SFRP5抑制BMP9诱导人脐带间充质干细胞成骨分化的实验研究

目的:探讨脂肪因子分泌型卷曲相关蛋白(secreted frizzled-related protein 5,SFRP5)对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)成骨分化的影响。方法:将人脐带间充质干细胞根据不同的处理因素分为4组:对照组、BMP9组、BMP9+SFRP5组和SFRP5组;分别在3天、5天和7天进行碱性磷酸酶(alkaline phosphatase,ALP)活性读数,7天进行ALP染色,21天进行茜素红染色检测钙盐沉积及油红O染色;收集不同组的细胞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,获取的标本进行HE、Masson染色,Alcian blue染色及油红O染色检测。Western blot检测成骨分化相关蛋白Runx2和OPN的表达。结果:BMP9组的ALP活性读数和染色结果和茜素红染色结果均较对照组增加,而BMP9+SFRP5组则较BMP9组降低;BMP9处理后出现少量脂滴,而BMP9+SFRP5组脂滴明显增加,SFRP5组脂滴最多;裸鼠皮下注射成骨模型的观察结果显示,对照组和SFRP5组没有形成肉眼可见的皮下包块,BMP9组和BMP9+SFRP5组能生成异位骨;4周后观测大体标本以及进行MicroCT检测,发现BMP9+SFRP5组的骨密度值小于BMP9组(P0.05)。HE、Masson染色,Alcian blue染色结果显示,BMP9组的骨分化程度大于BMP9+SFRP5,油红O染色结果示BMP9+SFRP5组有较多的成脂分化;SFRP5能抑制BMP9诱导的Runx2、OPN的蛋白质表达。结论:SFRP5抑制BMP9诱导的人脐带间充质干细胞成骨分化。

SFRP5 Inhibites Osteogenic Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Induced by BMP9

Objectives: To determine the role of SFRP5 in bone morphogenetic proteins 9 (BMP9)—mediated osteogenesis in human umbilical cord-derived mesenchymal stem cells(hUC-MSCs). Methods: The hUC-MSCs are divided into control group,BMP9 group,BMP9+SFRP5 group and SFRP5 group. Alkaline phosphatase (ALP) activity was carried out on 3, 5 and 7 days respectively; while ALP staining on 7 days. Aizarin red staining and oil red O staining were detected on 21 days. Different groups of cells were collected for subcutaneous injection in nude mice. After 4 weeks, the ectopic bone formation was analyzed by micro-CT scanning. The specimens were stained with H.E. staining, Masson staining, Alcian blue staining and oil red O staining. Osteogenesis differentiation related proteins expression of Runx2 and OPN were detected by Western blot. Results: Compared with control, the ALP activity and alizarin red staining were higher than BMP9 group; and BMP9+SFRP5 group was lower than that of BMP9 group. A small quantity of lipid droplets was detected in BMP9 group; and lipid droplets increased significantly in BMP9+SFRP5 group and SFRP5 group. Ectopic bone formation subcutaneously in nude mice was observed in BMP9 group and BMP9+SFRP5 group. The bone density of BMP9+SFRP5 group was lower than BMP9 group(P<0.05). The osteogenetic differentiation in BMP9 group was greater than that in BMP9+SFRP5 group by H.E. staining, Masson staining and Alcian blue staining, and more adipogenesis in BMP9+SFRP5 group by oil red O staining. SFRP5 inhibited the protein expression of Runx2 and OPN induced by BMP9. Conclusion: SFRP5 can inhibit the osteogenic differentiation of human umbilical cord mesenchymal stem cells induced by BMP9.