中国血液净化

中国血液净化 ›› 2021, Vol. 20 ›› Issue (11): 746-750.doi: 10.3969/j.issn.1671-4091.2021.11.006

• 基础研究 • 上一篇    下一篇

赖氨酸甲基转移酶SET8 介导损伤调节自噬基因调控血管平滑肌细胞凋亡的研究

张东雪1,刘兰1,张胜雷1,崔立文1,冯青燕1   

  1. 1河北医科大学第四医院肾内科
  • 收稿日期:2021-07-05 修回日期:2021-08-12 出版日期:2021-11-12 发布日期:2021-10-29
  • 通讯作者: 张胜雷 lei06352511@126.com E-mail:125657481@qq.com
  • 基金资助:
    河北省医学科学研究重点课题(20180513、20190702)

Lysine methyltransferase SET8 mediates damage regulated autophagy modulator 1 (DRAM1 ) to regulate apoptosis of vascular smooth muscle cells

  1. 1Departments of Nephrology, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050000, China
  • Received:2021-07-05 Revised:2021-08-12 Online:2021-11-12 Published:2021-10-29

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摘要： 【摘要】目的探讨赖氨酸甲基转移酶SET8(lysine methyltransferase SET8)介导损伤调节自噬基因(damage-regulated autophagy modulator 1，DRAM1)调控血管平滑肌细胞(vascular smooth muscle cells, VSMCs)凋亡的作用及机制。方法体外原代培养VSMCs，采用脂质体转染法处理细胞，将细胞分为3 组，正常组，SET8-NC 组(转染对照NS-shRNA 质粒)，SET8-shRNA 组(转染SET8-shRNA 质粒)。采用MTT比色法检测各组细胞增殖情况；原位末端标记法TUNEL 染色检测细胞凋亡情况；Western blot 检测各组细胞SET8、DRAM1、B 细胞淋巴瘤/白血病-2(bcelllymphoma2，Bcl-2)、Bcl-2 相关x 蛋白(Bcl-2 assaciated X protein，Bax)的表达情况。为验证DRAM1 对VSMCs 凋亡的影响，转染DRAM1 高表达，将细胞分为3 组，正常组，DRAM1-NC 组，DRAM1 组。检测各组细胞增殖凋亡情况及DRAM1、Bax 和Bcl-2的蛋白表达情况。结果①与正常组和SET8-NC 组比较，MTT 结果显示SET8-shRNA 组细胞于12h、24h、36h、48h 增殖降低(F=267.649，P＜0.001；F=477.534，P＜0.001；F=28.938，P=0.001；F=145.849，P＜0.001)，TUNEL 染色
结果显示SET8-shRNA 组绿色荧光颗粒显著增多。Western blot 结果显示，与正常组和SET8-NC 组比较，SET8- shRNA 组SET8、Bcl- 2 表达降低(F=34.247,P=0.001；F=39.218,P＜0.001)，DRAM1、Bax 表达升高(F=84.517,P＜0.001；F=15.570,P=0.004)。②与正常组和DRAM1-NC 组比较，MTT 结果显示DRAM1 组细胞于12h、24h、36h、48h 增殖降低(F=347.170，P＜0.001；F=74.621，P＜0.001；F=56.427，P＜0.001；F=393.547，P＜0.001)，TUNEL 染色结果显示DRAM1 组绿色荧光颗粒显著增多。Western blot 结果显示，与正常组和DRAM1-NC 组比较，DRAM1 组DRAM1 和Bax 表达升高(F=26.456,P=0.001；F=25.217,P=0.001)，Bcl-2 表达降低(F=626.70,P＜0.001)。结论SET8 低表达可促进血管平滑肌细胞发生凋亡，可能机制是通过上调DRAM1表达，促进凋亡蛋白Bax 表达，进而引起血管平滑肌细胞发生凋亡。

关键词: 血管平滑肌细胞, 赖氨酸甲基转移酶SET8, 自噬调节因子1, 凋亡

Abstract: 【Abstract】Objective To investigate the role and mechanism of lysine methyltransferase SET8 mediated damage regulated autophagy modulator 1 (DRAM1) in regulating the apoptosis of vascular smooth muscle cells (VSMCs). Methods VSMCs were cultured in vitro and treated with liposome transfection. The cells were divided into three groups: normal group, SET8-NC group (Transfection of control NS shRNA plasmid), and SET8-shRNA group (Transfection of SET8 shRNA plasmid). MTT colorimetry was used to detect cell proliferation in each group, and apoptosis was detected by in situ end labeling and TUNEL staining, and Western blot was used to detect the protein expression of SET8, DRAM1, B-cell lymphoma / leukemia-2 (Bcelllymphoma2,
Bcl-2), bcl-2 associated X protein (Bcl-2 Associated X protein, Bax) in each group. In order to verify the effect of DRAM1 on the apoptosis of VSMCs, we transfected DRAM1 to make it highly expressed, then divided the cells into three groups: the normal group, the DRAM1-NC group, and the DRAM1 group. The proliferation and apoptosis of each group and the protein expression of DRAM1, Bax and Bcl-2 were detected. Results ①Compared with the normal group and the SET8-NC group, MTT results showed that the proliferation of cells in set8 shRNA group decreased significantly at 12h, 24h, 36h and 48h (F=267.649, P＜0.001; F= 477.534，P＜0.001; F=28.938，P=0.001; F=145.849，P＜0.001), and the TUNEL staining results showed that the apoptosis of the SET8-shRNA group was significantly increased. Western blot results showed that, compared with the normal group and the SET8-NC group, the expression of SET8 and Bcl-2 in the SET8-shRNA group decreased (F=34.247, P=0.001; F=39.218, P＜0.001), and the expression of DRAM1 and Bax increased significantly (F=84.517, P＜0.001; F=15.570, P=0.004). ② Compared with the normal group and the DRAM1-NC group, MTT results showed that the proliferation of cells in DRAM1 group decreased significantly at 12h, 24h, 36h and 48h (F= 347.170, P＜0.001; F=74.621, P＜0.001; F=56.427, P＜0.001; F=393.547, P＜0.001). TUNEL staining showed that the green fluorescent particles in DRAM1 group increased significantly. Western blot results showed that, compared with the normal group and the DRAM1- NC group, the expression of DRAM1 and Bax in the DRAM1 group was increased (F=26.456, P=0.001; F=25.217, P=0.001), and the expression of Bcl- 2 was significantly decreased (F=626.70, P＜0.001). Conclusion The low expression of SET8 can promote the apoptosis of vascular smooth muscle cells. The possible mechanism is that the low expression of SET8 promotes the expression of DRAM1 and promote the expression of apoptotic protein Bax, which in turn causes the apoptosis of vascular smooth muscle cells.

Key words: Vascular smooth muscle cells, Lysine methyltransferase SET8, Damage-regulated autophagy modulator 1, Apoptosis

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