摘要
目的:在不同浓度的葡萄糖培养条件下,探讨内皮细胞传代过程中活性氧(ROS)的代谢异常及其对单核细胞趋化蛋白-1(MCP-1)表达的影响。方法:人脐静脉内皮细胞培养和传代后,选取第2、6代细胞分别在5.5mmol/L或16.5mmol/L葡萄糖培养基中培养,以培养0、12、24、48h为各时间观察点。流式细胞术检测细胞ROS含量,半定量逆转录聚合酶链式反应检测MCP-1的表达。结果:(1)第2代细胞经5.5mmol/L葡萄糖培养,ROS水平相对稳定。经16.5mmol/L葡萄糖培养,ROS则呈时间依赖性增加,培养24h和48h后分别较加入葡萄糖培养前增加56.5%和69.2%。同葡萄糖浓度培养下,各时间点第6代细胞ROS检测值均较第2代细胞有显著意义的增加(均P<0.01)。(2)第2代细胞经5.5mmol/L葡萄糖培养24h后MCP-1 mRNA表达开始上升,可见MCP-1 mRNA表达与时间相关。高葡萄糖培养与低葡萄糖相比,同时间点的MCP-1 mRNA表达均有显著性增强(P<0.01或P<0.05)。同浓度葡萄糖作用下,第6代细胞MCP-1 mRNA表达总体上高于第2代细胞。结论:高浓度葡萄糖培养在进一步提高传代内皮细胞ROS量的同时,促进了MCP-1 mRNA的表达,提示高糖促MCP-1表达作用可能也是通过ROS增加介导的。
Objective To study the relationship between abnormal changes of reactive oxygen species （ROS） and the expression of monocyte chemoattractant protein-1 （MCP-1） in passage vein endothelial cells when these cells were exposed to high glucose and the underlying mechanisms of passage cells＇ dysfunction under high glucose. Methods The human umbilical vein endothelial cells that isolated from newborn umbilical cord were cultured and verified as vascular endothelial cells by immunohistochemistry in vitro. Passage cells were divided into 4 groups： 5.5 mmol/L P2 group, passage 2 cells cultured in 5.5 mmol/L glucose; 5.5 mmol/L P6 group, passage 6 cells cultured in 5.5 mmol/L glucose; 16.5 mmol/L P2 group, passage 2 cells cultured in 16.5 mmol/L glucose; 16.5 mmol/L P6 group, passage 6 cells cultured in 16.5 mmol/L glucose. The endpoints for culture in each group were designed as 0, 12, 24 and 48 h. Levels of ROS were studied with flow cytometry and MCP-1 mRNA expression was assayed by reverse-transcription polymerase chain reaction. Results The levels of ROS stable in 5.5 mmol/L group were increased time-dependently in 16.5 mmol/L P2 group. For example, the levels of ROS were 56.5% or 69.2% times more at the time point of 24 h or 48 h than at the time point of 0 h in 16.5 mmol/L P2 group. The levels of ROS in 16.5 mmol/L P2 group increased significantly than in 5.5 mmol/L P2 group at the time point of 24 h and 48 h （all P 〈 0.05 ）. ROS also increased significantly in passage 6 cells compared to passage 2 cells in every point of time as these cells were treated by 5.5 mmol/L glucose or 16.5 mmol/L glucose. The expression of MCP-1 was increased time- dependently and dose-dependently by glucose at the point of 12, 24 and 48 h. The expressions of MCP-1 were also increased significantly in passage 6 cells compare to passage 2 cells in every point of time. Conclusion The expression of MCP-1 increased by high glucose is probably mediated by increased formation of ROS in passage VECs.
出处
《实用医学杂志》
CAS
2008年第17期2927-2930,共4页
The Journal of Practical Medicine
关键词
内皮细胞
葡萄糖
细胞培养技术
免疫组织化学
多聚酶链反应
Endothelial cells Glucose Cell culture techniques Immunohistochemistry Polymerase chain reaction
