目的：观察多球壳菌素(ISP-1)对体外培养的正常大鼠肾小球系膜细胞(GMC)的促凋亡作用, 研究ISP-1对细胞周期调节蛋白基因表达谱和凋亡相关蛋白Bax、Bcl-2表达的影响。方法：体外培养大鼠GMC, 加入100μmol/L ISP-1干预, 分别于作用6、12、24、48 h后以流式细胞术检测GMC凋亡, Hoechst33258/PI染色观察凋亡细胞形态变化, 琼脂糖DNA凝胶电泳法观察凋亡细胞核小体DNA的断裂现象, 并通过SuperArray Real-Time PCR细胞周期基因芯片测定ISP-1对细胞周期调节蛋白基因表达谱的影响, Western blotting法观察Bax和Bcl-2蛋白的表达。结果：ISP-1可显著诱导GMC凋亡, 并呈-定的时间依赖性, 作用48 h后细胞凋亡最显著, Hoechst33258/PI荧光染色法可见亮蓝色的凋亡小体, 琼脂糖凝胶电泳可见凋亡梯度的出现;PCR细胞周期基因芯片检测结果发现ISP-1可显著上调GMC Rad51、Atm、Brcal、Caspase3、cyclinA2、cyclinC、chek1、cyclinB1、cyclinB2、cyclinD2、cyclinF、Cdc25a和P27等-些与DNA损伤、凋亡和细胞周期调节蛋白相关的基因的表达;Western blotting发现ISP-1可显著上调GMC Bax蛋白表达, 下调Bcl-2蛋白表达。结论：ISP-1可时间依赖性诱导体外培养的正常大鼠GMC凋亡, 其机制与影响细胞周期调节蛋白及凋亡相关基因的表达有关。

Objective: To observe the effects of myriocin(ISP-1)on improvement of glomerular messangial cell(GMC)apoptosis and on gene expression profiles of cell cycle regulatory proteins in GMC. Methods: Rat GMC was cultured with 100 umol/LISP-1 for 6, 12, 24, and 48h, apoptosis was evaluated through flow cycometry, Hoechst33258/PIfluorescence stainigand DNA fragmentation analysis was carried out under Sepharose electrophoresis to observe thechanges of apoptosis and DNA fragmentation. Gene expression profilesof cellcycle regulatory proteinswere detected by Super Array Real-Time PCR microarray analysis. The expressionof Bax and 13cl-2 proteins was investigated byWestern blotting. Results: ISP-1 could significantly induce GMC apoptosisin atime-dependent manner, whichwas the most significant after 48 h. Apoptosis bodysof GMC were observedby Hoechst/PIfluorescence staining, and a typical ladder pattern was identified in DNA electrophoresis. SuperArray Real-Time PCRmicroarray analysis revealed that ISP-1 couldup-regulatethe expression ofgenes involved in DNAdamage, apoptosis and cell cycle regulation, such as Rad51, Atm, Brcal, caspases, cyclinA2, cyclinC, chekl, cyclinBl, cyclinB2, cyclinD2, cyclinF, Cdc25a, and P27. Western blotting showed that ISP-1 could significantly up-regulate Baxprotein expression and down-regulate Bcl-2 protein expression, respectively. Conclusion: ISP-1 could induce GMC apoptosis in a time-dependent manner, propabably through influencing gene expression of cell cycle regulatory proteins and apoptosis proteins.

国家自然科学基金资助项目(30472268);湖北省科技攻关项目(2007AA301B44)