BMP9诱导人脐带间充质干细胞体内外成骨分化的作用研究

目的:探讨人骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)在体内外诱导人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)成骨分化的作用研究。方法:设立Ad-BMP9处理组和Ad-GFP对照组感染hUC-MSCs,两组细胞分别于3天、5天、7天进行ALP活性检测,14天后采用免疫组织化学染色检测骨钙素(Osteocalcin,OCN)、骨桥蛋白(Osteopotin,OPN)的表达情况,21天后茜素红染色检测矿化结节的形成;然后收集不同分组hUC-MSC用于裸鼠皮下注射成骨模型的建立,4周后取出离体骨进行Micro-CT扫描和分析,并进行H&E、Masson Trichrome、Alcain Blue染色。结果:BMP9处理组的ALP活性和矿化结节形成明显高于对照组,免疫组化染色结果显示BMP9诱导组的OCN、OPG的阳性表达明显高于对照组;裸鼠皮下注射成骨模型的观察结果显示,空白对照组没有形成肉眼可见的皮下包块,仅感染Ad-BMP9的hUC-MSCs能生成异位骨,且形成的异位骨骨量明显,骨密度平均值为396.05±0.60;H&E染色结果显示BMP9诱导生成的异位骨中形成部分成熟的骨基质和骨小梁,Masson Trichrome染色结果显示BMP9明显诱导hUC-MSCs的基质矿化作用,Alcain Blue染色结果显示BMP9明显诱导hUC-MSCs的软骨内成骨作用。结论:BMP9成功诱导人脐带间充质干细胞的体内外成骨作用,为临床骨组织工程的细胞疗法提供了明确的可行性。

BMP9 Induced the Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in vitro and vivo

Objective:To investigate the effect of Bone morphogenetic proteins 9 on induced osteogenic differentiation of human umbilical cord mesenchymal stem cells in vitro and in vivo.Methods:BMP9 gene-recombinant adenovirus was infected into hUC-MSCs and paralleled with the Ad-GFP as negative control group. Cells in two groups were detected in 3d, 5d, 7d of alkaline phosphatase activity;Immunohistochemical staining was used to calculated the expression of osteocalcin and osteopontin after 14d;Alizarin red staining was used to observe the formation of mineralized nodules after 21d;Then collect both groups of infected hUC-MSCs for the establishment of ectopic osteogenic model through nude mice subcutaneous injection, removed the ectopic induced bone tissue after 4 weeks and present to Micro-CT scan and analysis, H&E staining, Masson Trichrome staining, Alcain Blue staining.Results:It is shown that ALP activity and mineralized nodule formation of BMP9 group was significantly higher than that in control group, Immunohistochemical staining showed positive expression of OCN, OPG in BMP9 group was significantly higher than that of control group; the results of ectopic osteogenic model showed that the control group had no visible subcutaneous mass, Only Ad-BMP9 infected hUC-MSCs can observed the formation of ectopic bone obviously, average value of bone mineral density is 396.05+0.60; H&E staining results showed that the generation of mature bone matrix and bone trabecula in the BMP9 induced ectopic bone tissue, Masson Trichrome staining results showed that BMP9 induced of matrix mineralization, Alcain Blue staining results showed that BMP9 induced endochondral osteogenic effect. Conclusion:The BMP9 significantly induces osteogenic differentiation of hUC-MSCs in vitro and in vivo, to provide the feasibility for clinical cell therapy in bone tissue engineering.