胰岛素铁硒传递蛋白(ITS)促进软骨组织形成的作用研究.docVIP

 胰岛素铁硒传递蛋白(ITS)促进软骨组织形 成的作用研究# 刘瑾春，刘霞，肖苒，曹谊林** 5 10 15 20 25 30 35 40 （中国医学科学院整形外科医院，北京 100144） 摘要：目的 研究胰岛素铁硒传递蛋白（ITS）在软骨细胞培养过程中对细胞增殖和软骨细胞 外基质表达影响，为组织工程化软骨培养方案的改进提供参考。 方法 P2 猪耳软骨细胞， 单 层 培 养 或 离 心 形 成 聚 集 体 (pellets) ， 根 据 培 养 液 不 同 分 为 两 组 ： 常 规 培 养 组 (DMED+10%FBS)和 ITS 组(DMED+10%FBS+1%ITS)，分别培养 4 周，1，2，3，4 周取材 检测，比较两组 pellets 的大体形态，组织学，二型胶原免疫组化染色和软骨特异基因的表 达。结果 MTT 曲线显示 ITS 可以促进软骨细胞增殖，细胞外基质糖胺多糖（GAG）染色（甲 苯胺蓝染色）和 Col免疫组织化学染色在 ITS 组明显强于对照组，软骨特异基因 Sox9,Col 在两组的表达相当，ITS 组 Aggrecan 的表达在第 2，3，4 周强于对照组。结论 ITS 可以通 过促进软骨细胞增殖，促进细胞外基质分泌从而能更好的促进组织工程软骨的形成。 关键词：胰岛素铁硒传递蛋白；软骨组织工程；软骨细胞 中图分类号：Q81 生物工程学 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM （ITS）ON CARTILAGE FORMATION OF CHONDROCYTE PELLETS CULTURED IN VITRO LIU Jinchun, LIU Xia, XIAO Ran, CAO Yilin (Plastic Surgery Hospital, Chinese Academy of Medical Science, Beijing 100144) Abstract: In this paper the effects of Insulin-Transferrin-Selenium（ITS）on cartilage formation of chondrocyte pellets cultured in vitro was investigated. Methods Chondrocytes were cultured in Dulbecco’s modi?ed Eagle’s medium with 10% FBS (control group), and Dulbecco’s modi?ed Eagle’s medium with 10% FBS plus 1% ITS (ITS group). Proliferation of chondrocytes in monolayer culture was evaluated by MTT assay. Chondrocyte pellets were cultured in the two groups for 4 weeks. The chondrocyte pellets were harvested at week 1, 2, 3 and 4, and were evaluated with histological, immunohistochemical analysis, and gene expression analysis by semi-quantitative PCR. Results MTT assay showed that ITS could significantly stimulated proliferation of chondrocytes. Staining of Toluidine blue and immunohistochemical staining of type Ⅱ collagen(Col Ⅱ) in pellets of ITS group were stronger than that in control group. Gene expression of Sox9 and ColⅡ were comparable in the two groups. Gene expression of ColⅠwas inhibited and Aggrencan was increased by ITS. Conclusion: ITS could improve proliferation of chondrocytes and stimulate deposition of extracellular matrix of chondrocyte