体外分离共培养诱导大鼠骨髓间充质干细胞定向分化为子宫韧带成纤维细胞的研究

摘要

目的:探讨大鼠骨髓间充质干细胞(BMSCs)定向诱导分化为子宫韧带成纤维细胞的方法.方法:利用差速贴壁法体外分离纯化大鼠 BMSCs,待细胞至70%～80%融合时传代.取成年雌性大鼠盆底韧带,自行分离传代并冻存,经复苏后获得子宫韧带成纤维细胞.实验组将稳定传代3次的大鼠 BMSCs 和大鼠子宫韧带成纤维细胞间接共培养3、6、12天,对照组仅培养BMSCs 3、6、12天,用免疫组化法、实时荧光定量 PCR 分别检测两组Ⅰ、Ⅲ型胶原蛋白和 mRNA 的表达.结果:成功分离培养出大鼠 BMSCs 和子宫韧带成纤维细胞.间接共培养6、12天,BM-SCs Ⅰ、Ⅲ型胶原蛋白和 mRNA 的表达增高,与对照组相比差异有统计学意义(P＜0.05).结论:间接共培养法可以促进 BMSCs 合成Ⅰ、Ⅲ型胶原蛋白,成功诱导分化为子宫韧带成纤维细胞.%Objective: To explore the method of culture rat bone marrow mesenchymal stem cells (BMSCs) and induction differentiation into uterine ligament fibroblasts.Methods: BMSCs of rat were isolated by differential adherence method in vitro.Cells were incubated for passage when 70％～80％ cells were confluent.Uterine ligament tissue of adult female rats were obtained, incubated and frozen for use.Uterine ligament fibroblasts were obtained following resuscitation.BMSCs in the experimental group following passage three times, after non-contact co-culture with ligament fibroblasts for 3,6 and 12 days, the transcriptional activities of collagen type Ⅰ , Ⅲ were assessed by real time RT-PCR.At the similar time-points, the protein levels of collagen Ⅰ , Ⅲ in the BMSCs were detected by immunohistochemical analysis.BMSCs in the control group culture for3, 6 and 12 days were detected by the same method.Results: BMSCs and uterine ligament fibroblasts were successfully isolated and cultured.Compared with control groups, the expressions of type Ⅰ and type Ⅲ collagens and mRNA in BMSCs were enhanced after induced for 6 and 12 days( P ＜0.05).Conclusions: Indirect co-culture with uterine ligament fibroblasts may promote the synthesis of collagen types Ⅰ , Ⅲ by the rat BMSCs, and induce differentiate into uterine ligament fibroblasts.