共培养系统下软骨细胞对骨髓间质干细胞向软骨细胞分化的诱导作用

 目的观察共培养系统下正常软骨细胞和关节炎软骨细胞对骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)向软骨细胞分化的促进作用。方法分离新西兰兔 BMSCs及正常软骨细胞。制作兔膝关节炎模型,提取兔关节炎软骨细胞。将 BMSCs与低熔点琼脂糖复合成凝胶块,构建软骨细胞-BMCSs共培养系统,分为正常软骨细胞 P0-BMSCs组、正常软骨细胞 P3-BMSCs组、关节炎软骨细胞 P0-BMSCs组、关节炎软骨细胞 P3-BMSCs组及 BMSCs组(对照组)。在 3、7、14天取材行实时定量 PCR、糖胺聚糖含量、细胞活性检测及组织切片观察。结果(1)正常软骨细胞 P0-BMSCs组的 II 型胶原基因表达增强,在 3、7、14天分别为对照组的 5.1、7.2、11.2倍; 正常软骨细胞 P3-BMSCs组 I 、 II 型胶原及蛋白聚糖基因表达均未见增强; 关节炎软骨细胞 P0-BMSCs组蛋白聚糖基因表达增强,在 14天为对照组的 7.8倍; 关节炎软骨细胞 P3-BMSCs组 I 型胶原基因表达水平在三个时间点均低于对照组。(2)正常软骨细胞 P0-BMSCs组糖胺聚糖含量为对照组的 2.59倍。除关节炎软骨细胞 P0-BMSCs组外,其余三组与对照组比较差异均有统计学意义。阿新蓝染色各组均为阳性,正常软骨细胞 P0-BMSCs组的细胞及细胞外基质蓝染最深。结论兔正常 P0软骨细胞与兔关节炎 P0软骨细胞能够有效促进 BMSCs向软骨细胞分化,正常 P3软骨细胞的促分化作用微弱,而关节炎 P3软骨细胞不具有促分化作用。

Chondrogenesis of bone marrow mesenchymal stem cells in co-culture system with chondrocyte

Objective To explore the effect of osteoarthritis (OA) chondrocytes and normal chondrocytes on the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) in self designed co-culture system. Methods Rabbit BMSCs and chondrocytes were isolated and expanded in vitro. OA chondrocytes were harvested from the rabbit of established osteoarthritis model. We made a BMSCs-low melting agarose constructs, then put it onto the self-made 6 well plates lattice assembly for co-culture with chondrocytes. The groups were divided into Normal P0-BMSCs, Normal P3-BMSCs, OA PO-BMSCs, OA P3-BMSCs and BMSCs (control) group. At 3, 7, 14 day culture, cultured cells in all groups were collected for real-time PCR, glycosaminoglycan (GAG) content, cytoactive detection, and histological observation. Results Type Ⅱ collagen gene expression was up-regulated in group Normal PO-BMSCs, which showed 5.1-, 7.2-, 11.2-fold increase over that of control group at 3, 7, 14 days, respectively. Type Ⅰ, Ⅱ collagen and aggrecan gene expressions were not obviously up-regulated. In group OA P3-BMSCs, type Ⅰ collagen gene expression level lower than the control group in 3, 7, 14 day. In normal PO-BMSCs, GAG content showed 2.59-fold increase over that of the control group. GAG content of group OA PO-BMSCs and control group showed no significant differences.Others groups showed significant differences in comparison with the control group (P＜0.05). Alcian stain showed positive in all groups. The normal PO-BMSCs group showed the darkest blue-stained. Conclusion Rabbit normal PO chondrocytes and rabbit OA P0 chondrocytes significantly enhanced chondrogenic differentiation of rabbit BMSCs. The secreted morphogens of the rabbit normal P3 chondrocytes and rabbit OA P3 chondrocytes do not have a significant effect on chondrogenic differentiation of rabbit BMSCs.