摘要
目的：探讨晚期糖基化终末产物（AGE）对足细胞凋亡的影响,及氧化应激在其中的作用。方法：小鼠足细胞株由美国纽约西奈山医学院Peter Mundel教授馈赠。用钙磷脂结合蛋白Ⅴ-荧光异硫氰酸盐（FITC）和碘化物（PI）标记细胞,采用荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。Dharmacon On TargetPlus SMARTpool si RNA试剂和Amaxa RNAi nucleofection试剂盒成功转染si RNA到足细胞。绿荧光蛋白载体证明转染的有效性,分别采用Western Blot和实时定量PCR（RT-PCR）方法来检测si RNA转染足细胞后AGE受体蛋白（RAGE）靶基因蛋白质和mRNA的表达。用LS50B型荧光分光光度计测活性氧,根据波长485nm在530nm发射的荧光来判断活性氧（ROS）的产生。观察活性氧的清除剂N-乙酰基-半胱氨酸（NAC）能否减少AGE-BSA诱导的足细胞凋亡。结果：AGE引起足细胞的凋亡呈剂量依赖性,随AGE浓度的增大,凋亡的发生率逐渐升高;RAGE siRNA能减少60%~70%RAGE mRNA和蛋白质的表达;ROS的清除剂NAC可明显减少AGE-BSA引起的ROS的产生和足细胞凋亡。结论：AGE与RAGE作用后活性氧产生增加,活性氧的增加可能是AGE引起足细胞凋亡的途径之一,可通过抗氧化减少ROS的产生延缓糖尿病肾病的进展。
Objective： To study the effect of AGE - RAGE interaction on podocyte apoptosis by reactive oxygen species （ROS）. Methods： Conditionally immortalized murine podocytes were obtained from Dr Peter Mundel. Fluorescence - activated cell sorting （FACS） was used for the quantification of apoptotic and necrotic podocytes after Annexin V - fluorescein isothiocyanate （FITC） and propidium iodide（PI） labeling as described in the manufacturer＇s protocol. Gene- specific siRNA was used to knock down RAGE gene. Western- blotting and Quantitative one step real - time polyrnerase chain reaction （PCR）were applied to test.the effects of siRNA for RAGE mRNA and protein expression. We incubated podocytes with N- acetyl- cysteine（NAC）,a ROS scavenger. Luminescence spectrometer Perkin - Elmer LS50B was used for the measurement of emitted fluorescence at 530 nm with exci- tation wavelength of 485 nm. Results：AGE exposure causes podocyte apoptosis in a dose- dependent manner. The siRNA was able to reduce the RAGE mRNA level and protein expression by 60 %- 70 %. AGE- RAGE interaction triggers ROS production, leading to podocyte apoptosis. Inhibition of ROS production with NAC can protect podocytes from apoptosis. Conclusion： Reduction of ROS production,AGE burden and RAGE expression are important therapeutic approaches to slow down the progression of diabetic nephropathy.
出处
《中国中西医结合肾病杂志》
2008年第10期864-866,I0002,共4页
Chinese Journal of Integrated Traditional and Western Nephrology
关键词
足细胞
晚期糖基化终末产物
活性氧
凋亡
Podocyte Advanced glycation endproducts Reactive oxygen species Apoptosis
