摘要
背景:关节软骨碎块后能增强其修复能力,其中具体机制尚不明确。目的:成年关节软骨碎块化后转化生长因子β1的表达与软骨细胞迁移关系的研究。方法:收集30只12-15周龄的SPF级成年SD雄性大鼠后肢膝关节(共60膝),采用划格法获取碎块关节软骨颗粒(约1 mm×1 mm×1 mm)和块状软骨(直径约5 mm,厚度约2 mm),复合明胶海绵碎屑及纤维蛋白胶制作体外培养模型。实验分3组,即碎块组、整块组、抑制剂组。碎块组和整块组均采用普通细胞培养液进行培养,抑制剂组采用添加外源性转化生长因子β1抑制剂Decori和普通细胞培养液培养。Transwell实验检测细胞迁移;鬼笔环肽染色观察细胞微丝骨架F-actin的变化;实时荧光定量PCR和Western blotting检测转化生长因子β1 mRNA和蛋白的表达;使用分子对接技术探索转化生长因子β1和F-actin之间的关系。结果与结论:①Transwell实验结果表明,与整块组相比,碎块组细胞通过matrigel基质胶的数量明显增多(P<0.05);与碎块组相比,抑制剂组细胞通过matrigel基质胶的数量明显减少(P<0.05);②鬼笔环肽染色结果表明,与整块组相比,碎块组的F-actin环状结构明显减少,逐渐呈现细丝状结构,细胞中应力纤维明显减少,细胞间隙明显减小,软骨损伤逐渐恢复;与碎块组相比,抑制剂组F-actin环状结构明显增多,细胞间隙明显增大;③与整块组相比,碎块组转化生长因子β1 mRNA和蛋白的表达水平明显上升(P<0.05);与碎块组相比,抑制剂组转化生长因子β1 mRNA和蛋白的表达水平明显下降(P<0.05);④分子对接模拟中转化生长因子β1与F-actin结合形成稳定的复合体,主要以氢键作用力为主;⑤提示转化生长因子β1在成年关节软骨碎块化后存在过表达的现象,并且过表达的转化生长因子β1能够促进软骨细胞迁移,促进软骨损伤的修复。
BACKGROUND:Fragments of articular cartilage can enhance bone repair ability,the specific mechanism of which is still unclear.OBJECTIVE:To investigate the relationship between transforming growth factorβ1(TGF-β1)expression and chondrocyte migration in adult fragmented articular cartilage.METHODS:Hind limbs(60 knees)from 30 male Sprague-Dawley rats of 12-15 weeks old,SPF grade,were aseptically collected,and the articular cartilage particles(about 1 mm×1 mm×1 mm)and massive cartilage blocks(approximately 5 mm in diameter and 2 mm in thickness)were obtained by cross-cutting method.in vitro culture model of composite gelatin sponge crumb and fibrin glue was established.There were three experimental groups:fragment group,whole block group,and inhibitor group.Both the fragment group and the whole group were cultured in a common cell culture medium,and the inhibitor group was treated with an exogenous TGF-β1 inhibitor,Decori,and a common cell culture solution.Transwell assay was used to detect cell migration.Phalloidin staining was used to observe the change of F-actin.The expression of TGF-β1 mRNA and protein was detected by qRT-PCR and western blot,respectively.Molecular docking technique was used to explore the relationship between TGF-β1 and F-actin.RESULTS AND CONCLUSION:Transwell assay results showed that the number of cells passing through matrigel increased significantly in the fragment group compared with the whole block group(P<0.05).Compared with the fragment group,the number of cells passing through matrigel in the inhibitor group was significantly reduced(P<0.05).The phalloidin staining results indicated that the F-actin ring structures were significantly reduced in the fragment group compared with the whole block group.Filamentous structures gradually formed with stress fibers in the cells being significantly reduced,the intercellular space beining significantly reduced,and cartilage damage recovering gradually.Compared with the fragment group,the F-actin ring structures were significantly increased
作者
吴聪
贾全忠
刘伦
Wu Cong;Jia Quanzhong;Liu Lun(Department of Orthopedics,Sichuan Mianyang 404 Hospital,Mianyang 621000,Sichuan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2022年第8期1167-1172,共6页
Chinese Journal of Tissue Engineering Research
