间充质干细胞复合藻酸盐凝胶体内成软骨分泌Ⅱ型胶原的代谢功能特征

Compound alginate gelatin and bone marrow mesenchymal stromal cells for the in vivo construction of chondrogenesis secreting type Ⅱ collagen

目的:观察以藻酸盐为支架材料,以经体外软骨向诱导的大鼠骨髓间充质干细胞为种子细胞异位成软骨的可能性。方法:实验于2004-01/2006-02在四川大学口腔生物医学工程教育部重点实验室完成。将32只雄性SD大鼠随机数字表法分为实验组和对照组,每组16只。实验组分离培养自体骨髓间充质干细胞,传至第3代后经转化生长因子β等诱导因子诱导10d后与藻酸钠复合,并滴加氯化钙使其成凝胶状后注入大鼠背部皮下;对照组只注入藻酸盐。术后4,8周分别半数处死动物,取材进行Ⅱ型胶原表达的免疫组织化学和原位杂交观察。结果:纳入大鼠32只,均进入结果分析。实验组在术后4,8周均见Ⅱ型胶原免疫组化及原位杂交阳性,而对照组Ⅱ型胶原免疫组化和原位杂交均为阴性。结论:在蛋白水平和分子水平上证明以骨髓间充质干细胞为种子细胞,以藻酸盐为支架材料构建的组织工程化软骨具备了软骨分泌Ⅱ型胶原的代谢功能特点,表明该方法是一种较理想的组织工程软骨构建方法。

AIM：To investigate the feasibility of chondrogenesis by alginate gelatin as the mesenchymal stromal cells （BMSCs） as seed cells chondrogenicly induced in vitro. METHODS： The experiment was conducted in the Key Laboratory of Oral Biomedical Engineering of Ministry of Education, Sichuan University between January 2004 and February 2006. Thirty-two adult male SD rats were randomly assigned to experimental arid control groups with 16 animals in each group. After isolated and cultured to the third passage, the BMSCs of the experimental group ware induced by transforming growth factor β and other inducing factors for 10 days and combined with algin, which was added calcium chloride to form alginate gelatin, and injected into the hypodermic tissue of the becks of rats. The control group was only injected alginate. Half of the animals ware killed at the 4^th and 8^th weeks after the operation, respectively to perform the immunohistochemical examination and in situ hybridization for type Ⅱ collagen. RESULTS： All 32 rats ware involved in the result analysis. In the experimental group, type Ⅱ collagen was found in the examination and in situ hybridization positive at the 4^th arid 8^th weeks after the operation, while in the control group, it presented negative. CONCLUSIONS： It is proved both in the level of protein and gene expression that the compound constructed by alginate gelatin as scaffold arid BMSCs as seed cells could express type Ⅱ collagen, which is the metabolizing characteristic of cartilage, indicating it is an ideal approach for the construction of tissue engineered cartilage.