胰岛素样生长因子-1对rhBMP-2诱导下成纤维细胞增殖及成骨转化的影响

Effect of IGF-Ⅰ on the proliferation and osteoblast conversion of fibroblast stimulated by rhBMP-2

目的探讨胰岛素样生长因子-1(IGF-1)在重组人骨形态蛋白2(rhBMP-2)诱导成纤维细胞(Fb)转化为成骨细胞过程中是否具有促进作用。方法从成年新西兰大白兔背部皮肤中分离、纯化提取成纤维细胞,然后培养。将细胞分为空白对照组、rhBMP-2干预组和IGF-1+rhBMP-2干预组。空白对照组:常规培养液培养,不加入任何诱导剂及干预因子;rhBMP-2干预组:常规培养液中加入100μg/L的rhBMP-2;IGF-1+rhBMP-2干预组:常规培养液中加入100μg/L的rhBMP-2与终浓度为50μg/L的IGF-1。定期在倒置显微镜下观察Fb生长状况,并采用四甲基偶氮唑蓝(MTT)检测细胞增殖情况,同时行碱性磷酸酶(ALP)活性检测、茜素红染色及钙钴染色评价其成骨分化的能力。结果与其他2组相比,IGF-1+rhBMP-2组细胞增殖速度快,周期短,MTT法检测示细胞生长旺盛,而空白对照组与rhBMP-2干预组之间无显著性差异。rhBMP-2干预组ALP表达高于空白对照组;而IGF-1+rhBMP-2组ALP表达明显高于其他2组。茜素红染色空白对照组未见明显钙化结节,rhBMP-2干预组细胞可见红色钙化结节,而IGF-1+rhBMP-2组钙化结节数目明显较rhBMP-2干预组多,面积增大。钙钴染色:rhBMP-2干预组和IGF-1+rhBMP-2干预组细胞胞浆中均可见浅棕色至棕黑色的细胞颗粒,但以IGF-1+rhBMP-2干预组棕黑色颗粒较多。空白对照组未见明显阳性细胞。结论 Fb在体外一定条件下经过诱导剂诱导后,可向成骨细胞方向转化。IGF-1可以促进Fb在rhBMP-2诱导下的增殖及成骨表型表达。

Objective It is to discuss whether IGF-Ⅰ can accelerate the osteogenesis process of fibroblast（Fb） intervened by rhBMP-2 in vitro.Methods Fb were isolated and purified from back skin in adults of New Zealand rabbits and then were incubated.The cells were divided into 3 groups.The control group was cultured in the routine media,and wasn＇t given any induction agent and interruption factor;the rhBMP-2 group was cultured in the routine media,and was given 100 μg/L rhBMP-2;IGF-I＋rhBMP-2 group was cultured in the routine media,and was given 100 μg/L rhBMP-2 and 50 μg/L IGF-I.The growth of Fb was observed under microscope,and the cell proliferation was detected by MTT assay.Alkaline phosphatase（ALP） activity test and alizarin red staining were used to evaluate the ability of osteogenesis.Results Compared with the other two groups,the cell proliferation of IGF-I＋rhBMP-2 group was quicker and the cell growth cycle time was obviously shorter,while MTT assay detection showed a good growth of Fb.There was no difference between the control group and the rhBMP-2 group.Compareed with the control group,the expression of ALP was more in the rhBMP-2 group,and was the most in the IGF-I＋rhBMP-2 group.The control group had no obvious calcified nodules,and some little in the rhBMP-2 group,but there were more and bigger calcified nodules in the IGF-I＋rhBMP-2 group.After calcium-cobalt staining,in the rhBMP-2 group and IGF-I＋rhBMP-2 group there were some cell particles stained from light brown to brownish-black in cytoplasm,which were more in the latter group,but no positive cells were found in the control blank group.Conclusion Fb can transform to osteoblast under certain condition and inducing factor in vitro.IGF-I can advance the proliferation and osteogenic phenotype expression of Fb stimulated by rhBMP-2.