TGF-β3对成骨细胞的增殖和成骨能力的影响及其机制初探

The effect and primary mechanism of transforming growth factor-β3 on the proliferation and osteogenic capability of osteoblasts

目的观察不同浓度的转化生长因子-β3(transforming growth factor-β3,TGF-β3)对成骨细胞增殖和成骨能力的影响及其机制。方法分离获得新西兰幼兔颅盖骨成骨细胞,纯化并鉴定后用不同浓度的TGF-β3(0.1、1、10、100μg/L)诱导成骨细胞,CCK-8法检测增殖活性,定量检测法测定碱性磷酸酶(ALP)含量;免疫细胞化学染色观察Ⅰ型胶原Α1(COL-1A1)、Runt相关骨形成蛋白-2(Runx-2)和骨钙素(OCN)蛋白表达;qPCR法检测ALP、骨桥蛋白(OPN)、骨涎蛋白(BSP)、成骨细胞特异性转录因子(Osx)和Smad4基因的表达;Western blotting法检测Smad2/3蛋白的表达情况。结果成功分离获得并鉴定成骨细胞;10、100μg/L的TGF-β3能明显促进成骨细胞增殖(P<0.05);10μg/L TGF-β3组ALP含量始终高于对照组(P<0.05);免疫细胞化学染色结果显示10、100μg/L的TGF-β3组各蛋白表达均强于对照组;qPCR结果示10、100μg/L的TGF-β3能促进ALP、OPN、BSP、Osx和Smad4基因的表达(P<0.05);Western blotting结果示1~100μg/L的TGF-β3组Smad2/3蛋白表达高于对照组(P<0.05)。结论10~100μg/L的TGF-β3可促进成骨细胞的增殖和成骨能力,浓度为10μg/L时效果最显著,其诱导成骨作用通过Smad依赖通路实现。

Objective To explore the effect of transforming growth factor-β3(TGF-β3)in different doses on the proliferation and osteogenic capability of osteoblasts and its mechanism.Methods Osteoblasts were isolated from the calvarium of neonatal New Zealand white rabbit,purified and identified and induced by different doses(0.1,1,10,100μg/L)of TGF-β3.Cell proliferation of each group was tested by CCK-8 assay and ALP activity was measured.The expression levels of COL-1 A1,Runx-2 and OCN proteins were examined by immunohistochemical staining method.Relative expression of ALP,OPN,BSP,Osx and Smad4 genes were measures by qPCR.Western blotting method was used to test the expression of Smad2/3 protein.Results The osteoblasts were isolated and identified successfully.10μg/L and 100μg/L TGF-β3 can improve the proliferation of osteoblasts.The ALP activity in 10μg/L group was higher than the control group(P<0.05).Immunohistochemical staining results showed the protein expression in 10μg/L and 100μg/L TGF-β3 group was stronger than the control group.The ALP,OPN,BSP,Osx and Smad4 gene expression in 10μg/L and 100μg/L TGF-β3 group was higher than the control group(P<0.05).The Smad2/3 protein expression in 1-100μg/L TGF-β3 group was higher than the control group(P<0.05).Conclusion TGF-β310-100μg/L can improve the proliferation and osteogenic ability of osteoblasts and this effect is more obvious when the dose is 10μg/L.This effect is produced through activating Smad-dependent pathway.