O_2·^-和H_2O_2表达上调对2型糖尿病患者血小板异常活化的影响

Effect of Elevated O_2·^- and H_2O_2 Expression on Abnormal Activation of Platelets in Patients with Type 2 Diabetes Mellitus

【目的】探讨血小板O_2·^-和H_2O_2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O_2·^-、胞浆O_2·^-和H_2O_2水平;用NADH/PMS和H_2O_2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O_2·^-、H_2O_2水平明显高于正常对照组(P<0.05),而胞浆O_2·^-水平差异无统计学意义。通过流式分析H_2O_2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O_2·^-、H_2O_2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 statistic difference 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 O2 ·-and H2O2 level increased,thus regulating aberrant activation of diabetic platelet.