大鼠急性胰腺炎外分泌细胞自噬中miR-148a的作用研究

doi:10.3969/j.issn.1000-8535.2021.04.001

广州医药 ›› 2021, Vol. 52 ›› Issue (4): 1-4.DOI: 10.3969/j.issn.1000-8535.2021.04.001

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大鼠急性胰腺炎外分泌细胞自噬中miR-148a的作用研究

陈希妍, 张建新, 孔令宇, 牛丽丹, 李广鹏, 朱秀英, 石金河

新乡医学院第一附属医院急诊科(卫辉 453100)

出版日期:2021-07-20 发布日期:2021-11-23
通讯作者: 石金河,E-mail:sjh4402901@163.com
基金资助:
2016年河南省组织再生重点实验室开放课题项目(KFKT16003),国家自然科学基金青年基金(81502313)

The role of miR-148a on autophagy in exocrine cells of rat acute pancreatitis

CHEN Xiyan, ZHANG Jianxin, KONG Lingyu, NIU Lidan, LI Guangpeng, ZHU Xiuying, SHI Jinhe

Department of Emergency, the First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, China

Online:2021-07-20 Published:2021-11-23

摘要/Abstract

摘要： 目的探讨miR-148a对大鼠急性胰腺炎细胞模型中细胞自噬的影响。方法选取培养AR42J细胞,细胞分为4组,即正常对照组、模型组、miR-148a mimics组及miR-148a阴性对照组。利用Lipofectamine 2000转染miR-148a mimics及阴性对照miR-148a至AR42J细胞,继续培养48 h后,利用浓度为200 μmol的牛磺胆酸钠盐(TLCs)刺激以上两组及模型组AR42J细胞20 min,正常对照组不做处理,然后提取各组细胞蛋白及RNA。利用RT-qPCR检测各组细胞中miR-148a的表达;利用CCK8实验检测各组细胞的活性;利用ELISA法检测各组细胞培养液中炎性因子IL-6,IL-1β及TNF-α的含量;利用Western blot检测自噬相关的基因Beclin1、LC3Ⅰ、 LC3Ⅱ的表达。结果 RT-qPCR结果显示,与正常对照组相比较,模型组心肌细胞中miR-148a mRNA的表达降低,而miR-148a mimics组细胞中miR-148a mRNA的表达显著升高;CCK-8实验结果显示,转染miR-148a mimics至细胞后,可提高模型细胞的活性;ELISA实验结果显示,与模型组相比较,转染miR-148a mimics至细胞后,细胞培养液中炎性因子IL-6,IL-1β及TNF-α的含量显著降低;Western blot结果显示,与模型组相比较,转染miR-148a mimics至细胞后,可降低细胞中Beclin1的表达,降低LC3Ⅱ/LC3Ⅰ的比率。结论利用miR-148a mimics提高TLCs刺激的细胞模型中的miR-148a表达后,细胞中Beclin1的表达降低,LC3Ⅱ/LC3Ⅰ的比率降低,抑制了细胞自噬,降低了炎性因子IL-6、IL-1β、TNF-α的释放,从而提高了细胞的活性,miR-148a可通过调节模型细胞的自噬而发挥细胞保护作用。

关键词: 急性胰腺炎, miR-148a, 自噬, 细胞活性

Abstract: Objective To investigate the effect of miR-148a on autophagy in rat acute pancreatitis cell model. Methods AR42J cells were cultured and divided into 4 groups: normal control group, model group, miR-148a mimics group and miR-148a negative control group. miR-148a mimics and miR-148a negative control were transfected to AR42J cells with Lipofectamine 2 000, then cells were cultured for 48 h. The AR42J cells were stimulated with sodium taurocholate (TLCs) at a concentration of 200 μmol for 20 min, the normal control group was not treated, then the protein and RNA were extracted in each group. The expression of miR-148a was detected by RT-qPCR in each group. The activity of cells was detected by CCK8 assay in each group. The contents of IL-6, IL-1β and TNF-α in the cell culture medium were detected by ELISA. Western blot was used to detect the expression of autophagy related genes Beclin1, LC3Ⅰ and LC3Ⅱ. Results RT-qPCR results showed that the expression of miR-148a mRNA in model group was significantly lower than that in normal control group, while the expression of miR-148a mRNA in miR-148a mimics group was significantly higher than that in normal control group. The results of CCK-8 assay showed that miR-148a could significantly increase the activity of model cells stimulated by TLCs. The results of ELISA showed that the contents of IL-6, IL-1β and TNF-α in cell culture medium were significantly decreased after miR-148a mimics transfection, compared with the model group. Western blot showed that miR-148a mimics could significantly decrease the expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ, compared with the model group. Conclusion miR-148a mimics was used to enhance the expression of miR-148a in cells model stimulated by TLCs, the expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ were decreased, and the autophagy was inhibited. The release of IL-6, IL-1β and TNF-α was decreased and the activity of cells was increased. miR-148a plays a cellular protective role by regulating autophagy in model cells.

Key words: Acute pancreatitis, miR-148a, Autophagy, Cell activity

陈希妍, 张建新, 孔令宇, 牛丽丹, 李广鹏, 朱秀英, 石金河. 大鼠急性胰腺炎外分泌细胞自噬中miR-148a的作用研究[J]. 广州医药, 2021, 52(4): 1-4.

CHEN Xiyan, ZHANG Jianxin, KONG Lingyu, NIU Lidan, LI Guangpeng, ZHU Xiuying, SHI Jinhe. The role of miR-148a on autophagy in exocrine cells of rat acute pancreatitis[J]. Guangzhou Medical Journal, 2021, 52(4): 1-4.

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大鼠急性胰腺炎外分泌细胞自噬中miR-148a的作用研究

The role of miR-148a on autophagy in exocrine cells of rat acute pancreatitis

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