戊二酰辅酶A脱氢酶基因沉默及高浓度赖氨酸对BRL肝细胞活性的影响

发布时间：2018-03-23 04:12

本文选题：戊二酰辅酶A脱氢酶　切入点：细胞活性　出处：《中国当代儿科杂志》2017年09期 　论文类型：期刊论文

【摘要】：目的探讨戊二酰辅酶A脱氢酶(GCDH)基因沉默和赖氨酸代谢物蓄积对肝细胞活性的影响。方法将BRL肝细胞分为正常对照组、阴性对照组和GCDH沉默组。构建含靶向沉默GCDH基因的sh RNA慢病毒载体,分别用该病毒和阴性对照病毒感染GCDH沉默组和阴性对照组BRL肝细胞。感染后细胞再用含5 mmol/L赖氨酸培养基培养。免疫荧光技术检测慢病毒感染效率;Western blot法检测GCDH蛋白表达水平;MTT法检测细胞活性,Hoechest 33342染色检测细胞凋亡,Western blot法检测细胞凋亡的经典指标Caspase3水平。结果构建的慢病毒可有效沉默肝细胞GCDH表达(P0.01)。MTT及Hoechest 33342染色检测各组间细胞活性及细胞凋亡比较差异无统计学意义(P0.05)。Caspase3蛋白表达在各组间比较差异亦无统计学意义(P0.05)。结论 GCDH基因沉默和赖氨酸代谢物蓄积对肝细胞无明显损伤作用。
[Abstract]:Objective to investigate the effects of glutaryl coenzyme A dehydrogenase (GCDH) gene silencing and lysine metabolite accumulation on hepatocyte activity. Methods BRL hepatocytes were divided into normal control group. Negative control group and GCDH silencing group. The sh RNA lentivirus vector containing the target silencing GCDH gene was constructed. The hepatocytes of GCDH silencing group and negative control group were infected with the virus and the negative control group respectively. After infection, the cells were cultured in 5 mmol/L lysine medium. The efficiency of lentivirus infection was detected by immunofluorescence technique and GCDH was detected by Western blot method. The level of protein expression was determined by MTT assay and the activity of cells was detected by Hoechest 33342 staining. Western blot assay was used to detect the Caspase3 level of apoptosis. Results the constructed lentivirus could effectively silence the expression of GCDH in hepatocytes and detect the expression of GCDH in hepatocytes by Hoechest 33342 staining. There was no significant difference in cell activity and apoptosis between the two groups. There was no significant difference in the expression of P0.05N. Caspase3 between the two groups. Conclusion GCDH gene silencing and lysine metabolite accumulation have no significant damage to hepatocytes.
【作者单位】：华中科技大学同济医学院附属同济医院儿科;
【分类号】：R725.9

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