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、Collagen Ⅱ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基因感染率最高.BMSCs迁移和成软骨分化能力检测结果显示:与空白组比较,sh-SDF-1组细胞向划痕区域迁移的细胞数量、酸性黏多糖及Collagen Ⅱ蛋白相对表达量显著减少(P＜0.05);补肾壮筋汤组和sh-SDF-1+补肾壮筋汤组迁移的细胞数量、阿利新蓝染色及Collagen Ⅱ蛋白相对表达量显著增多(P＜0.05).细胞免疫荧光显示:与空白组比较,sh-SDF-1组细胞SDF-1、CXCR4蛋白相对表达量显著减少;补肾壮筋汤组和sh-SDF-1+补肾壮筋汤组细胞SDF-1、CXCR4蛋白相对表达量显著增多(P＜0.05).qPCR结果显示:与空白组比较,sh-SDF-1组归巢关键调控因子的mRNA转录水平降低(P＜0.05),补肾壮筋汤组归巢关键调控因子的mRNA转录水平升高(P＜0.05).结论 补肾壮筋汤可通过上调SDF-1/CXCR4轴促进小鼠BMSCs归巢保护关节软骨.
Mechanism of Bushen Zhuangjin Decoction to Promote BMSCs Homing and Protect Articular Cartilage in Mice by the SDF-1/CXCR4 Axis
Objective To investigate the mechanism by which Bushen Zhuangjin Decoction(BZD)regulates the SDF-1/CX-CR4 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 CollagenⅡ and 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);com-pared with the model group,the annular defect between the femoral condyles was improved and the arrangement of chondrocytes was slightly disordered in the BZD group.The immunofluorescence staining of joint tissues showed that compared with the sham group,the relative expression of SDF-1 protein increased in the model group(P＜0.05);compared with the model group,the relative expression level of SDF-1 increased in the BZD group(P＜0.05).qPCR and Western blot results showed that compared with the sham group,the mRNA transcription level and protein relative expression level of key homing regulatory factors(SDF-1,CXCR4,MIP-1α,MCP-1,MIP-1β,RANTES,VEGF,G-CSF,NCAM-1,MMP-2)increased in the model group(P＜0.05);compared with the model group,the mRNA transcription level and relative protein expression level of key homing regulatory factors increased in the BZD group(P＜0.05).(2)In the cell experiment,BMSCs were identified by flow cytometry:CD44 and CD105 were positively expressed,while CD34 was negatively expressed.When the MOI value was 100,the infection rate of SDF-1 gene was the highest.The results of BMSCs migration and chondrogenic differentiation showed that the number of cells migrating to the scratched area,the relative expression of Collagen Ⅱ protein,and acid mucosaccharide of sh-SDF-1 cells were significantly reduced compared with the blank group.The number of migrated cells,Alcian blue staining and the relative expression of Collagen Ⅱ protein significantly increased in the BZD group and the sh-SDF-1+BZD group(P＜0.05).Immunofluorescence finding showed that compared with the blank group,the relative protein expression of SDF-1 and CXCR4 in the sh-SDF-1 group was significantly reduced(P＜0.05),while the relative protein expression of SDF-1 and CXCR4 significantly increased in the BZD group and the sh-SDF-1+BZD group(P＜0.05).qPCR results showed that compared with the blank group,the mRNA transcription level of key homing regulatory factors decreased in the sh-SDF-1 group(P＜0.05),while those increased in the BZD group(P＜0.05).Conclusion BZD can pro-mote the homing of BMSCs to protect articular cartilage in mice by upregulating the SDF-1/CXCR4 axis.
万方数据
维普
