沉默钙非依赖型磷脂酶A2β基因对间歇性高糖诱导胰岛微血管内皮细胞凋亡的影响

Silencing calcium-independent phospholipase A2β gene on microvascular islet endothelial cell apoptosis induced by intermittent high glucose

目的 通过小分子干扰（siRNA）技术沉默大鼠胰岛微血管内皮细胞（IMECs）的钙非依赖型磷脂酶A2（iPLA2）基因,研究不同葡萄糖浓度对IMECs iPLA2表达的影响及其与细胞凋亡之间的关系.方法 通过分离纯化大鼠胰岛,获取IMECs细胞,采用随机数余数分组法将其分为正常糖组（5 mmol/L）、恒定高糖组（28 mmol/L）及间歇高糖组（每24小时轮换培养于5mmol/L或28 mmol/L葡萄糖）;通过实时定量聚合酶链反应（RT-PCR）检测葡萄糖浓度分别为恒定的5 mmol/L、28 mmol/L以及每24小时轮换的5 mmol/L或28 mmol/L培养条件下的IMECs iPLA2的基因表达;合成iPLA2的RNA干扰序列并转染至IMECs中,通过RT-PCR验证其是否转染成功;采用细胞DNA片段化检测和膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V/PI）流式细胞仪检测不同葡萄糖浓度条件下IMECs的凋亡.组间两两比较采用LSD检验,凋亡率采用随机区组设计的两因素ANOVA分析.结果 RT-PCR检测显示各组细胞iPLA2基因表达逐渐减弱,依次为1.90±0.23、0.90±0.05及0.23±0.03,且各组间差异有统计学意义（P＜0.05）.成功合成并转染iPLA2 siRNA后,IMECs间歇性葡萄糖培养组较正常糖组及恒定高糖组DNA片段化更为明显,间歇高糖组流式细胞凋亡率较正常糖组升高,差异有统计学意义（44.9±2.7比3.1±1.0,P＜0.05）.结论 间歇性高糖环境可抑制IMECsiPIA2基因表达,且通过小分子干扰技术抑制iPLA2基因表达能够促进细胞的凋亡,提示iPLA2表达减弱可能参与了间歇性高糖诱导的IMECs凋亡过程.

Objective To study the calcium-independent phospholipase A2 （iPLA2）expression of islet microvascular endothelial cells （IMECs） and its relationships with apoptosis in different concentrations of glucose by silencing （iPLA2） genes of rat pancreatic IMECs using small interference technology.Methods Rat IMECs are successfully separated and purified,then divided into three groups such as normal glucose,constant high glucose and intermittent high glucose group by the method of random number remainder.Detecting the expression of IMECs iPLA2 under different concentrations of glucose such as 5 mmol/L,28 mmol/L and a intermittent one of 5 mmoL/L or 28 mmol/L by RT-PCR; synthesizing iPLA2 RNA interference sequences （siRNA） and transfecting it to IMECs,usiug RT-PCR to verify the transfection; determining the IMECs apoptosis rate in normal and intermittent high glucose by DNA fragmentation and Annexin V/PI flow cytometry.Differences between groups were compared with LSD test,and ANOVA,single factor randomized block design,was used to analyse the apoptosis rate.Results The expression of iPLA2 significantly weakened under intermittent high glucose from 1.90 ± 0.23 to 0.90 ± 0.05 and 0.23 ± 0.03,compared with under normal glucose and under constant high glucose; successfully synthesized and transfected iPLA2 siRNA,after silencing iPLA2 gene,the DNA fragmentation became more apparent and the flow cytometry apoptosis significantly increased under intermittent high glucose conditions （44.9 ± 2.7）,compared with under normal glucose （3.1 ± 1.0,P ＜ 0.05）.Conclusions The expression of IMECs iPLA2 would be suppressed in intermittent high glucose condition.Inhibiting the iPLA2 gene expression by small interfering technology can promote apoptosis,suggesting that iPLA2 expression weakening may be involved in the IMECs apoptosis induced by intermittent high glucose.