O2·-和H2O2表达上调对2型糖尿病患者血小板异常活化的影响

摘要

【目的】探讨血小板O2·-和H2O2水平变化导致2型糖尿病患者血小板异常活化的分子机制。【方法】检测2型糖尿病患者和正常对照组的血小板4项临床参数；应用免疫荧光技术观察血小板形态结构的变化；应用流式细胞术测定两组血小板内线粒体O2·-、胞浆O2·-和H2O2水平；用NADH/PMS和H2O2分别处理正常对照组血小板，流式细胞术检测血小板的活化阳性百分率；应用Western Blot检测两组血小板内过氧化氢酶（Catalase）、超氧化物歧化酶2型（Mn-SOD）的表达差异。【结果】2型糖尿病组平均血小板体积（MPV）明显高于正常对照组（P<0.001），而血小板数量（PLT）、血小板分布宽度（PDW）、血小板比积（PCT）差异均无统计学意义；免疫荧光显示2型糖尿病组血小板形态结构较正常对照组发生改变，流式细胞术检测血小板的线粒体O2·-、H2O2水平明显高于正常对照组（P<0.05），而胞浆O2·-水平差异无统计学意义。通过流式分析H2O2和NADH/PMS体系明显促进血小板的活化（P<0.01）。Western Blot结果显示2型糖尿病组血小板Catalase的表达下调，而Mn-SOD的表达及活性无差异。【结论】糖尿病血小板内Catalase表达下调可能是导致线粒体O2·-、H2O2水平升高的原因，从而调控糖尿病血小板的异常活化。%[Objective]To investigate the molecular mechanism of abnormal platelet activation induced by platelet O2 ·- and H2O2 levels in type 2 diabetes mellitus.[Methods]The platelet parameters in patients with type 2 diabetic patients and normal controls were measured;Immunofluorescence technique was used to observe the platelet morphology changing;Flow cytometry was used to detect platelet intracellular O2 ·- and H2O2 content in two groups,then with platelets in normal controls treated with NADH/PMS system and H2O2 respectively,platelet activation positive percentage was observed. Standard Western blot analysis protocols were used to detect expression difference of Catalase and type 2 super-oxide dismutase(Mn-SOD)in platelets.[Results]The MPV in the group of type 2 diabetic patients was significantly higher than in the normal control group(P < 0.001),but there was no statisticdifference in PLT,PDW,PCT between two groups. Immunofluorescence results showed that morphology of platelets in type 2 diabetic patients changed contrast to normal group. Through flowcytometry detection,the content of mitochondrial O2·-and H2O2 of platelet in type 2 diabetic patients were obviously higher than in normal group(P<0.05),whereas no significant difference in cytoplasmic O2·-. We adopted NADH/PMS system and H2O2 to treat platelets of normal group,heightened activated positive percentage were observed which described O2 ·- and H2O2 can significantly promote platelet activation(P<0.01). Western blot results showed that expression of Catalase in platelet of type 2diabetes patients decreased,while the expression and activity of Mn-SOD had no difference.[Conclusion]It is diabetic platelet Catalase expression decreased that may lead to Diabetic platelet mitochondrial O 2 ·- and H2O2 level increased ,thus regulating aberrant activation of diabetic platelet.