Gao Jin-Zhi, Zhang Cai, Yi Qin, Ying Yan-Qin, Luo Xiao-Ping
Department of Pediatrics, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2017 Sep;19(9):1014-1019. doi: 10.7499/j.issn.1008-8830.2017.09.016.
To investigate the effect of glutaryl-CoA dehydrogenase (GCDH) gene silencing and accumulation of lysine metabolites on the viability of hepatocytes.
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.
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).
GCDH gene silencing and accumulation of lysine metabolites may not cause marked hepatocyte injury.
探讨戊二酰辅酶A脱氢酶（GCDH）基因沉默及赖氨酸代谢产物蓄积对肝细胞活力的影响。
将BRL细胞分为正常对照组、阴性对照组和GCDH沉默组。构建沉默GCDH基因的shRNA慢病毒载体，分别用该慢病毒和阴性对照病毒感染GCDH沉默组和阴性对照组的BRL肝细胞，然后在含5 mmol/L赖氨酸的培养基中培养。采用免疫荧光法检测慢病毒感染效率。采用蛋白质免疫印迹法检测GCDH蛋白表达。采用MTT法评估细胞活力。采用Hoechest33342染色检测细胞凋亡。采用蛋白质免疫印迹法检测细胞凋亡指标Caspase-3的表达。
构建的慢病毒有效沉默了肝细胞中的GCDH基因（P<0.01）。MTT法和Hoechest 33342染色显示各组细胞活力和凋亡无显著差异（P>0.05）。各组Caspase-3蛋白表达也无显著差异（P>0.05）。
GCDH基因沉默及赖氨酸代谢产物蓄积可能不会导致明显的肝细胞损伤。
