CAJ | 학술논문

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50 mg/L),分别于8h、24 h和48 h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100 mL/L FBS的RPMI 1640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-4 mRNA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,相邻细胞间足突相互连接.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0-actin-4 mRNA各时间点表达比较,8h差异无统计学意义(P＞0.05),24 h、48 h差异有统计学意义(P均＜0.01),实验组在24 h、48 h后α-actin-4 mRNA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24 h后细胞质中α-actin-4分布明显减少,刺激48 h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.
목적 관찰표령매소(PAN)대족세포α-보기동단백4(α-actin-4)mRNA표체화분포적영향,탐토α-actin-4여족세포손상적관계.방법 장체외배양신소구족세포계(MPC5)수궤분위실험조여대조조.실험조채용PAN자격(제량위50 mg/L),분별우8h、24 h화48 h관찰세포형태,제취총RNA화세포면역조직화학관찰α-actin-4적분포.대조조용100 mL/L FBS적RPMI 1640배양액배양.응용Image J도상연건처리세포도상,계산2조수궤선택적30개세포재상술3개시간점200배경하적세포상대면적,실시정량형광취합매련반응(RT-PCR)화간접면역형광법검측α-actin-4 mRNA표체화분포변화.결과 대조조족세포생장정황량호,포체정불규칙성형,여증식상태족세포비교,세포체화세포핵균현저증대,자포체신출수지양돌기,상린세포간족돌상호련접.실험조세포포체축소,족돌회축、소실,세포간실거상호련접.2조간0-actin-4 mRNA각시간점표체비교,8h차이무통계학의의(P＞0.05),24 h、48 h차이유통계학의의(P균＜0.01),실험조재24 h、48 h후α-actin-4 mRNA표체승고.대조조α-actin-4재포질내정세사상균균분포,재족돌중정방사상분포;8h후재실험조α-actin-4압력사섬유변단,배렬문란,PAN자격24 h후세포질중α-actin-4분포명현감소,자격48 h후출현세포질분포결실.결론 α-actin-4표체분포적이상여PAN손상족세포적시간정정상관,α-actin-4시족세포손상궤제중적일개중요분자.
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.