不同状态软骨细胞在共培养系统下对BMSCs向软骨细胞诱导分化作用的研究

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

[目的]观察不同代次正常软骨细胞和关节炎软骨细胞对琼脂糖-BMSCs向软骨细胞分化的促进作用。[方法]分离新西兰兔BMSCs,正常软骨细胞。制作兔关节炎模型,提取兔关节炎软骨细胞。将BMSCs和低熔点琼脂糖复合成凝胶块,放在自制的六孔板网格架上,构建兔软骨细胞-BMCSs共培养系统。在3、7、14 d取各组琼脂糖-BMSCs凝胶块进行实时定量PCR、GAG含量检测。[结果]兔关节炎模型制作成功,关节面色泽较灰暗,关节软骨粗糙。Normal P0-BMSCs组的II型胶原基因表达明显增强,Normal P3-BMSCs组Ⅰ、Ⅱ型胶原及蛋白聚糖基因表达均未见明显增强,OA P0-BMSCs组蛋白聚糖基因表达明显增强,OA P3-BMSCs组I型胶原基因表达水平在3个时间点均低于对照组。Normal P0-BMSCs组的GAG含量为5.7±0.49μg/mg(湿重),较对照组有明显上升。OA P0-BMSCs组GAG含量与对照组相比差异无统计学意义(P>0.05),其余三组与对照组相比差异均具有统计学意义(P<0.05)。[结论]兔正常P0软骨细胞与兔关节炎P0软骨细胞所分泌的形态发生素能够有效促进BMSCs向软骨细胞分化,而兔正常P3软骨细胞的促分化作用微弱,兔关节炎P3软骨细胞不能促进BMSCs向软骨细胞分化。

[Objective]To explore the effect of osteoarthritis chondrocytes and normal chondrocyte on chondrogenesis of bone mesenchymal stem cells（BMSCs） in self designed co-culture system. [Methods]BMSCs and chondrocytes were isolated from New Zealand rabbits.Six-month old rabbit was used to establish osteoarthritis model,and osteoarthritis chondrocytes was isolated for further experiments.BMSCs-low melting agarose constructs were put onto the self-made 6 well plates lattice assembly.Real-time PCR,1,9-dimethylmethylene blue（DMMB） test were performed at 3、7、14 days in all groups,respectively.[Results]Rabbit osteoarthritis model was successfully established.Articular surface was gray and rough.Type Ⅱ collagen gene expression was up-regulated in group Normal P0-BMSCs.Type Ⅰ,Ⅱ collagen and aggrecan gene expression was not obviously up-regulated.TypeI collagen gene expression level of group OA P3-BMSCs was lower than control group in 3、7、14 days.GAG content in group Normal P0-BMSCs was 5.7±0.49 μg/（mg/wet weight）more than that in other groups.GAG content in group OA P0-BMSCs and control group were of no statistical difference（P0.05）.There were significant differences between experimental groups and control group（P0.05）.[Conclusion]Rabbit normal P0 chondrocytes and rabbit OA P0 chondrocytes secreted morphogens enhanced chondrogenic differentiation of rabbit BMSCs.Rabbit normal P3 chondrocytes and rabbit OA P3 chondrocytes secreted morphogens do not have a significant effect on chondrogenic differentiation of rabbit BMSCs.