骨髓间充质干细胞与关节软骨细胞Transwell共培养诱导形成软骨

Transwell co-culture of bone marrow mesenchymal stem cells and articular chondrocytes induces cartilage formation

背景:研究证实,关节软骨细胞具有分泌诱导因子促进骨髓间充质干细胞向关节软骨细胞分化的能力,但至今未见两种细胞运用Transwell共培养诱导骨髓间充质干细胞形成软骨的报道。目的:观察在Transwell共培养系统中共培养后,关节软骨细胞诱导骨髓间充质干细胞向关节软骨样细胞转化的能力。方法:骨髓间充质干细胞来源于4周龄SD大鼠的股骨及胫骨干骨髓,关节软骨细胞来源于4周龄SD大鼠的正常股骨头表面的关节软骨。分别吸取第3代骨髓间充质干细胞和关节软骨细胞,按1:1的细胞比例分别植于Transwell共培养系统中,下室植入骨髓间充质干细胞,上室植入关节软骨细胞。同时设置相同浓度单纯骨髓间充质干细胞对照组,分别在含胎牛血清的DMEM培养基中培养,相差显微镜下观察细胞的增殖和基质合成情况,并对各组细胞爬片进行Ⅱ型胶原免疫组化染色及氨基聚糖含量检测。结果与结论:共培养组骨髓间充质干细胞数量增多,细胞外基质合成丰富,其基质能被Ⅱ型胶原免疫组化染色,细胞染色呈现黄色。氨基聚糖含量随着诱导时间增加而增多。提示关节软骨细胞分泌物具有促进骨髓间充质干细胞向关节软骨样细胞转化的能力,与此同时,骨髓间充质干细胞还能分泌促进组织细胞修复的细胞因子,使共培养中的软骨细胞功能得以加强。由此可见,软骨细胞与骨髓间充质干细胞共培养诱导具有独特的优势。

BACKGROUND：Articular chondrocytes （ACs） have the ability of promoting bone marrow mesenchymal stem cells （BMSCs） to differentiate to ACs via the secretion of inducing factors,but the study of forming ACs using Transwell to co-culture has not been reported now.OBJECTIVE：To investigate the ability of promoting BMSCs differentiation to ACs after co-culturing in Transwell.METHODS：BMSCs were obtained from femoral and tibial shaft of SD rats aged 4 weeks,ACs were obtained from the surface o normal femoral head of SD rats aged 4 weeks.The third passage of BMSCs and ACs were harvested and placed into Transwel co-culture system at a ratio of 1：1,BMSCs were placed in the bottom and ACs in the upper.Meanwhile BMSCs at the same concentration served as control group,cultured in DMEM containing 10% fetal bovine serum.Cell proliferation and matrix synthesis were observed under phase contrast microscopy,cell-seeded cover slips in each group were analyzed for glucose amino glycan content detection and type Ⅱ collagen immunohistochemical staining.RESULTS AND CONCLUSION：The number of co-cultured BMSCs increased,synthesis of extracellular matrix was rich,the matrix can be stained yellow by type Ⅱ collagen immunohistochemical staining.Glucose amino glycan content increased with the induction time increasing.The secretions of ACs have the ability of promoting BMSCs to transform to ACs.Meanwhile,BMSCs can secrete cell factors to stimulate tissue restoring,thus strengthening the function of ACs in Transwell.ACs and BMSCs co-culture inducing has its unique advantages.