扩张型心肌病致病基因LMNA新突变E82K对细胞周期影响的实验研究

Effects of a novel familial dilated cardiomyopathy associated LMNA gene mutation E82K on cell cycle of HEK293 cells

目的探讨国人家族性扩张型心肌病 LMNA 致病基因中发现的新突变 E82K 位点对细胞周期的影响。方法构建野生型及突变型 LMNA 基因的真核表达载体并与空载体分别转染HEK293细胞,抗生素筛选得到稳定转染的细胞株,用0.8 mmol/L 过氧化氢诱导对照组以及分别转染空载体、野生型 LMNA 基因和突变型 LMNA 基因的细胞组24 h,流式细胞仪检测各组细胞的细胞周期。结果转染突变型 LMNA 基因的细胞细胞周期被阻滞于G0/G1期,而其他3组均被阻滞于G2/M 期。结论 LMNA 基因 E82K 突变可以阻止过氧化氢诱导的细胞周期向 G2/M 期积聚。

Objective To investigate the effect of a novel LMNA gene mutation E82K found in a Chinese family with dilated cardiomyopathy on cell cycle of HEK293 cells. Methods （ 1 ） Human wild type full-length LMNA gene cDNA was subcloned into eukaryotic expression vector pTracer-CMV and point mutation was introduced into the cDNA. LMNA gene wild type and mutant E82K LMNA gene were transfected into HEK293 cells respectively and stable cell lines resistant to antibiotic were obtained 4 weeks later. （2） Cell cycle changes were analyzed by flow cytometry in HEK293 cells transfected with wild type and mutant E82K LMNA gene and empty vector in the presence of 0. 8 mmol/L H2 02. Results Cell circle was arrested at G0/G1 phase in the cells transfected with mutated E82K LMNA gene and at G2/M phase in other cell groups in the presence of H2O2. Conclusion Cell circle was arrested at G0/G1 phase in the cells transfected with E82K LMNA gene in the presence of H2O2 in HEK293 cells.