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中华老年骨科与康复电子杂志

中华老年骨科与康复电子杂志 ›› 2022, Vol. 08 ›› Issue (03) : 152 -158. doi: 10.3877/cma.j.issn.2096-0263.2022.03.005

基础研究

二甲胺四环素对小胶质细胞激活状态影响的研究
左安俊1, 欧振飞1, 王天瑞1, 丁磊1, 李天予1, 于腾波1,†()   
  1. 1. 266100　青岛大学附属医院运动医学科
  • 收稿日期:2021-11-15 出版日期:2022-06-05
  • 通信作者: 于腾波
  • 基金资助:
    国家自然科学基金资助项目(31872310)

The effect of minocycline on the activation state of microglia

anjun Zuo1, zhenfei Ou1, tianrui Wang1, lei Ding1, tianyu Li1, tengbo Yu1,†()   

  1. 1. The Affiliated Hospital of Qingdao University, Department of Sports Medicine, Qingdao 266100, China
  • Received:2021-11-15 Published:2022-06-05
  • Corresponding author: tengbo Yu
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左安俊, 欧振飞, 王天瑞, 丁磊, 李天予, 于腾波. 二甲胺四环素对小胶质细胞激活状态影响的研究[J/OL]. 中华老年骨科与康复电子杂志, 2022, 08(03): 152-158.

anjun Zuo, zhenfei Ou, tianrui Wang, lei Ding, tianyu Li, tengbo Yu. The effect of minocycline on the activation state of microglia[J/OL]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2022, 08(03): 152-158.

目的

探究二甲胺四环素对大鼠小胶质细胞M1、M2激活状态的研究。

方法

取10只Wistar大鼠脑组织，选取其小胶质细胞，以LPS及IL-4产生M1激活组和M2激活组，确定Notch信号通路对小胶质细胞M1激活途径及M2激活途径的调控作用，通过不同浓度二甲胺四环素干预Notch信号通路，调控小胶质细胞的激活状态，观察细胞膜特异性抗原CD16/32及CD206蛋白表达和细胞培养上清中TNF-α、IL-12、IL-10水平。

结果

成功诱导M1激活组和M2激活组，确定Notch信号通路对M1激活途径及M2激活途径起调控作用，二甲胺四环素在0 μg/ml、5 μg/ml、10 μg/ml、20 μg/ml浓度中，促使小胶质细胞TNF-α、IL-12分泌逐渐减少，IL-10分泌逐渐增多，CD16/32蛋白表达逐渐降低，CD206蛋白表达逐渐增加。

结论

二甲胺四环素能够抑制小胶质细胞向M1型极化，且对M2型极化过程没有影响，可以改善脊髓损伤患者的预后。

关键词: 二甲胺四环素, 脊髓损伤, 小胶质细胞
Objective

To investigate the activation status of minocycline on microglia M1 and M2 in rats.

Methods

The brain tissue of 10 Wistar rats was used to select microglia cells, and LPS and IL-4 were used to produce M1-activated group and M2-activated group to determine the regulatory effect of Notch signaling pathway on M1-activated pathway and M2-activated pathway of microglia. The Notch signaling pathway was interfered with different concentrations of minocycline to regulate the activation state of microglia. The expression of cell membrane specific antigen CD16/32 and CD206 and the levels of TNF-α, IL-12 and IL-10 in cell culture supernatant were observed.

Results

The M1 activation group and M2 activation group were successfully induced, and the Notch signaling pathway was determined to regulate the M1 activation pathway and M2 activation pathway. Minocycline promoted the secretion of TNF-α and IL-12 in microglia at 0 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL concentrations. The secretion of IL-10 increased gradually, the expression of CD16/32 decreased gradually, and the expression of CD206 increased gradually.

Conclusion

Minocycline can inhibit the polarization of microglia to M1-type and has no effect on M2-type polarization process, which can improve the prognosis of SCI patients.

