Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (24): 3831-3837.doi: 10.12307/2023.194
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Wang Liping1, Li Jisheng2, Deng Li2, Wang Zhiqiang2, Liu Jinming1, Deng Chen2, Sun Lin2
Received:
2022-04-01
Accepted:
2022-06-06
Online:
2023-08-28
Published:
2023-01-19
Contact:
Sun Lin, MD, Chief physician, Department of Orthopedics, Third Hospital of Shanxi Medical University (Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital), Taiyuan 030032, Shanxi Province, China
About author:
Wang Liping, Master candidate, College of Basic Medicine, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
Supported by:
CLC Number:
Wang Liping, Li Jisheng, Deng Li, Wang Zhiqiang, Liu Jinming, Deng Chen, Sun Lin. Effects of high-mobility group box 1 on different subtypes of rat spinal reactive astrocytes after oxygen-glucose deprivation/restoration in vitro[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(24): 3831-3837.
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2.2 OGD/R损伤后星形胶质细胞活化为反应性星形胶质细胞 光镜下和细胞免疫荧光染色观察显示星形胶质细胞体积增大,划痕实验显示星形胶质细胞向损伤中心迁移,与对照组相比,氧糖剥夺后细胞的迁移能力增强,表明星形胶质细胞在损伤后成为一种更为活跃的状态,细胞的迁移能力增强,见图2。Western blot结果表明,OGD6 h/R6 h开始C3和S100A10表达增多,C3在OGD6 h/R12 h表达最多(P < 0.05),而S100A10在OGD6 h/R6 h表达最多(P < 0.05),表明星形胶质细胞在损伤早期活化为A2型反应性星形胶质细胞,在损伤后期活化为A1型反应性星形胶质细胞,见图3。为了使A2型反应性星形胶质细胞在整体的丰度增加,选择A2型反应性星形胶质细胞最多的时间点OGD6 h/R6 h作为后续实验的模型组。"
2.3 HMGB1对反应性星形胶质细胞的影响 以往研究已经证明OGD6 h/R2 h即可产生HMGB1,随着复氧复糖时间的增加HMGB1逐渐增多[14]。为了研究HMGB1对反应性星形胶质细胞的影响,实验选取慢病毒转染沉默HMGB1表达后进行OGD6 h/R6 h,以及氧糖剥夺6 h后加入丙酮酸乙酯继续复氧复糖6 h抑制HMGB1的表达,Western blot结果显示HMGB1干扰组及丙酮酸乙酯组的C3表达低于OGD6 h/R6 h组(P < 0.05),S100A10表达高于OGD6 h/R6 h组(P < 0.05),见图4。C3和S100A10在细胞质表达,DAPI染细胞核呈蓝色。细胞免疫荧光结果显示,与OGD6 h/R6 h组相比,HMGB1干扰组及丙酮酸乙酯组的C3平均荧光强度明显减弱(P < 0.05),S100A10平均荧光强度明显增强(P < 0.05),见图5。 结果表明抑制HMGB1后A1型反应性星形胶质细胞减少,A2型反应性星形胶质细胞增多。"
2.5 HMGB1对反应性星形胶质细胞影响的机制 为探究HMGB1对反应性星形胶质细胞影响的机制,氧糖剥夺6 h后加入CLI-095(TLR4抑制剂)和BAY 11-7082(NF-κB抑制剂)再复氧复糖6 h,Western blot结果显示与OGD6 h/R6 h组相比,CLI-095组TLR4表达减少(P < 0.05),CLI-095组和BAY 11-7082组NF-κB表达减少(P < 0.05),CLI-095组和BAY 11-7082组C3表达减少(P < 0.05),S100A10表达增多(P < 0.05),见图7。结果说明CLI-095可以有效抑制TLR4的表达,BAY 11-7082可以有效抑制NF-κB的表达,抑制TLR4可以降低NF-κB的表达,抑制TLR4和NF-κB可以使C3表达减少,S100A10表达增多。结果表明HMGB1可以与TLR4受体结合进一步激活NF-κB减少A1型反应性星形胶质细胞,增多A2型反应性星形胶质细胞。"
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