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目的:研究与盆底韧带成纤维细胞间接共培养对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化的影响,探索在体外条件下诱导BMSCs向韧带成纤维细胞分化微环境的创建并检测诱导分化细胞内弹性蛋白、LOX、Fibulin-5的表达。方法:选择7天SD大鼠,利用密度梯度离心法体外分离纯化大鼠骨髓间充质干细胞并对其进行鉴定;取大鼠盆底韧带组织,自行分离传代,获得大鼠盆底韧带成纤维细胞;取纯化的第四代盆底韧带成纤维细胞与纯化培养后的第四代骨髓间充质干细胞间接共培养;实时定量RT-PCR检测弹性蛋白、LOX及Fibulin-5在不同共培养天数BMSCs中mRNA的表达情况。结果:与相应天数阴性对照组相比,3天共培养组弹性蛋白、LOX、Fbulin-5 mRNA的表达量增加,但均无显著变化(P>0.05),6、12天共培养组mRNA的表达量均显著增高(P<0.05);与3天对照组相比,6天对照组和12天对照组的表达量均无显著变化(P>0.05);与3天共培养组相比,6天共培养组的表达量增高,但差异无统计学意义(P>0.05),12天共培养组的表达量均有显著增高(P<0.05)。结论:与盆底韧带成纤维细胞间接共培养可以促进BMSCs弹性蛋白、LOX、Fbulin-5mRNA的表达,这一结果为BMSCs作为韧带组织工程的种子细胞在体外诱导分化条件的探索提供了一条新的研究思路。
OBJECTIVE: To investigate the effect of indirect co-culture with pelvic ligament fibroblasts on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and to explore the mechanism of BMSCs differentiating into fibroblasts in vitro To create and detect the induced differentiation of intracellular elastin, LOX, Fibulin-5 expression. Methods: Sprague-Dawley rats were selected for 7 days. Rat bone marrow-derived mesenchymal stem cells were isolated and purified by density gradient centrifugation in vitro. The pelvic ligaments of rats were isolated and passaged separately to obtain rat pelvic ligament fibroblasts The purified fourth generation of pelvic ligament fibroblasts were cultured with the fourth generation of purified bone marrow mesenchymal stem cells co-culture; real-time quantitative RT-PCR elastin, LOX and Fibulin-5 in different co-culture days BMSCs mRNA expression. Results: Compared with the corresponding negative control group, the expression of elastin, LOX and Fbulin-5 mRNA increased in 3 days co-culture group, but there was no significant change (P> 0.05) (P <0.05). Compared with the 3-day control group, the expression levels in the 6-day control group and the 12-day control group had no significant changes (P> 0.05). Compared with the 3-day co-culture group, The expression level in 6-day co-culture group increased, but the difference was not statistically significant (P> 0.05). The expression level in 12-day co-culture group was significantly increased (P <0.05). CONCLUSION: Indirect co-culture with pelvic ligament fibroblasts can promote the expression of elastin, LOX and Fbulin-5 mRNA in BMSCs. This result provides a new method for exploring the differentiation of BMSCs as a seed cells in ligament tissue engineering in vitro Research ideas.