小鼠间质干细胞在基质凝胶与细胞微球中的成软骨分化

Extraculuar material promoted C3H10T1/2 chondrogenic differentiation than Pellet cultured in vitro

目的探讨细胞基质凝胶与细胞微球两种不同培养微环境对干细胞成软骨分化影响，分析软骨分化特点以优化培养微环境。方法采用透明质酸钠、硫酸软骨素及交联剂按照质量比1：1：2均匀混合后制成的细胞外基质凝胶，分别运用小鼠间质干细胞与基质凝胶共培养；离心沉淀形成细胞微球等方法进行成软骨诱导培养。在成软骨分化后1、2、3周行定量PCR检测分化相关基因表达；至终末分化后行组织切片及染色，对比细胞外基质形成及分化相关基因表达趋势，评价干细胞成软骨分化结果。结果干细胞与基质凝胶均匀混合后形成柱状胶体结构，细胞均匀分散其中；而细胞微球则最终形成具有一定韧性的灰白球状组织。细胞排列紧密且组织表面由纤维组织包裹。定量PCR结果示：启动因子SOX-9在两种培养体系中有表达，差异有统计学显著意义。在基质凝胶中，标志基因Aggrecan表达仅在第2周时至最高，而在细胞微球中持续表达，差异有统计学意义。Col-Ⅱal在基质凝胶中持续表达；在细胞微球早期分化表达显著，差异有统计学意义；Col-Xal表达在分化第2、3周时，以基质凝胶中为显著；在分化第1、2周时，以细胞微球分化为显著，差异有统计学意义；基质凝胶有利于Col-Ⅱal的表达且可持续维持软骨细胞肥大状态；细胞微球则有利于Aggrecan的表达；甲苯胺蓝染色凝胶中细胞外基质较细胞微球含量明显增多。结论基质凝胶培养有利于促进干细胞成软骨分化及维持分化表型；它具有模拟细胞外基质微环境、维持细胞空间结构并提高细胞生物活性等优势。

Objective In order to understand the chondrogenesis differentiation of mesenchymal stem cells in either hy- drogel or pellet cuhure, we applied the two methods and reveal the possible mechanism and for further investigation. Methods In C3H10T1/2 chondrogenic differentiation, we apply extracellular matrix hydrogel mixed the cell suspensions of freshly prepared （including scaling chondroitin sulfate, sodium hyaluronate synthesis and cross-linking agent） co-culture system and high cell densi- ty pellet formed by eentrifugation. Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3 （10 ng/ml）, dexamethasone （100 nmol/L）, aseorbic acid （50 ug/ml）, 1 ： 100 dilution ITS＋Premix and high glucose-DMEM medium with 0.2 vol- ume fraction fetal bovine serum. And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group. Histochemistry staining was utilized to identify extraeellular proteoglyean and real-time PCR was performed to assess gene expression of SOX9, collagen Ilal/Xal and aggreean for the 1st, 2nd and 3rd week respectively. Results In the hydrogel model for 3 weeks chondrogenic differentiation, the expression of master transcription factor SOX9 was upregulated in both culture mod- els. While the marker genes of collagen Ilal and collagen Xal were all promoted in hydrogel culture, the aggreean gene expression was peaked in pellet culture. In addition, immunocytochemistry analysis of the hydrogel and pellet for 3 week illustrated the ex- pression of extracellular matrix and more obviously in the hydrogel model. Conclusion In compared with pellet culture, the MSCs in the hydrogel were more likely promoted chondrogenesis leading to the eventual expression of marker genes. And the hy- drogel would be applied in regeneration of cartilage injury.