MST1 / 2 抑制剂促进大鼠脊髓损伤修复的实验研究
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1. 漯河医学高等专科学校基础医学部,河南 漯河 462000;2. 漯河医学高等专科学校第三附属医院,河南 漯河 462000

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MST1 / 2 inhibitor for spinal cord injury repair in rats
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1. Faculty of Basic Medicine, Luohe Medical College, Luohe 462000, China. 2. Luohe Medical College Third Affiliated Hospital, Luohe 462000

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    目的 探讨 MST1 / 2 抑制剂对大鼠脊髓损伤后 Yes 相关蛋白 YAP、神经生长相关蛋白 43、神经胶质纤维蛋白 GFAP、细胞凋亡因子 Caspase3 表达及运动功能恢复的影响。 方法 选取 105 只体重 180 ~ 200 g 成年雌性 SD 大鼠,对 35 只仅做椎板切除手术且不注射药物为假手术组,对 70 只大鼠建立脊髓顿挫损伤模型,分为生理盐水组、MST1 / 2 抑制剂组,腹腔注射生理盐水、1 mg / kg XMU-MP-1。 在手术后第 1、3、7、14、21、28 天,进行斜板实验及 BBB 运动功能评分,然后处死大鼠收集脊髓损伤区周围组织,进行免疫荧光染色和免疫印迹检测,观察各组大鼠脊髓组织中 YAP、GAP43、Caspase3、GFAP 表达变化情况。 结果 斜板实验与 BBB 运动功能评分结果显示,从术后第 7 天起,MST1 / 2 抑制剂组评分明显高于生理盐水组,一直持续到术后第 28 天,差异有统计学意义( P<0. 05)。 免疫印记结果显示,术后第 14 天 MST1 / 2 抑制剂组的 YAP 表达明显高于生理盐水组,差异有统计学意义(P< 0. 05);术后第 14 天 MST1 / 2 抑制剂组 GFAP、Caspase3 表达明显低于生理盐水组,同时发现 GAP43 出现表达,差异有统计学意义(P< 0. 05)。 荧光染色显示,MST1 / 2 抑制剂组较少炎症细胞浸润、形成神经组织结构框架,生理盐水组明显炎症细胞浸润、形成大量神经胶质瘢痕。免疫荧光染色结果显示,假手术组 YAP 在神经细胞中出现表达,生理盐水组和MST1 / 2 抑制剂组 YAP 在神经胶质细胞出现表达,且 MST1 / 2 抑制剂组 YAP、GAP43 阳性细胞数量明显高于其他两组,MST1 / 2 抑制剂组成熟肥大的星形胶质细胞数量明显少于生理盐水组;免疫荧光双标结果显示,YAP 与 GFAP 出现共同表达。 结论 脊髓损伤后大剂量应用 MST1 / 2 抑制剂能明显促进 YAP 表达,减轻炎症损伤反应,抑制神经细胞凋亡,改善脊髓损伤区微环境,促进星形胶质细胞形成原始神经组织结构框架,有利于神经突起再生修复,促进运动功能恢复。

    Abstract:

    Objective To investigate the effect of an MST1 / 2 kinase inhibitor on expression of YAP, GAP43,Caspase 3, and GFAP in rats with acute spinal cord contusion ( SCC). Methods A total of 105 adult female SD rats weighing 180 ~ 200 g were used, of which 35 rats were subjected to a laminectomy and no drug injection as the sham operation group, whereas 70 rats were subjected to spinal cord contusion and divided into normal saline and MST1 / 2 inhibitor groups injected with normal saline or 1 mg / kg XMU-MP-1, respectively. Western Blot and immunofluorescence were used to observe YAP, GAP43, Caspase3 and GFAP expression. BBB and inclined plane tests were used to observe motor functions in rats. Results The inclined plane test and BBB scoring showed the scores of the MST1 / 2 inhibitor group were significantly better than those of normal saline group from day 7 to 28 (P< 0. 05). Western Blot showed that YAP expression in the MST1 / 2 inhibitor group was significantly higher than that the normal saline group from day 14. GFAP and Caspase3 expression in the MST1 / 2 inhibitor group was significantly lower than that in the normal saline group from day 14.GAP43 expression was also observed in the MST1 / 2 inhibitor group from day 14. These differences were statistically significant (P< 0. 05). Immunofluorescence showed that the MST1 / 2 inhibitor group had less infiltration of inflammatory cells and formed a structural framework of nerve tissue, whereas the normal saline group had obvious infiltration of inflammatory cells and formed a large number of glial scars. Immunofluorescence also showed that YAP was expressed in nerve cells in the Sham operation group. Moreover, the number of YAP and GAP43-positive cells in the MST1 / 2 inhibitor group was significantly higher than that in the other two groups, and the number of mature and hypertrophic astrocyte cells in the MST1 / 2 inhibitor group was significantly lower than that in the normal saline group; double immunofluorescent staining showed that YAP and GFAP were co-expressed. Conclusions A high dose of the MST1 / 2 inhibitor decreased YAP expression, activated reactive astrocytes, reduced the inflammatory injury response, inhibited apoptosis of nerve cells, improved the microenvironment of the injured area, and promoted neurite regeneration and function recovery.

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徐纪伟,孙丹华,陈旭东,王宁,孙壕烨. MST1 / 2 抑制剂促进大鼠脊髓损伤修复的实验研究[J].中国实验动物学报,2022,30(7):942~948.

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  • 收稿日期:2022-04-23
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  • 在线发布日期: 2023-03-10
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