【摘要】 目的研究少汗型外胚层发育不良症EDA-A1基因突变体对人脐静脉内皮细胞(ECV)周期和增殖活性的影响。方法构建EDA-A1基因编码序列(CDS)突变体和野生型的真核表达载体pcDNA3.1(-)-EDA-A1-M/W,转染人脐静脉内皮细胞,逆转录PCR(RT-PCR)扩增EDA-A1基因,蛋白免疫印迹法(Western blot)法检测EDA-A1蛋白表达,MTT法、流式细胞术检测EDA-A1基因突变体对ECV细胞增殖和细胞周期的影响。结果 RT-PCR、Western blot结果显示,野生型组和突变体组ECV细胞表达EDA-A1基因及其蛋白,而空质粒转染组[pcDNA3.1(-)]和空白对照组细胞无表达。与对照组(空质粒租)相比,突变体组ECV细胞增殖活性下降,生长抑制率为45.70%(P<0.01),野生型细胞增殖活性无明显改变(P>0.05)。突变体组中有更多细胞进入G0/G1期、S期;野生型组S期细胞增多,G2/M期细胞明显减少(P<0.05)。结论突变体和野生型EDA-A1基因对ECV细胞增殖和细胞周期有不同的生物学功能。更多还原

【Abstract】 Objective To investigate the effect of ectodysplasin A(EDA-A1)gene of hypohidrotic ectodermal dysplasia on proliferation and cell cycle of human umbilical vein endothelial cell(ECV304).Methods Recombinant eukaryotic expression vectors pcDNA3.1(-)-EDA-A1-M/W(mutant,M and wild,W)containing the coding sequence were transected into ECV304 cells.EDAA1gene was amplified by reverse transcription polymerase chain reaction(RT-PCR),and the protein was detected by Western blot.Cell viability and cycle distribution were invested by MTT and Flow cytometry(FCM).Results The EDA-A1 gene and protein were detected respectively by RT-PCR and Western blot in ECV cells transfected with pcDNA3.1(-)-EDA-A1-M/W,but not in ECV cells transfected with plasmid pcDNA3.1(-)and cells without transection.And also,compared with control groups,EDAA1 gene mutant significantly decreased proliferation of ECV cells and its inhibition rate was 45.70%(P<0.01),whereas wild EDA-A1 gene did not cause such growth inhibition(P>0.05).A significant increase of the G0/G1 and S fraction was seen in the ECV cells of mutant group,compared with wild group with an accumulation in S phase and a concomitant decrease in G2/M phase population(P<0.05).Conclusion Mutant and wild EDA-A1 gene may have distinct biological functions on proliferation and cell cycle distribution of cultured human umbilical vein endothelial cell.更多还原