组织蛋白酶B过表达质粒和特异性siRNA表达质粒的构建与转染及对血管平滑肌细胞作用初步探讨  

Establishment of the cathepsin B over-expression group and gene-silent group and preliminary study of effect on the vascular smooth muscle cells by cathepsin B

作　　者：张涛[1] 羊镇宇[1] 薄小萍[1] 王强[1] 陈茂华[1] 李坚[1] 郭素峡[1] 冯健 尤华彦[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：《南京大学学报（自然科学版）》2012年第6期809-816,共8页Journal of Nanjing University（Natural Science）

摘　　要：构建并转染pcDNA3.1-cathepsin B和pSilencer2.1-cathepsin 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B全基因序列并构建pcDNA3.1-cathepsin B表达质粒,同时设计针对组织蛋白酶B靶序列的siRNA,按shRNA表达质粒的克隆策略,设计针对该序列的双链DNA序列并构建pSilencer2.1-cathepsin 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/act D(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B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B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-cathepsin B细胞组织蛋白酶B蛋白水平明显高于对照组,而转染pSilencer 2.1-cathepsin B细胞组织蛋白酶B蛋白水平明显低于对照组.噻唑蓝分析经TNF-α/actD诱导后细胞活性明显降低,与对照组(14.60%±1.34%)比较,沉默组(21.30%±2.37%)活性增加,而过表达组(9.98%±1.04%)较对照组明显下降,加入E64d、CA-074ME组(18.23%±1.05%,17.40%±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-α/act D诱导的血管平滑肌细胞凋亡具有促进作用.To establish the cathepsin B over expression group and the gene-silent group,and preliminarily study the role of cathepsin B in the process of vascular smooth muscle cells apoptosis. We cloned the cathepsin B gene sequence and constructed the pcDNA3. 1-cathepsin B plasmid, simultaneously designed the target mRNA sequence of cathepsin B and negative control, According to shRNA expression vector clone strategy, we designed the double-stranded DNA sequence and constructed pSilencer 2. 1 Cathepsin B plasmid. Then transfect Human aortic smooth muscle cells（ HA VSMC）by lipofeetion method. By TNF-α/act D induced apoptosis,we observe the role of cathepsin B in the process of vascular smooth muscle cells apoptosis by MTT assay and flow cytometry. Western blot showed the cathepsin B protein was significantly higher in the over-expression group than that in the control group, while the the silent group was significantly lower. After the intervention of TNF-α, MTT assay showed the HA-VSMC viability decreased significantly. As compared with the control group（ 14.60 %± 1.34 %）, the silent group（21.3 %± 2.37% ）significantly increased, while the over expression group significantly decreased（ 9.98 % ± 1.04% ）. The E64d and CA-074ME groups（ 18.23 % ± 1.05 %,17.4 % ± 1.03 % ）compared with the over-expression group increased sig nificantly, flow cytometry gets similar results. So cathepsin B can promote TNF -α induced apoptosis in vascular smooth muscle cells.

关 键 词：组织蛋白酶B 血管平滑肌细胞 小分子RNA干扰 细胞凋亡 

分 类 号：Q463[生物学—生理学]