【摘要】：目的 在單層培養(yǎng)系統(tǒng)中評價堿性成纖維細(xì)胞生長因子和骨形態(tài)發(fā)生蛋白-2單獨或聯(lián)合應(yīng)用后對大鼠關(guān)節(jié)軟骨細(xì)胞增殖和分化作用,并篩選出其體外軟骨細(xì)胞培養(yǎng)的最佳濃度,探索構(gòu)建組織工程軟骨的最適條件。 方法 取Wistar大鼠關(guān)節(jié)軟骨細(xì)胞體外單層培養(yǎng),培養(yǎng)至第三代軟骨細(xì)胞,以對數(shù)生長期細(xì)胞作為實驗細(xì)胞,分別加入不同濃度的b-FGF和BMP-2。A組設(shè)定為空白對照組,各實驗組根據(jù)b-FGF和BMP-2濃度分別6組,兩種因子濃度分別為1ng/ml、3ng/ml、5ng/ml、10ng/ml、20ng/ml、30ng/ml。加入生長因子培養(yǎng)24H、3d、5d后以四唑鹽(MTT)比色法測細(xì)胞相對數(shù)(OD值),計算每種因子濃度組490nm處的光吸收值的平均值,并轉(zhuǎn)換成細(xì)胞相對數(shù)。同樣分組,收集72H后的細(xì)胞爬片,用免疫細(xì)胞化學(xué)染色法檢測Ⅱ型膠原蛋白表達并行半定量分析。分別得出兩種因子對軟骨細(xì)胞增殖和分化作用的最佳濃度,然后分別用各因子最佳濃度單獨或聯(lián)合培養(yǎng)得出兩種因子是否具有協(xié)同效應(yīng)。 結(jié)果 b-FGF濃度在5ng/ml至30ng/ml范圍內(nèi)時可促進軟骨細(xì)胞的增殖,并以20ng/ml處理后OD值最高。BMP-2各濃度組對軟骨細(xì)胞的OD值相對其他組無明顯提高。b-FGF對軟骨細(xì)胞Ⅱ型膠原蛋白合成無明顯影響,而BMP-2可以顯著促進軟骨細(xì)胞膠原蛋白的合成,以20ng/ml為最佳濃度。兩種因子最佳濃度聯(lián)合作用軟骨細(xì)胞,其OD值和平均灰度值對比其余各組均有統(tǒng)計學(xué)意義,聯(lián)合作用軟骨細(xì)胞能延緩軟骨細(xì)胞的去分化現(xiàn)象。 結(jié)論 b-FGF能顯著促進前期軟骨細(xì)胞的增殖,對后期軟骨細(xì)胞作用較弱,最佳濃度為20ng/ml。BMP-2可以明顯促進軟骨細(xì)胞Ⅱ型膠原蛋白的合成,維持細(xì)胞表型,最佳濃度為20ng/ml。BMP-2與b-FGF的聯(lián)合應(yīng)用可以維持細(xì)胞的分化表型,進一步促進成軟骨作用。
[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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