L-精氨酸增强人凝血因子Ⅷ基因表达机制的研究  

Investigation on the mechanism of L-arginine improving human coagulant factor Ⅷ gene

作　　者：尹俊[1] 谢舜峰[2] 廖敬源[1] 张泽文[1] 沈友进[1] 林萍[1] 王学锋[3] 王鸿利[3] 

机构地区：[1]汕头大学医学院第二附属医院血液科,广东汕头515041 [2]汕头大学医学院第一附属医院普外科,广东汕头515041 [3]上海交通大学医学院附属瑞金医院上海血液学研究所,上海200025

出　　处：《诊断学理论与实践》2009年第3期303-308,共6页Journal of Diagnostics Concepts & Practice

基　　金：广东省自然科学基金资助项目(07008197)

摘　　要：目的:进一步探讨L-精氨酸能够增强人凝血因子Ⅷ(hFⅧ)基因在体外的表达机制。方法:扩增B结构域缺失的hFⅧcDNA(BDDhFⅧcDNA)的A1、A2、A3、C1和C2结构域基因,并将其作为启动子,分别插入含氯霉素乙酰转移酶(CAT)报告基因的载体pCAT3-Enhancer的启动子位点,构建成载体pA1-CAT3-Enhancer、pA2-CAT3-Enhancer、pA3-CAT3-Enhancer、pC1-CAT3-Enhancer和pC2-CAT3-Enhancer。再将各载体分别转染人脐静脉内皮细胞(HUVECs)后,加入L-精氨酸(终浓度10mmol/L)培养24h。实验设转染阴性对照(不含启动子)、转染阳性对照,各组再设加L-精氨酸及不加L-精氨酸的对照组(共14组)。采用体外细胞核连缀反应(run-onassay)检测CAT基因的转录,采用酶联免疫吸附试验(ELISA)检测各组HUVECs裂解液中CAT蛋白的含量。结果:在无L-精氨酸存在的情况下,转染阴性对照载体pCAT3-Basic、pA3-CAT3-Enhancer、pC1-CAT3-Enhancer和pC2-CAT3-Enhancer的HUVECs裂解液中均未检测到有CAT基因转录,CAT蛋白含量均为背景值,转染阳性对照载体pCAT3-Control的HUVECs裂解液中CAT基因有强转录,转染pA1-CAT3-Enhancer和pA2-CAT3-Enhancer的HUVECs裂解液中只能检测到微弱的CAT基因转录,CAT蛋白含量分别为(61.85±7.86)pg/106个细胞和(60.27±7.27)pg/106个细胞。在与L-精氨酸共培养24h后,转染pA2-CAT3-Enhancer的HUVECs裂解液中CAT基因的转录和蛋白表达显著增强,CAT蛋白含量达到(368.56±21.85)pg/106个细胞,显著高于未加L-精氨酸组(P<0.01),而转染其他各载体的HUVECs裂解液中CAT基因转录和表达的变化与未加L-精氨酸组间差异均无统计学意义。结论:hFⅧ的A1和A2结构域基因能作为启动子驱动CAT基因的转录和表达,而L-精氨酸可显著增强hFⅧA2结构域基因启动的CAT基因转录和表达。Objective To further investigate the mechanism of L-arginine in improving human coagulant factor Ⅷ gene expression. Methods Five functional domains of B domain deleted human coagulant factor Ⅷ cDNA （BDDhFⅧ cDNA） including fragments A1, A2, A3, C1 and C2 were amplified and inserted into pCAT3-Enhancer reporter vector, which contained chloramphenicol acetyl transferase （CAT）, to produce five plasmids pA1-CAT3-Enhancer, pA2-CAT3- Enhancer, pA3-CAT3-Enhancer, pC1-CAT3-Enhancer and pC2-CAT3-Enhancer. Within the constructed vectors, fragments A1, A2, A3, C1 and C2 served as promoter respectively, pCAT3-Basic, which was absent for both promoter and enhancer, and pCAT3-Control, which contained both promoter and enhancer, served as negative and positive control respectively. Human umbilical vein endothelial cells （HUVECs） were isolated and transfected with these seven plasmids respectively and incubated with L-arginine （final concentration 10 mmol/L） for 24 hours. Nucleoli were then isolated and underwent run-on assay to determine the transcription of CAT, and HUVECs lysate was collected for determining the concentration of CAT with enzyme linked immunosorbent assay （ELISA）. Results There was no transcription and only a background expression of CAT gene from HUVECs transfected with pCAT3-Basic, pA3-CAT3-Enhancer, pC1-CAT3-Enhancer and pC2-CAT3-Enhancer. While an intense mRNA and CAT concentration were displayed from HUVECs transfected with pCAT3-Control, however, only weak mRNA and CAT concentration were observed from HUVECs transfected with pAI-CAT3-Enhancer and pA2-CAT3-Enhancer, the concentrations of CAT were （61.85±7.86） pg/10^6 cells and （60.27±7.27） pg/10^6 cells respectively. In the lysate of HUVECs incubated with L-argine, obviously augmented mRNA and CAT concentration were only observed from HEVECs transfected with pA2-CAT3-Enhancer, the concentration of CAT was （368.56±21.85） pg/10^6 cells （P〈0.01, compared with those without L-arginine incubation）, and no statistic

关 键 词：L-精氨酸 凝血因子Ⅷ 启动子 氯霉素乙酰转移酶 人脐静脉内皮细胞 

分 类 号：R554.1[医药卫生—血液循环系统疾病]