Hcdc14A在体外糖尿病环境下人脑血管内皮细胞损伤中的作用

Role of Hcdc14A in the in vitro diabetes environment-induced human brain vascular endothelial cell injury

目的 观察体外糖尿病环境如高糖、高游离脂肪酸（FFA）和低氧刺激对人脑血管内皮细胞（HBVEC）人类细胞分裂周期蛋白14A（Hcdc14A）和相关周期蛋白表达及细胞增殖、凋亡的影响,进而探讨糖尿病诱发脑血管内皮细胞损伤的新机制.方法 分别采用不同条件的高糖、FFA和低氧刺激HBVEC,RT-PCR检测Hcdc14A的mRNA表达,Western印迹法检测Hcdc14A及相关周期蛋白周期素B、周期素D、周期素E和p53的蛋白表达,XXT法检测细胞增殖率,流式细胞仪测定细胞凋亡率.结果 不同浓度葡萄糖（10、15和25 mmol/L）和FFA（50、100及200 μmol/L）分别孵育HBVEC 72 h,或以25 mmol/L葡萄糖、200μmol/L FFA分别刺激24、48和72 h,均呈剂量和时间依赖性地降低Hcdcl4A的mRNA和蛋白表达（均P＜0.05）,同时下调周期素B、周期素D和周期素E的蛋白表达（均P＜0.05）,上调p53蛋白表达（均p＜0.05）,低氧刺激也显示相似结果.联合刺激（葡萄糖25 mmdl/L＋FA 200 μmol/L）48 h后,再进行低氧刺激24 h后,与正常对照组、高糖组、高FFA组及低氧组相比,高糖＋高FFA＋低氧联合刺激组显著下调HBVEC的Hcdc14A mRNA和蛋白表达（均P＜0.05）,同时下凋周期素B、周期素D和周期素E的蛋白水平（均P＜0.05）,并上调p53蛋白水平（均P＜0.05）.与正常对照组、高糖组、高FFA组及低氧组相比,高糖＋高FFA＋低氧联合刺激组抑制HBVEC增殖的作用最为明显（40.7％±12.1％对100.0％±10.2％、67.5％±12.1％ 、55.3％±11.2％和64.4％ ±11.3％,均P＜0.05）,并使细胞凋亡率明显增加（22.5％±1.77％对5.7％±0.83％、9.3％±0.95％、16.3％±1.69％和16.3％ ＋1.15％,均P＜0.05）.结论 细胞周期调控蛋白Hcdc14A可能在糖尿病诱发脑血管内皮细胞损伤中起重要作用,可能是参与细胞损伤的主要信号通路.

Objective To investigate the effects of the in vitro diabetes environment such as high glucose,high free fatty acid （FFA）,and hypoxia on human cell dlivision cycle protein 14A （Hcdcl4A） and related cyclins expressions,cell proliferation and apoptosis in human hrain vascular endothelial cells（HBV EC）,and to explore the new mechanisn of HBVEC injury induced by diabetes.Methods HBVECs were stimulated respectively with high glucose,high FFA,and hypoxia under different conditions.The mRNA and protein levels of Hcdcl4A were detected by RT-PCR and Western blot.At the same time,the protein levels of cyclin B,cyclin D,cyclin E,and p53 were detected by Western blot.The cell proliferation was tested by XTT.The cell apoptosis was detected by AnnexinV/ PI.Results After HBVECs were incubated with various concentrations of glucose （10,15,and 25 mmol/L） and FFA （50,100,and 200 μmoL/L） or with 25 mmol/L glucose and 200 μmol/L FFA for 24,48,and 72 h,respectively,the mRNA and protein expressions of Hcdc14A were dose-and time-dependently decreased,along with decreased cyclin B,cyclin D,and cyclin E protein expressions and increased p53 protein expression （all P〈0.05）.Treatment with hypoxia showed a similar result.Compared with control,high glucose,FFA,and hypoxia,the combined intervention of high glucose ＋ FFA ＋ hypoxia significantly downregulated the mRNA and protein （all P〈0.05） expressions of Hcdc14A,along with decreased cyclin B,cyclin D,and cyclin E protein expressions and increased p53 protein expression（all P〈0.05）.Compared with control,high glucose,FFA,and hypoxia,the combined intervention of above stimuli obviously reduced the proliferation of HBVECs （40.7% ± 12.1% vs 100.0% ± 10.2%,67.5% ±12.1%,55.3% ± 11.2%,and 64.4% ± 11.3%,all P〈0.05） and increased the cell apoptosis （22.5% ± 1.77% vs 5.7%±0.83%,9.3% ±0.95%,16.3% ±1.69%,and 16.3% ±1.15%,all P〈0.05）.Conclusion The findings suggest that Hcdc14A may play an important role in diabetes-induced injury of brain vascular endothelial cells,and seems to be one of the major signaling pathways involved in cell injury.