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

Effect of glutaryl-CoA dehydrogenase gene silencing and high-concentration lysine on the viability of BRL hepatocytes

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shRNA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（P < 0.01）。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（P > 0.05）。Caspase3蛋白表达在各组间比较差异亦无统计学意义（P > 0.05）。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 effect of glutaryl-CoA dehydrogenase (GCDH) gene silencing and accumulation of lysine metabolites on the viability of hepatocytes. Methods BRL cells were divided into normal control group, negative control group, and GCDH silencing group. The shRNA lentiviral vector for silencing GCDH gene was constructed, and the BRL hepatocytes in the GCDH silencing group and the negative control group were infected with this lentivirus and negative control virus respectively, and then cultured in a medium containing 5 mmol/L lysine. Immunofluorescence assay was used to measure the infection efficiency of lentivirus. Western blot was used to measure the expression of GCDH protein. MTT assay was used to evaluate cell viability. Hoechest33342 staining was used to measure cell apoptosis. Western blot was used to measure the expression of Caspase-3, an index of cell apoptosis. Results The lentivirus constructed effectively silenced the GCDH gene in hepatocytes (P < 0.01). MTT assay and Hoechest 33342 staining showed no significant differences in cell viability and apoptosis between groups (P > 0.05). There was also no significant difference in the expression of Caspase-3 protein between groups (P > 0.05). Conclusions GCDH gene silencing and accumulation of lysine metabolites may not cause marked hepatocyte injury.