上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

血管紧张素Ⅱ对大鼠肾小管上皮细胞程序性坏死的影响

王鱼璇，甘华   

  1. 重庆医科大学附属第一医院肾内科， 重庆 400016
  • 出版日期:2015-06-28 发布日期:2015-07-30
  • 作者简介:王鱼璇（1989—）， 女， 硕士生; 电子信箱: 236594590@qq.com。

Effects of angiotensin Ⅱ on necroptosis of rat renal tubular epithelial cells

WANG Yu-xuan, GAN Hua   

  1. Department of Nephrology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Online:2015-06-28 Published:2015-07-30

PDF (PC)

1675

摘要：

目的 探讨血管紧张素Ⅱ（AngⅡ)对大鼠肾小管上皮细胞(NRK-52E)程序性坏死的影响。方法 体外合成RIP3 siRNA干扰片段，用siRNA-mate转染试剂转染NRK-52E细胞，Western blotting验证RIP3蛋白表达。MTT法测细胞存活率，流式细胞仪测细胞凋亡率，荧光酶标仪测细胞中的活性氧(ROS)，实时定量PCR和Western blotting检测RIP1和RIP3 mRNA和蛋白的表达。结果 RIP3 siRNA转染NRK-52E后，与对照组比较，RIP3蛋白表达明显下降(P<0.01)。与对照组比较，AngⅡ组存活率下降，凋亡率及ROS、RIP1和RIP3表达明显升高(P<0.01)。与AngⅡ组比较，Z-VAD提高细胞存活率，降低凋亡率和ROS水平(P<0.01)，上调RIP1和RIP3表达(P<0.01)；Nec-1和RIP3 siRNA降低坏死率(P<0.01)，对凋亡率无明显影响(P>0.05)，并能降低ROS、RIP1和RIP3的表达(P<0.01)。结论 AngⅡ能诱导大鼠肾小管上皮细胞发生程序性坏死，与RIP1、RIP3及ROS生成有关。

关键词: 程序性坏死, 血管紧张素Ⅱ, 肾小管上皮细胞, RIP1, RIP3, 活性氧

Abstract:

Objective To explore the effects of angiotension Ⅱ (Ang Ⅱ) on the necroptosis of rat renal tubular epithelial cells (NRK-52E). Methods The small interfering RNA (siRNA) targeted at the RIP3 mRNA was synthesized in vitro and was transfected by the transfection reagent siRNA-mate to NRK-52E. The protein expression of RIP3 was detected by the Western blotting. The cell viability was evaluated by the MTT assay. The cell apoptosis rate was determined by the flow cytometer. The intracellular reactive oxygen species (ROS) level was detected by the fluorescent microplate reader. The mRNA and protein expressions of RIP1 and RIP3 were detected by the qPCR and Western blotting. Results Compared with the control group, the protein expression of RIP3 significantly decreased after RIP3 siRNA was transfected by NRK-52E (P<0.01). Compared with the control group, the cell viability of the AngⅡ group decreased, while the apoptotic rate, ROS level, and expressions of RIP1 and RIP3 significantly increased (P<0.01). Compared with the AngⅡ group, Z-VAD increased the cell viability, decreased the apoptotic rate and ROS level (P<0.01), and up-regulated the expressions of RIP1 and RIP3 (P<0.01). Nec-1 and RIP3 siRNA decreased the necrosis rate (P<0.01), had no significant effect on the apoptotic rate (P>0.05), and decreased the ROS level and expressions of expressions of RIP1 and RIP3 (P<0.01). Conclusion AngⅡ can induce the necroptosis of rat renal tubular epithelial cells, which is relevant to the generation of RIP1, RIP3 and ROS.

Key words: necroptosis, angiotensionⅡ, renal tubular epithelial cell, RIP1, RIP3, reactive oxygen species