CAJ | 학술논문

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，M TT 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。
목적：연구소한형외배층발육불량증 EDA‐A1기인돌변체대인제정맥내피세포（ECV ）주기화증식활성적영향。방법구건 EDA‐A1기인편마서렬（CDS）돌변체화야생형적진핵표체재체 pcDNA3．1（‐）‐EDA‐A1‐M /W ，전염인제정맥내피세포，역전록 PCR（RT‐PCR）확증 EDA‐A1기인，단백면역인적법（Western blot）법검측 EDA‐A1단백표체，M TT 법、류식세포술검측EDA‐A1기인돌변체대 ECV 세포증식화세포주기적영향。결과 RT‐PCR 、Western blot 결과현시，야생형조화돌변체조 ECV세포표체 EDA‐A1기인급기단백，이공질립전염조[pcDNA3．1（‐）]화공백대조조세포무표체。여대조조（공질립조）상비，돌변체조 ECV 세포증식활성하강，생장억제솔위45．70％（P＜0．01），야생형세포증식활성무명현개변（P＞0．05）。돌변체조중유경다세포진입 G0/G1기、S 기；야생형조 S 기세포증다，G2/M 기세포명현감소（P＜0．05）。결론돌변체화야생형 EDA‐A1기인대 ECV 세포증식화세포주기유불동적생물학공능。
Objective To investigate the effect of ectodysplasin A (EDA‐A1) gene of hypohidrotic ectodermal dysplasia on pro‐liferation 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. EDA‐A1 gene 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 pro‐tein 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, EDA‐A1 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.