联合应用生长因子促骨髓间充质干细胞向韧带样细胞分化

背景：利用生长因子诱导骨髓间充质干细胞定向分化成韧带样细胞并促其分泌胶原蛋白是构建组织工程化韧带的关键。生长因子碱性成纤维细胞生长因子和转化生长因子β1在细胞定向分化过程中都起着十分重要的作用。目的：采用转化生长因子β1和碱性成纤维细胞生长因子诱导体外培养的兔骨髓间质干细胞,转化为韧带样细胞,并研究此种韧带样细胞的生物特性。方法：自幼兔四肢骨抽取骨髓分离纯化骨髓间充质干细胞并培养、增殖。采用10μg/L转化生长因子β1和25μg/L碱性成纤维细胞生长因子对骨髓间质干细胞进行诱导分化,分为空白组、转化生长因子β1组、碱性成纤维细胞生长因子组、转化生长因子β1＋碱性成纤维细胞生长因子组。观察生长因子对骨髓间质干细胞生长、形态的影响,使用MTT法绘制细胞生长曲线,使用天狼腥红染色法定量对比骨髓间质干细胞分泌胶原蛋白量。把转化生长因子β1＋碱性成纤维细胞生长因子联合诱导的骨髓间质干细胞使用BrdU荧光染色标记,然后移植到脱细胞真皮基质上,观察细胞增殖分布情况,并与空白组对照。结果与结论：转化生长因子β1＋碱性成纤维细胞生长因子联合组,细胞形态优于空白组及单一因子组,细胞增殖率、胶原分泌量也较高。脱细胞真皮基质上,转化生长因子β1＋碱性成纤维细胞生长因子组细胞增殖分布情况明显优于空白组。提示联合使用生长因子转化生长因子β1和碱性成纤维细胞生长因子刺激兔骨髓间质干细胞,能够促使兔骨髓间质干细胞定向转化为韧带样细胞,对组织工程前交叉韧带的构建具有积极意义。

A combined use of growth factors promotes ligament cell-like cell differentiation of bone marrow mesenchymal stem cells

BACKGROUND： A key to construction of tissue-engineered ligament is to induce bone marrow mesenchymal stem cells into ligament cells like cells using growth factors and to promote the differentiated cells to secrete collagen protein. Growth factors basic fibroblast growth factor and transforming growth factor β1 play an important role in oriented differentiation of cells. OBJECTIVE： To in vitro induce rabbit bone marrow mesenchymal stem cells to differentiate into ligament cell-like cells using basic fibroblast growth factors and transforming growth factor β1 and investigate the biological characteristics of the ligament cell-like cells. METHODS： Bone marrow mesenchymal stem cells were harvested and cultured. Then these cells were induced using 25 μg/L basic fibroblast growth factors and 10 μg/L transforming growth factor β1. Four groups were designated： namely blank control group, transforming growth factor β1 group, basic fibroblast growth factor group and transforming growth factor β1 ＋ basic fibroblast growth factor group （combination group）. Effects of both growth factors on the growth and morphology of bone marrow mesnchymal stem cells were investigated. Cell growth curve was drawn using MTT assay. The mount of collagen secreted by bone marrow mesenchymal stem cells was quantified using Picro-Sirius red staining. After BrdU fluorescent staining, bone marrow mesenchymal stem cells induced by transforming growth factor β1 combined with basic fibroblast growth factor were transplanted onto acellular dermal matrix to determine cell proliferation and distribution. Comparisons were made between the combination group and blank control group. RESULTS AND CONCLUSION： Cell morphology in the combination group, as well as cell proliferation rate and collagen secretion amount, was superior to that in the blank control group, transforming growth factor β1 group, or basic fibroblast growth factor group. These findings suggest that transforming growth factor β1 combined with basic fibroblast growth factor can promote bone marrow mesenchymal stem cells to differentiate into ligament cell-like cells, which exhibits important significance for construction of tissue-engineered anterior cruciate ligament.