siRNA抑制Caspase-3表达对内毒素诱导肝细胞凋亡的影响

发布时间：2018-03-01 00:32

本文关键词： 小干扰RNA 半胱氨酸蛋白酶内毒素肝细胞细胞凋亡细胞增殖　出处：《山东医药》2017年34期 　论文类型：期刊论文

【摘要】：目的观察利用小干扰RNA(siRNA)抑制Caspase-3表达对内毒素诱导肝细胞凋亡的影响。方法针对Caspase-3的DNA序列843~1 634 bp设计引物,常规分子克隆至慢病毒表达载体p Lenti-7.1,同时设置空载体为阴性对照;利用Lipofectamine 2000转染至293T细胞,48 h后收获抑制Caspase-3表达病毒质粒V-Caspase-3和空载体病毒质粒V-control。将肝细胞HL7702随机分为观察组和对照组,分别加入V-Caspase-3、V-control培养4 h,均与终浓度为20 mg/L的内毒素共培养12 h。采用Western blotting法检测Caspase-3蛋白,流式细胞术测算细胞凋亡率,原位末端标记技术测算细胞凋亡指数(AI),MTT法观察细胞增殖活力。结果观察组HL7702细胞Caspase-3相对表量为17.32±2.16,低于对照组的62.14±4.36(P0.01);观察组HL7702细胞凋亡率为28.75%,低于对照组的72.67%(P0.01);观察组HL7702细胞AI为23.52±1.24,低于对照组的54.26±1.87(P0.01);观察组HL7702细胞增殖率为65.47%±6.24%,高于对照组的41.39%±4.87%(P0.01)。结论通过siRNA抑制Caspase-3表达能有效降低内毒素诱导的HL7702细胞凋亡,从而增强细胞增殖活性。
[Abstract]:Objective to observe the effect of small interference RNA-siRNAs (siRNAs) on lipopolysaccharide induced hepatocyte apoptosis. Methods A primer was designed for the DNA sequence of Caspase-3 (843 ~ 1 634 BP), which was cloned into lentivirus expression vector pLenti-7.1, and empty vector was set as negative control. After transfection of Lipofectamine 2000 into 293T cells for 48 h, Caspase-3 expression plasmid V-Caspase-3 and empty vector virus plasmid V-controlwere harvested. HL7702 in hepatocytes was randomly divided into two groups: observation group and control group. V-Caspase-3 V-control was added for 4 h and co-cultured with endotoxin for 20 mg/L for 12 h. Caspase-3 protein was detected by Western blotting assay and apoptosis rate was measured by flow cytometry. Results the relative surface volume of Caspase-3 of HL7702 cells in the observation group was 17.32 卤2.16, which was lower than that of the control group (62.14 卤4.36) P0.01.The apoptotic rate of the HL7702 cells in the observation group was 28.75 and was lower than that in the control group (P 0.01). The AI of HL7702 cells was 23.52 卤1.24, which was lower than that of the control group (54.26 卤1.87P0.01), and the proliferative rate of HL7702 cells in the observation group was 65.47% 卤6.24g, which was higher than that in the control group (41.39% 卤4.87p 0.01). Conclusion the inhibition of Caspase-3 expression by siRNA can effectively reduce the apoptosis of HL7702 cells induced by endotoxin. Thus, the cell proliferation activity was enhanced.
【作者单位】：河南医学高等专科学校;
【基金】：河南省高等学校重点科研项目计划(15B310006)
【分类号】：R575

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