BMP-2与b-FGF对大鼠关节软骨细胞体外增殖和分化的作用

发布时间：2019-01-01 18:32

【摘要】：目的 在单层培养系统中评价碱性成纤维细胞生长因子和骨形态发生蛋白-2单独或联合应用后对大鼠关节软骨细胞增殖和分化作用,并筛选出其体外软骨细胞培养的最佳浓度,探索构建组织工程软骨的最适条件。 方法 取Wistar大鼠关节软骨细胞体外单层培养,培养至第三代软骨细胞,以对数生长期细胞作为实验细胞,分别加入不同浓度的b-FGF和BMP-2。A组设定为空白对照组,各实验组根据b-FGF和BMP-2浓度分别6组,两种因子浓度分别为1ng/ml、3ng/ml、5ng/ml、10ng/ml、20ng/ml、30ng/ml。加入生长因子培养24H、3d、5d后以四唑盐(MTT)比色法测细胞相对数(OD值),计算每种因子浓度组490nm处的光吸收值的平均值,并转换成细胞相对数。同样分组,收集72H后的细胞爬片,用免疫细胞化学染色法检测Ⅱ型胶原蛋白表达并行半定量分析。分别得出两种因子对软骨细胞增殖和分化作用的最佳浓度,然后分别用各因子最佳浓度单独或联合培养得出两种因子是否具有协同效应。 结果 b-FGF浓度在5ng/ml至30ng/ml范围内时可促进软骨细胞的增殖,并以20ng/ml处理后OD值最高。BMP-2各浓度组对软骨细胞的OD值相对其他组无明显提高。b-FGF对软骨细胞Ⅱ型胶原蛋白合成无明显影响,而BMP-2可以显著促进软骨细胞胶原蛋白的合成,以20ng/ml为最佳浓度。两种因子最佳浓度联合作用软骨细胞,其OD值和平均灰度值对比其余各组均有统计学意义,联合作用软骨细胞能延缓软骨细胞的去分化现象。 结论 b-FGF能显著促进前期软骨细胞的增殖,对后期软骨细胞作用较弱,最佳浓度为20ng/ml。BMP-2可以明显促进软骨细胞Ⅱ型胶原蛋白的合成,维持细胞表型,最佳浓度为20ng/ml。BMP-2与b-FGF的联合应用可以维持细胞的分化表型,进一步促进成软骨作用。
[Abstract]:Objective to evaluate the effects of basic fibroblast growth factor and bone morphogenetic protein-2 on proliferation and differentiation of rat articular chondrocytes in monolayer culture system. The optimal concentration of chondrocytes in vitro was selected to explore the optimal conditions for constructing tissue engineered cartilage. Methods the articular chondrocytes of Wistar rats were cultured in vitro and cultured to the third generation chondrocytes. The cells of logarithmic growth phase were used as experimental cells, and b-FGF and BMP-2.A groups with different concentrations were added as blank control group. According to the concentration of b-FGF and BMP-2 in each group, the concentration of the two factors was 1ng / ml, 3ng / ml, 5ng / ml, 10ng / ml, 20ng / ml, 30ng / ml, respectively. The relative number of cells (OD) was measured by tetrazolium (MTT) colorimetry for 5 days. The average value of light absorption at 490nm in each factor concentration group was calculated and converted into the relative number of cells. The cell climbing slices after 72 H were collected and the expression of type 鈪，

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