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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,h UC-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h UC-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h UCMSC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AdBMP9的h UC-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h UC-MSCs的基质矿化作用,Alcain Blue染色结果显示BMP9明显诱导h UC-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。
Objective: To investigate the effect of bone morphogenetic proteins 9 (BMP9) on osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells (h UC-MSCs) in vitro and in vivo. Methods: Ad-BMP9-treated group and Ad-GFP control group were infected with h UC-MSCs. ALP activity was detected on day 3, day 5 and day 7 respectively. Immunohistochemical staining was used to detect osteocalcin Osteocalcin (OCN) and osteopontin (OPN) were detected. Alzheimer’s staining was used to detect the formation of mineralized nodules 21 days later. Different groups of h UCMSCs were collected for subcutaneous injection into osteoblasts in nude mice. 4 After weeks, the isolated bone was removed for Micro-CT scanning and analysis, and H & E, Masson Trichrome, Alcain Blue staining. Results: The ALP activity and the formation of mineralized nodules in BMP9-treated group were significantly higher than those in control group. Immunohistochemical staining showed that the expression of OCN and OPG in BMP9-induced group was significantly higher than that in control group. The results showed that there was no macroscopic subcutaneous mass in the blank control group. Only hBac-MSCs infected with AdBMP9 could produce heterotopic bone, and the amount of ectopic bone formed was obviously, the average BMD was 396.05 ± 0.60; H & E The results of staining showed that some mature bone matrix and trabecular bone were formed in the ectopic bone induced by BMP9. Masson Trichrome staining showed that BMP9 obviously induced the matrix mineralization of h UC-MSCs. Alcain Blue staining showed that BMP9 obviously induced hUC Endochondral ossification of MSCs. Conclusion: BMP9 successfully induced human umbilical cord mesenchymal stem cells in vitro and in vivo osteogenesis, which provided a clear feasibility for clinical bone tissue engineering.