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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 construct and transfect the pcDNA3.1-cathepsin B and pSilencer2.1-cathepsin B plasmids and establish the cathepsin B overexpression and gene silencing cell lines to explore the effect of cathepsin B on the apoptosis of vascular smooth muscle cells.Through RT- PCR amplification of cathepsin B gene sequence and construct pcDNA3.1-cathepsin B expression plasmid, designed for cathepsin B target sequence siRNA, according to the shRNA expression plasmid cloning strategy, the design of the sequence of double-stranded DNA sequence The pSilencer2.1-cathepsin B plasmid was constructed and transfected into human aortic smooth muscle cells (HA-VSMCs) by lipofectamine 2000. The overexpressing cathepsin B and gene silencing strains were established by screening and identification of TNF-α / act D Actinomycin D) induced apoptosis. The effects of cathepsin B on vascular smooth muscle cells were observed by cell viability assay and flow cytometry. The effects of cathepsin inhibitor (E64d) and cathepsin B-specific inhibitor (CA) -074ME) were used to observe the change of cell activity.The results showed that the protein level of cathepsin B in pcDNA3.1-cathepsin B transfected cells was significantly higher than that in control group, while pSilencer 2.1-cathepsin B cells transfected The protein level of cathepsin B was significantly lower than that of the control group, and the activity of cells in the silencing group (21.30% ± 2.37%) was increased compared with the control group (14.60% ± 1.34%) after being induced by TNF-α / actD (9.98% ± 1.04%) was significantly lower than that of the control group. The activity of E64d and CA-074ME group (18.23% ± 1.05%, 17.40% ± 1.13%) was higher than that of the overexpression group Similar results were obtained, indicating that cathepsin B promotes TNF-α / act D-induced vascular smooth muscle cell apoptosis.