基于SDF-1/CXCR4轴探讨补肾壮筋汤促进小鼠BMSCs归巢和保护关节软骨的机制研究  

Mechanism of Bushen Zhuangjin Decoction to Promote BMSCs Homing and Protect Articular Cartilage in Mice by the SDF-1/CXCR4 Axis

作　　者：黄艳峰 马德尊 付长龙 叶锦霞 黄云梅 李西海 HUANG Yanfeng;MA Dezun;FU Changlong;YE Jinxia;HUANG Yunmei;LI Xihai(Academy of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China;Fujian Key Laboratory of Integrative Medicine on Geriatrics,Fuzhou,Fujian 350122,China;School of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China)

机构地区：[1]福建中医药大学中西医结合研究院,福建福州350122 [2]福建省中西医结合老年性疾病重点实验室,福建福州350122 [3]福建中医药大学中西医结合学院,福建福州350122

出　　处：《康复学报》2024年第1期44-54,共11页Rehabilitation Medicine

基　　金：国家自然科学基金项目(82074461);福建省中青年教师教育科研项目(JAT231042);福建省科学技术厅引导性项目(2021Y0032);陈可冀中西医结合发展基金资助项目(CKJ2023004)。

摘　　要：目的探讨补肾壮筋汤调控SDF-1/CXCR4轴促进小鼠BMSCs归巢和保护关节软骨的作用机制,为膝骨关节炎(KOA)的康复治疗提供实验依据。方法①动物实验中,选用8周龄SPF级C57BL/6雄性小鼠30只,采用随机数字表法分为假手术组、模型组、补肾壮筋汤组,每组10只,干预12周。采用micro-CT、HE染色观察各组软骨形态变化;激光共聚焦显微镜观察骨组织SDF-1荧光强度;qPCR、Western blot检测各组归巢相关调控因子mRNA转录水平和蛋白相对表达量。②细胞实验中,选用4周龄SPF级C57BL/6雄性小鼠,采用全骨髓贴壁法提取原代BMSCs,流式细胞术对所提取的细胞进行鉴定后,筛选最佳慢病毒MOI值;随机将细胞分为空白组、空载组、补肾壮筋汤组、sh-SDF-1组、sh-SDF-1+补肾壮筋汤组5组,采用划痕实验观察各组BMSCs迁移情况;阿利新蓝、CollagenⅡ免疫细胞学染色观察各组细胞成软骨分化能力;激光共聚焦显微镜观察各组SDF-1、CXCR4的荧光强度;qPCR检测各组归巢相关调控因子mRNA转录水平。结果①动物实验中,关节组织形态学结果(micro-CT、HE染色)显示:与假手术组比较,模型组股骨髁间可见一圆形缺损,骨皮质失连,软骨细胞缺失;与模型组比较,补肾壮筋汤组股骨髁间环形缺损好转,软骨细胞排列稍紊乱。关节组织免疫荧光显示:与假手术组比较,模型组SDF-1蛋白相对表达量升高;与模型组比较,补肾壮筋汤组SDF-1的蛋白相对表达量升高(P<0.05)。qPCR与Western blot结果显示:与假手术组比较,模型组归巢关键调控因子(SDF-1、CXCR4、MIP-1α、MCP-1、MIP-1β、RANTES、VEGF、G-CSF、NCAM-1、MMP-2)的mRNA转录水平和蛋白相对表达量升高(P<0.05);与模型组比较,补肾壮筋汤组归巢关键调控因子mRNA转录水平和蛋白相对表达量升高(P<0.05)。②细胞实验中,BMSCs经细胞流式术鉴定:CD44、CD105呈阳性表达,CD34呈阴性表达。当病毒MOI值为100时,SDF-1基因感染率�Objective To investigate the mechanism by which Bushen Zhuangjin Decoction(BZD)regulates the SDF-1/CXCR4 axis to promote the homing of bone marrow mesenchymal stem cells(BMSCs)and protect articular cartilage in mice,thereby providing experimental basis for the rehabilitation treatment of knee osteoarthritis(KOA).Methods(1)In the animal experiment,30 SPF male C57BL/6 mice,8 weeks old,were selected and randomly divided into sham group,model group and BZD group,with 10 mice in each group.Intervention in each group lasted for 12 weeks.The morphological changes of the cartilage in each group were observed by micro-CT and hematoxylin-eosin staining.The fluorescence intensity of SDF-1 in bone tissue was observed by laser confocal microscopy.qPCR and Western blot were used to detect mRNA transcription level and protein relative expression level of homing-related regulatory factors in each group.(2)In the cell experiment,4-week-old SPF male C57BL/6 mice were selected and the primary BMSCs were extracted by the whole bone marrow adherence method.After the extracted cells were identified by flow cytometry,the optimal lentivirus MOI value was screened.The cells were randomly divided into five groups:blank group,empty vector group,BZD group,sh-SDF-1 group and sh-SDF-1+BZD group.The migration of BMSCs in each group was observed by cell scratch test.The chondrogenic differentiation ability of cells in each group was observed by immunocytological staining of CollagenⅡand Alcian blue.The fluorescence intensity of SDF-1 and CXCR4 in each group was observed by confocal laser microscope.mRNA transcription level of homing-related regulatory factors were detected by qPCR.Results(1)In the animal experiment,the joint histomorphologic findings(micro-CT and hematoxylin-eosin staining)showed that compared with the sham group,there was a circular defect between the femoral condyles,with cortical separation and loss of chondrocytes in the model group(P<0.05);compared with the model group,the annular defect between the femoral condyles was

关 键 词：软骨损伤 补肾壮筋汤 BMSCS SDF-1/CXCR4 归巢 

分 类 号：R285.5[医药卫生—中药学]