，观察细胞活性改变.实验结果表明转染pcDNA3. 1-cathcpsin B细胞组织蛋白酶
B蛋白水平明显高于对照组，而转染pSilcncer 2. 1-cathcpsin B细胞组织蛋白酶B蛋白水平明显低于对
照组.嚷哩蓝分析经TNF-翻actD诱导后细胞活性明显降低，与对照组(14.60%士1. 34 %)比较，沉默组
(21. 30%士2.37%)活性增加，而过表达组((9. 98%士1.04%)较对照组明显下降，加入E64d,CA-074ME
组(18. 23%士1.05%,17.40%士1. 13%)较过表达组活性增加，流式细胞术定量检测得到相似结果.表
明组织蛋白酶B对TNF-翻act I}诱导的血管平滑肌细胞凋亡具有促进作用.
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