嘌呤霉素损伤小鼠肾小球足细胞系中足细胞α-辅肌动蛋白4的表达与分布  

Distribution and expression of α-actin-4 in puromycin aminonueleoside injured mouse podocyte cell line

作　　者：王辉阳[1] 于力[1] 李晓艳[1] 于生友[1] 张瑶[1] 郝志宏[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院儿科,510180

出　　处：《中华实用儿科临床杂志》2014年第17期1296-1300,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（81273205）;国家科技支撑计划项目（2012BA103802）;广东省科技计划项目（20118020800002）

摘　　要：目的观察嘌呤霉素（PAN）对足细胞α-辅肌动蛋白4（α-actin-4）mRNA表达和分布的影响，探讨α-actin-4与足细胞损伤的关系。方法将体外培养肾小球足细胞系（MPC5）随机分为实验组与对照组。实验组采用PAN刺激（剂量为50mg／L），分别于8h、24h和48h观察细胞形态，提取总RNA和细胞免疫组织化学观察α-actin-4的分布。对照组用100mL／LFBS的RPMI1640培养液培养。应用Image J图像软件处理细胞图像，计算2组随机选择的30个细胞在上述3个时间点200倍镜下的细胞相对面积，实时定量荧光聚合酶链反应（RT—PCR）和间接免疫荧光法检测α-actin-4mRNA表达和分布变化。结果对照组足细胞生长情况良好，胞体呈不规则星形，与增殖状态足细胞比较，细胞体和细胞核均显著增大，自胞体伸出树枝样突起，相邻细胞问足突相互连接。实验组细胞胞体缩小，足突回缩、消失，细胞间失去相互连接。2组间α-ctin-4mRNA各时间点表达比较，8h差异无统计学意义（P＞0．05），24h、48h差异有统计学意义（P均＜0．01），实验组在24h、48h后α-actin-4mRNA表达升高。对照组α-actin-4在胞质内呈细丝状均匀分布，在足突中呈放射状分布；8h后在实验组α-actin-4压力丝纤维变短，排列紊乱，PAN刺激24h后细胞质中α-actin-4分布明显减少，刺激48h后出现细胞质分布缺失。结论α-actin-4表达分布的异常与PAN损伤足细胞的时间呈正相关，α-actin-4是足细胞损伤机制中的一个重要分子。Objective To observe the expression and distribution of α-actin-4 mRNA through puromycin aminonueleoside（PAN） injury podocyte, and discuss the relation between α-actin-4 and podocyte damage. Methods Podocytes were cultured in vitro,and 2 groups were set up：control group and PAN stimulation group. The control group was cultured with concentration of 100 mL/L FBS RPMI 1640 nutrient solution culture,while the PAN group was cultivated to PAN（50 mg/L） treatment, and cell morphology ,extraction of total RNA of α-actin-4 were observed in 8 h,24 h and 48 h. The podocyte morphology was observed and pictures were taken through phase-contrast microscope, then the differences of morphology and areas between the 2 groups were analyzed. The distribution and mRNA expression of α-actin-4 were detected by indirect immunocyto-fluorescence and real-time quantitative PCR,respectively. Results The well-developed podocyte arborization was formed after the in vitro induction, and the PAN treatment led to the podocyte foot process retraction and effacement together with the mouse podocyte cell line shrinkage and the loss of cell contact. The above time point α-actin-4 mRNA expressions between the 2 groups were compared, and there was no significant difference in 8 h （P 〉0. 05 ）, but significant difference was found in 24 h ,48 h, α-actin-4 higher mRNA expression ,with statistical significance（ all P 〈0.01 ）. α-actin-4 in the control group had thin filaments evenly distributed in the cytoplasm, but a radioactive distribution in foot process. In the experimental group, α-actin-4 pressure silk fiber was shorter,with disordered arrangement, and PAN stimulus after 24 h, α-actin-4 distribution in cytoplasm was decreased significantly, while cytoplasmic distribution was missing after 48 h. Conclusions The abnormal of distribution and mRNA expression of α-actin-4 is time-related to the PAN injury podocyte, and α-actin-4 is an important part of podocyte damage mechanism.

关 键 词：足细胞 足细胞α-辅肌动蛋白4 嘌呤霉素 

分 类 号：R726.9[医药卫生—儿科]