钙非依赖型磷脂酶A_2对过氧化氢诱导胰岛微血管内皮细胞凋亡的影响

Effect of calcium-independent phospholipase A_2 on islet microvascular endothelial cell apoptosis induced by hydrogen peroxide

目的用过氧化氢(H_2O_2)诱导大鼠胰岛微血管内皮细胞(IMECs)凋亡,观察钙非依赖型磷脂酶A_2(iPLA_2)对其凋亡的影响。方法 siRNA技术抑制IMECs iPLA_2表达,DNA ladder法检测H_2O2诱导后的DNA片段,Annexin V-FITC/PI双染色检测细胞凋亡率,DIOC_6(3)染色检测细胞线粒体膜电位。结果 (1)siRNA-iPLA_2可明显抑制IMECs内iPLA_2 mRNA的表达。(2)H_2O_2诱导后细胞DNA片段化程度随诱导时间的增加而增多,抑制iPLA_2表达后细胞的梯状条带明显增亮。(3)干扰组和未干扰组、干扰阴性对照组相比凋亡率明显升高(P均<0.01)。(4)与未干扰组比较,干扰组细胞线粒体膜电位明显下降(P<0.1)。结论抑制iPLA_2表达可促进H_2O_2诱导的IMECs凋亡。

Objective To observe the effect of calcium-independent phospholipase A2 （iPLA2） on islet microvascular endothelial cells （IMECs） apoptosis induced by hydrogen peroxide （H2 O2）. Methods siRNA of calcium-independent phospholipase A2 was transfected in IMECs to inhibit the expression of iPLA2. For detection of the apoptosis, apoptotic DNA ladder were analyzed and an Annexin V-FITC/PI staining kit was used with flow cytometry. Mitochondrial membrane potentials were determined by flow cytometry with labeled DIOC6 （3） reagent. Results Compared with controls （normal IMECs and negative siRNA IMECs）,the DNA fragmentations of siRNA- iPLA2 IMECs were more apparent, and the apoptotic rate at 300μmol/L H2O2 were significantly higher [ 43.87±4.13% （iPLA2-siRNA2）, 32.94± 3.90%（N group）, 35.23 ± 4. 70% （NC-siRNA） （P〈0.01）3. Mitochondrial membrane potentials were lower in siRNA-iPLA2 IMECs than other groups. Conclusion Inhibition of iPLA2 may promote the apoptosis of the IMECs induced by H2O2.