Key words: Minocycline, Spinal cord injury, Microglial
图1~2 细胞培养。图1 M1激活组；图2 M2激活组
图3 M1激活组与M2激活组中TNF-α、IL-12、IL-10基础水平比较 注：“*”表示P<0.01
图4 M1激活组与M2激活组CD16/32及CD206基础水平比较 注："*"表示P<0.01
表1 M1激活组与M2激活组中TNF-α、IL-12、IL-10基础水平比较（±s，pg/ml）
组别 IL-10 IL-12 TNF-α
M1激活组 27.38±1.76 4.89±0.08 89.36±2.06
M2激活组 54.47±1.73 2.31±0.03 44.78±1.74
t值 -19.002 54.354 28.605
P值 <0.01 <0.01 <0.01
表2 M1激活组与M2激活组CD16/32及CD206基础水平比较（%，±s）
组别 CD16/32 CD206
M1激活组 22.48±1.78 14.04±0.53
M2激活组 11.01±2.53 21.24±1.26
t值 10.721 -4.428
P值 <0.01 <0.01
图5 Notch信号通路调控下IL-12、TNF-α、IL-10水平比较 注："*"表示P<0.01
图6 Notch信号通路调控下细胞膜CD16/32及CD206蛋白表达水平比较 注："*"表示P<0.01
表3 Notch信号通路调控下IL-12、TNF-α、IL-10水平比较（±s，pg/ml）
组别 IL-10 TNF-α IL-12
DAPT+M1激活组 13.81±1.61 61.13±3.74 3.86±0.07
M1激活洗脱+DAPT组 25.85±2.70 82.58±2.55 4.59±0.14
t值 -6.646 -8.228 -8.014
P值 <0.01 <0.01 <0.01
DAPT+M2激活组 48.39±3.40 20.81±1.70 1.27±0.15
M2激活洗脱+DAPT组 36.81±1.95 39.08±1.51 2.50±0.21
t值 5.113 -13.926 -8.342
P值 <0.01 <0.01 <0.01
表4 Notch信号通路调控下细胞膜CD16/32及CD206蛋白表达水平比较（±s，%）
组别 CD16/32 CD206
DAPT+M1激活组 32.38±1.68 11.37±1.42
M1激活洗脱+DAPT组 40.04±0.89 19.88±0.90
t值 -6.973 -8.761
P值 <0.01 <0.01
DAPT+M2激活组 11.51±1.19 43.04±2.69
M2激活洗脱+DAPT组 22.54±2.23 30.54±0.61
t值 -7.567 7.863
P值 <0.01 <0.01
图7 不同浓度二甲胺四环素对细胞存活率影响
图8 不同浓度二甲胺四环素对TNF-α、IL-12、IL-10水平影响比较 注："*"表示与前组相比有差异P<0.01
图9 不同浓度二甲胺四环素对CD16/32及CD206蛋白水平影响比较 注："*"表示与前组相比有差异P<0.01
表5 不同浓度二甲胺四环素对TNF-α、IL-12、IL-10水平影响比较（±s，pg/ml）
组别 TNF-α IL-12 IL-10
LPS 79.22±1.53bcd 5.13±0.72bcd 13.87±1.80bcd
二甲胺四环素（5 μg/mL）+LPS 60±1.32acd 3.86±0.83acd 26.68±1.89acd
二甲胺四环素（10μg/mL）+LPS 40.45±1.80abd 2.36±0.59abd 40.46±2.33abd
二甲胺四环素（20μg/mL）+LPS 19.67±1.09abc 1.29±0.11abc 53.14±1.88abc
F值 923.855 388.462 219.759
P值 <0.01 <0.01 <0.01
表6 不同浓度二甲胺四环素对CD16/32及CD206蛋白水平影响比较（±s，%）
组别 CD16/32 CD206
LPS 43.51±2.10bcd 12.48±0.64bcd
二甲胺四环素（5 μg/ml）+LPS 35.43±6.81acd 23.45±1.21acd
二甲胺四环素（10μg/ml）+LPS 23.60±0.54abd 31.96±1.42abd
二甲胺四环素（20μg/ml）+LPS 11.34±1.18abc 45.13±5.72abc
F值 45.095 62.324
P值 <0.01 <0.01
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