miR-494负调控ROCK1、PTEN抑制急性胰腺炎细胞凋亡的机制研究  

The mechanism of miR-494 negatively regulating ROCKI and PTEN in inhibiting apoptosis of acute pancreatitis cells

作　　者：冯辉[1] 孙江利[1] 牛泽群[1] 王立明[1] 裴红红[1] 潘龙飞[1] Feng Hui;Sun Jiangli;Niu Zequn;Wang Liming;Pei Honghong;Pan Longfei(Emergency Medicine Department,the Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China)

机构地区：[1]西安交通大学第二附属医院急诊科,710004

出　　处：《中华急诊医学杂志》2021年第10期1210-1215,共6页Chinese Journal of Emergency Medicine

基　　金：陕西省中医药科学技术研究课题(2019-ZZ-JC031);陕西省重点研发计划项目(2019SF-116);西安交通大学第二附属医院自由探索项目[2020YJ(ZYTS)359]。

摘　　要：目的探讨miR-494负调控ROCKI、PTEN进而抑制胰腺细胞凋亡并参与急性胰腺炎发生发展的机制研究方法雨蛙素处理大鼠胰腺腺泡AR42J细胞,ELISA法检测细胞培养上清液中淀粉酶、TNF-α、IL-1、IL-6水平以验证急性胰腺炎细胞模型:RT-PCR检测正常AR42J细胞(对照组)、急性胰腺炎细胞模型(模型组)中的miR-494表达水平。流式细胞法检测对照组、转染阴性对照miRNA的急性胰腺炎细胞模型(阴性对照组)以及转染miR-494的急性胰腺炎细胞模型(miR-494转染组)的细胞凋亡情况Western Blot检测对照组、阴性对照组以及miR-494转染组中促凋亡蛋白ROCKI、PTEN的表达情况。结果雨蛙素处理AR42J细胞8 h、12 h时的细胞培养上清液中的淀粉酶、TNF-α、IL-1、IL-6水平均显著高于0h及对照组,差异均有统计学意义(P<0.05).提示模型构建成功。急性胰腺炎细胞模型构建后8h、12h、24h的miR-494表达水平均显著高于4 h时及对照组,差异均有统计学意义(P<0.05)。模型组的细胞凋亡比例显著高于对照组,miR-494转染组的细胞凋亡比例显著低于模型组,差异均有统计学意义(P<0.05)。miR-494转染组ROCKI、PTEN蛋白的表达水平均显著低于模型组和阴性对照组,差异均有统计学意义(P<0.05),结论急性胰腺炎发生时过表达的miR-494能够抑制促凋亡蛋白表达,从而抑制胰腺腺泡细胞凋亡并促进急性膜腺炎发生发展。Objective To explore the mechanism of miR-494 negatively regulating ROCKI and PTEN in inhibiting apoptosis of pancreatic cells and participating in the occurrenee and development of acute pancreatitis.Methods Pancreatic acinar cells AR42J from rats were treated by caerulein,and then the levels of amylase,tumor necrosis factor alpha(TNF-α),interleukin 1(IL-1)and IL-6 in the supernatant of cell culture were detected by ELISA to verify the cell model of acute pancreatitis.RT-PCR was used to detect the expression of miR-494 in normal AR42J cells(control group)and acute pancreatitis cell model(model group).Flow cytometry was used to detect the apoptosis of the control group,negative control miRNA transfected acute pancreatitis cell model(negative control group)and miR-494 transfected acute pancreatitis cell model(miR-494 transfection group).Western blot was used to detect the expression of ROCKI and PTEN in the control group,negative control group and miR-494 transfection group.Results The levels of amylase,TNF-α,IL-1 and IL-6 in the supernatant of AR42J cells treated with caerulein for 8 h and 12 h were significantly higher than those at 0 h and the control group(P<0.05),indicating that the model was successfully constructed.The expression levels of miR-494 at 8 h,12 h and 24 h after the establishment of acute pancreatitis cell model were significantly higher than those at 4 h and the control group(P<0.05).The apoptosis rate of the model group was significantly higher than that of the control group(P<0.05),and the apoptosis rate of the miR-494 transfection group was significantly lower than that of the model group(P<0.05).The expression levels of ROCK1 and PTEN in the miR-494 transfection group were significantly lower than those in the model group and negative control group(P<0.05).Conclusions When acute pancreatitis occurs,overexpression of miR-494 can inhibit the expression of pro-apoptotic protein,thus inhibiting the apoptosis of pancreatic acinar cells and promoting the development of acute pancreatitis.

关 键 词：急性胰腺炎 凋亡 miR-494 促凋亡蛋白 

分 类 号：R576[医药卫生—消化系统]