摘要
目的:观察同型半胱氨酸诱导培养人脐静脉内皮细胞表达RANTESmRNA及蛋白,并分析其机制。方法:实验于2004-11/2005-11在新乡医学院分子生物研究室完成。①采用酶消化法分离脐静脉内皮细胞。②当脐静脉内皮细胞生长至汇合状态时,随机分为对照组、同型半胱氨酸组和二硫代氨基甲酸吡咯烷预处理组。对照组用无血清培养基、同型半胱氨酸组用含0.1mmol/L同型半胱氨酸的无血清培养基培养,分别孵育8h;二硫代氨基甲酸吡咯烷预处理组用含100μmol/L二硫代氨基甲酸吡咯烷的无血清培养基培养20min后,再加入0.1mmol/L的同型半胱氨酸孵育8h。③采用斑点杂交检测脐静脉内皮细胞中RANTESmRNA的表达。④采用Westernblot检测脐静脉内皮细胞中RANTES蛋白的表达。⑤用免疫细胞化学方法检测正常对照组和同型半胱氨酸组核因子κBP65蛋白的表达。结果:①脐静脉内皮细胞中RANTESmRNA的表达:图像分析显示,同型半胱氨酸组、二硫代氨基甲酸吡咯烷预处理组的积分吸光度值分别为18790和16420,分别为对照组(积分吸光度值为12680)的1.48倍和1.29倍。②脐静脉内皮细胞中RANTES蛋白的表达:图像分析显示,同型半胱氨酸组RANTES蛋白印迹条带的积分吸光度值为0.7942,而二硫代氨基甲酸吡咯烷预处理组的积分吸光度值为0.5013,分别是对照组(0.2930)的2.72倍和1.72倍。③脐静脉内皮细胞中核因子κBP65蛋白的表达:在对照组脐静脉内皮细胞中,核因子κBP65蛋白的表达以细胞质为主;同型半胱氨酸刺激后,细胞核染色的阳性率明显增加,由对照组的8.3%上升到43.5%(P<0.01)。结论:同型半胱氨酸能诱导人脐静脉内皮细胞表达RANTESmRNA和蛋白,核因子κB可能参与了该过程。同型半胱氨酸可能通过诱导内皮细胞表达RANTES在动脉粥样硬化形成中起作用。
AIM： To investigate whether homocysteic acid could induce cultured human umbilical vein endothelial cells to express regulated upon activation, normal T cell expressed and secreted （RANTES） and protein, and analyze the mechanism by which the expression of RANTES was induced, METHODS： The experiment was conducted in Molecular Biology Laboratory of Xinxiang Medical University from November 2004 to November 2005. ①The human umbilical vein endothelial cells were separated with pancreatin digestion. ②When the human umbilical vein endothelial cells were confluent, they were divided into three groups including control group, homocysteic acid group, and pyrrolidine dithiocarbamate pretreatment group at random. The human umbilical vein endothelial cells of the control group were cultured with the medium without serum, and the homocysteic acid group was exposed to homocysteic acid at a concentration of 0.1 mmol/L The two groups mentioned above were incubated for 8 hours, respectively. The pyrrolidine dithiocarbamate pretreatment group was incubated with pyrrolidine dithiocarbamate at a concentration of 100 μmol/L for 20 minutes at first, and then exposed to homocysteic acid at a concentration of 0.1 mmol/L for 8 hours.③The expression of RANTES mRNA in human umbilical vein endothelial cells were detected with dot blot hybridization. ④The expression of RANTES protein in human umbilical vein endothelial cells was determined with Western blot method. ⑤The expressions of NF-κB P65 protein in the control and homocysteic acid groups were detected with immunocytochemical method. RESULTS： ①The expression of RANTES mRNA in human umbilical vein endothelial cells： The image analysis showed, in human umbilical vein endothelial cells treated with homocysteic acid or pyrrolidine dithiocarbamate, the integral absorbance values were 18 790 and 16 420, which were 1.48-fold and 1.29-fold as much as that of the control group （12 680）, respectively. ②The expression of RANTES protein in human umbilical vein endot
出处
《中国临床康复》
CSCD
北大核心
2006年第45期88-90,I0007,共4页
Chinese Journal of Clinical Rehabilitation
基金
河南省科技厅自然科学基金资助项目(0511042301)~~
