局部应用霉酚酸酯抑制成纤维细胞增殖及迁移预防硬膜外纤维化  

Topical application of mycophenolate mofetil prevents epidural fibrosis by inhibiting the proliferation and migration of fibroblasts

作　　者：朱东明 张振[1] 张杰[2] 颜连启[3] Zhu Dongming;Zhang Zhen;Zhang Jie;Yan Lianqi(Dalian Medical University,Dalian 116044,Liaoning Province,China;The Second Xiangya Hospital of Central South University,Changsha 410012,Hunan Province,China;Northern Jiangsu People’s Hospital,Yangzhou 225001,Jiangsu Province,China)

机构地区：[1]大连医科大学,辽宁省大连市116044 [2]中南大学湘雅二医院,湖南省长沙市410012 [3]苏北人民医院,江苏省扬州市225001

出　　处：《中国组织工程研究》2022年第16期2551-2556,共6页Chinese Journal of Tissue Engineering Research

基　　金：江苏省医学创新团队课题(CXTDB2017004),项目参与者:颜连启。

摘　　要：背景:研究发现,椎板切除术后的硬膜外纤维化主要由成纤维细胞增殖及迁移造成,而霉酚酸酯能够抑制成纤维细胞的增殖和迁移。目的:探讨局部应用霉酚酸酯预防椎板切除术后硬膜外纤维化的作用及其可能机制。方法:(1)细胞实验:分别以0,0.01,0.1,1,10,100μmol/L的霉酚酸酯溶液处理成纤维细胞24 h,采用CCK-8法检测细胞活性,选择较适宜的处理浓度进行后续的细胞实验。分别以0,0.1,1,10μmol/L的霉酚酸酯溶液处理成纤维细胞24 h,Ed U和细胞周期实验检测细胞增殖情况,划痕实验与Transwell细胞迁移实验检测细胞迁移能力,Western blot检测细胞增殖相关蛋白(细胞核增殖抗原、Cyclin D1)与迁移相关蛋白(α-微管蛋白、黏着斑蛋白)的表达量。(2)动物实验:取48只成年SD大鼠,构建大鼠椎板切除模型,随机分4组:对照组将浸透生理盐水的棉片置于术后骨缺损区域,低、中、高浓度组分别将浸透2.5,5,10 g/L霉酚酸酯溶液的棉片置于术后骨缺损区域,术后4周取术区椎体进行组织学分析。结果与结论:(1)细胞实验结果:CCK-8检测显示,霉酚酸酯呈浓度依赖性抑制成纤维细胞的活性;Ed U实验显示,霉酚酸酯呈浓度依赖性抑制成纤维细胞的增殖;流式细胞仪检测显示,0.1μmol/L的霉酚酸酯溶液使细胞周期停滞在G0/G1期;划痕实验与Transwell实验显示,霉酚酸酯呈浓度依赖性抑制成纤维细胞的迁移能力;Western blot检测显示,霉酚酸酯呈浓度依赖性抑制成纤维细胞增殖相关蛋白与迁移相关蛋白的表达;(2)动物实验结果:组织学观察显示,随着霉酚酸酯浓度增加,术区纤维化组织中成纤维细胞数量与胶原生成逐渐减少;(3)结果表明,椎板切除术后局部应用霉酚酸酯可有效预防硬膜外纤维化的发生,可能是通过抑制成纤维细胞的增殖和迁移发挥作用。BACKGROUND: Studies have shown that epidural fibrosis after laminectomy is mainly caused by the proliferation and migration of fibroblasts, and mycophenolate mofetil can inhibit the proliferation and migration of fibroblasts. OBJECTIVE: To explore the effect of topical application of mycophenolate mofetil in preventing epidural fibrosis after laminectomy and its possible mechanism.METHODS:(1) Cell test: Fibroblasts were separately treated with 0, 0.01, 0.1, 1, 10, and 100 μmol/L mycophenolate mofetil solution for 24 hours. Cell viability was detected by CCK-8 assay, and the appropriate treatment concentration was selected for subsequent cell experiments. Fibroblasts were separately treated with 0, 0.1, 1, 10 μmol/L mycophenolate mofetil solution for 24 hours. EdU assay and cell cycle assay were used to detect cell proliferation. Wound scratch assay and transwell assay were used to detect cell migration. Western blot assay was used to detect the expression of cell proliferation-related proteins(nuclear proliferation antigen, Cyclin D1) and migration-related proteins(α-tubulin, vinculin).(2) Animal test: Forty-eight adult SD rats were selected to construct rat laminectomy model and randomly divided into four groups. In the control group, cotton pads soaked with normal saline were placed on the postoperative bone defect area. In the low, medium, and high concentration groups, cotton pads soaked with 2.5, 5, and 10 g/L mycophenolate mofetil solution were placed on the postoperative bone defect area. Four weeks after the operation, the vertebral body of the operative area was taken for histological analysis. RESULTS AND CONCLUSION:(1) Cell test results: CCK-8 assay showed that mycophenolate mofetil inhibited the viability of fibroblasts in a concentrationdependent manner. EdU assay showed that mycophenolate mofetil inhibited the proliferation of fibroblasts in a concentration-dependent manner. Flow cytometry showed that 0.1 μmol/L mycophenolate mofetil solution blocked the cell cycle in G0/G1 phase. Wound scratch

关 键 词：材料 霉酚酸酯 成纤维细胞 增殖 迁移 硬膜外纤维化 动物 小鼠 实验 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]