摘要
目的:观察丹参酮ⅡA对高糖诱导的大鼠血管平滑肌细胞增殖的作用,分析该作用与丝裂原活化蛋白激酶信号传导通路的关系。方法:实验于2004-01/2005-06在东南大学医学院发育与疾病相关基因实验室完成。①取大鼠胸主动脉,用组织贴壁法原代培养大鼠血管平滑肌细胞并传代,实验使用第4～8代细胞。②按实验分组干预细胞,正常对照组,培养液含5mmol/L葡萄糖;高糖组,培养液含30mmol/L葡萄糖;高糖对照组,培养液含30mmol/L葡萄糖+10g/L二甲亚砜;丹参酮ⅡA组,培养液含30mmol/L葡萄糖+0.5mg/L丹参酮ⅡA;U0126组,培养液含30mmol/L葡萄糖+25μmol/LU0126。③用二步法测定0.125,0.25,0.5,1.0,2.0,5.0,10.0mg/L丹参酮ⅡA对血管平滑肌细胞的毒性。④BrdU掺入DNA的ELISA法测定细胞内DNA合成水平。⑤同位素示踪法测定丝裂原活化蛋白激酶活性。结果:①高糖诱导的大鼠血管平滑肌细胞呈现快速增殖状态。②当丹参酮ⅡA剂量大于1.0mg/L时,有一定的细胞毒性。四甲基噻唑蓝法测定结果表明1.0,2.0,5.0,10.0mg/L丹参酮ⅡA组血管平滑肌细胞线粒体脱氢酶活性明显低于正常对照组(分别为0.654±0.023,0.580±0.032,0.465±0.041,0.376±0.053,0.840±0.085,P<0.01),表明此浓度可造成细胞功能障碍,有明显的细胞毒性。③随着丹参酮ⅡA作用浓度的增加,细胞内BrdU掺入DNA的量逐渐下降,0.03125mg/L丹参酮ⅡA与高糖对照组相比差异有显著性意义(分别为0.958±0.086,1.059±0.047,P<0.05),0.0625,0.125,0.25,0.5mg/L丹参酮ⅡA与高糖对照组相比差异有非常显著性意义(分别为0.865±0.058,0.781±0.099,0.738±0.071,0.616±0.028,1.059±0.047,P<0.01)。④高糖组血管平滑肌细胞内丝裂原活化蛋白激酶的活性相对于正常对照组显著升高[分别为(1048.59±94.78),(395.82±43.53)μkat/g,P<0.01],丹参酮ⅡA组和U0126组丝裂原活化蛋白激酶活性低于高糖组[分别为(450.55±50.47),(417.30±62.96),(1048.59±94.78)μkat/g,P<0.01]。结论:丹参酮ⅡA对高糖诱导的血管平滑肌细胞增殖具有明显抑制作用的机制可能是其部分阻断了丝裂原活化蛋白激酶信号传导通路。
AIM： To observe the effect of tanshinone Ⅱ A （TSN） on proliferation of vascular smooth muscle cells （VSMC） induced by high glucose and explore its reltitionship With mitogen activated protein kinase （MAPK） signal pathway, METHODS： The experiment was conducted in the Genetic and Developmental Biology Department, Southeast University from January 2004 to June 2005,①The aorta choracalis of rats was separated carefully to culture VSMC with the tissue adherence method, and the 4^th to 8^th generation cells were employing for the experiment.②Cells were divided into normal control group cultured in culture medium containing 5 mmol/L glucose; high glucose group, cultured in culture medium containing 30 mmol/L glucose; high glucose control group cultured in culture medium containing 30 mmol/L glucose and 10 g/L demasorb （DMS）; TSN group cultured in culture medium containing 30 mmol/L glucose and 0.5 mg/L TSN; U0126 group cultured in culture medium containing 30 mmol/L glucose and 25 μmol/L U0126,③The toxicity of TSN at 0.125, 0,25, 0.5, 1.0, 2.0, 5,0, 10,0 mg/L respectively on VSMC was assayed by MTT. ④DNA synthesis was analyzed by ELISA of BrdU. ⑤The activity of MAPK was measured by liquid scintillation counting. RESULTS： ①The proliferation of VSMC induced by high glucose appeared exceptional fast. ②TSN showed cell toxicity when its concentration was 〉1,0 mg/L. The MTT showed that mitochondrial dehydrogenase activity of VSMC in TSN groups at 1.0, 2.0, 5,0, 10.0 mg/L was obviously lower than the normal control group （0,654±0.023, 0.580±0.032, 0,465±0.041, 0.376±0,053, 0,840±0.085, P 〈 0,01）, it supposed that this concentration had already caused the cell functional obstruction and showed obvious cell toxicity. ③DNA synthesis analyzed by ELISA of BrdU was gradually dedined with the increase of TSN, and 0.031 25 mg/L TSN group had significant differences compared with the high glucose group （0.958±0.086, 1,059±0.047, P 〈 0.05）, and 0,0625, 0.125, 0.25, 0.5 mg/L TSN groups were significantly lower than the high glucose group （0,865 ±0.058, 0.781±0.099, 0.738±0.071, 0.616±0.028, 1.059±0.047, P 〈 0.01）. ④The activity of MAPK of VSMC in the high glucose group was obviously higher than that in the control group [ （1 048,59±94.78）, （395,82±43.53） μkat/g, P 〈 0.01], and the activity of MAPK in the TSN group and U0126 group were significantly lower than that in the high glucose group [（450.55±50.47）, （417.30±62.96）, （1 048.59±94.78） μkat/g, P 〈 0.01]. CONCLUSION： The inhibitory effect of TSN on proliferation of VSMC may be related to its blockage in MAPK signal pathway.
出处
《中国临床康复》
CSCD
北大核心
2006年第31期55-57,F0003,共4页
Chinese Journal of Clinical Rehabilitation
