盐酸四环素对大鼠骨髓间充质干细胞的成骨诱导分化活性

Tetracycline hydrochloride induces the osteogenic differentiation of rat bone marrow mesenchymal stem cells

背景:盐酸四环素不仅具有促进骨髓间充质干细胞增殖的作用,还具有极强的骨亲活性。研究盐酸四环素诱导骨髓间充质干细胞向成骨细胞分化,可为获得骨组织工程细胞提供新方法。目的:观察体外盐酸四环素对大鼠骨髓间充质干细胞向成骨细胞分化的作用。方法:骨髓全血细胞贴壁法分离培养大鼠骨髓间充质干细胞,通过细胞形态和骨髓间充质干细胞标志物CD34、CD45、CD90、CD29流式检测对骨髓间充质干细胞进行鉴定;取第3代骨髓间充质干细胞与盐酸四环素共培养,以含体积分数10%FBS的L-DMEM的完全培养基培养做对照组。通过检测碱性磷酸酶活性、Ⅰ型胶原和骨钙素免疫组织化学染色、茜素红矿化钙结节组织化学染色、Ⅰ型胶原蛋白和骨钙素的RT-PCR的基因表达,研究盐酸四环素对骨髓间充质干细胞的成骨诱导分化活性。结果与结论:①盐酸四环素能够持续促进骨髓间充质干细胞的碱性磷酸酶活性增加,与对照组比较具有显著性差异;②盐酸四环素诱导培养骨髓间充质干细胞21 d,Ⅰ型胶原蛋白和骨钙素染色表达阳性,茜素红染色出现红色钙结节,RT-PCR检测Ⅰ型胶原蛋白和骨钙素基因表达;对照组程阴性表达;③结果表明,盐酸四环素具有明显促进骨髓间充质干细胞成骨诱导分化的作用。

BACKGROUND:Tetracycline hydrochloride not only promotes the proliferation of bone marrow mesenchymal stem cells(BMSCs),but also has a strong bone activity.Studies on the osteogenic differentiation of BMSCs induced by tetracyclinehydrochloride will provide a new cell source for bone tissue engineering.OBJECTIVE:To observe the induction and differentiation activity of BMSCs into osteoblasts cultured by tetracyclinehydrochloride in vitro.METHODS:BMSCs were separated and expanded by whole bone marrow adherent method and identified by cellmorphology and BMSCs cell markers CD34,CD45,CD90,CD29.Passage3BMSCs were collected and cultured withtetracycline hydrochloride(experimental group)or cultured in L-DMEM containing10%fetal bovine serum(controlgroup).Osteogenic induction and differentiation activity of BMSCs cultured by tetracycline hydrochloride were detectedby alkaline phosphatase activity detection,collagen I and osteocalcin immunohistochemistry staining,alizarin redstaining of mineralized calcium nodules,and detection of collagen I and osteocalcin gene expression by RT-PCR.RESULTS AND CONCLUSION:Tetracycline hydrochloride continuously promoted alkaline phosphatase activity inthe BMSCs,significantly different from that in the control group.After21days of culture,positive expression ofcollagen I and osteocalcin proteins,red calcium nodules shown by alizarin red staining,and mRNA expression ofcollagen I and osteocalcin were observed in the experimental group,while the negative expression was found in thecontrol group.In conclusion,tetracycline hydrochloride has a significant role to promote the osteogenicdifferentiation of BMSCs.