经典Wnt信号通路对BMP9诱导干细胞骨向分化的影响及机制研究

发布时间：2018-08-25 14:43

【摘要】：目的：探讨经典Wnt信号通路与骨形态发生蛋白9（bone morphogenetic protein9，BMP9）诱导小鼠间充质干细胞C3H10T1/2细胞骨向分化的相互影响及分子机制。方法： 1、以小鼠间充质干细胞C3H10T1/2为目的细胞，，根据不同的处理因素将其分为4个组：GFP组、BMP9组、Wnt3a组、BMP9+Wnt3a组； 2、通过染色和定量测定早期成骨标志碱性磷酸酶（ALP）的活性； 3、应用Western blotting、Q-PCR检测晚期成骨标志骨钙蛋白（OC）、骨桥蛋白（OPN）的表达； 4、应用茜素红染色检测钙盐沉积的情况； 5、应用Western blotting、Q-PCR检测成骨转录因子Runx2的表达； 6、应用异位成骨动物模型进行进一步验证二者对干细胞成骨分化的相互影响。结果： 1、在加入处理因素后第7天进行ALP染色和活性测定，发现Wnt3a+BMP9组的ALP活性比BMP9组、Wnt3a组明显增高（P＜0.01）。 2、第14天进行茜素红染色观测钙盐沉积，结果发现从大体标本可以看到从GFP组、Wnt3a组、BMP9组、BMP9+Wnt3a组颜色有递增的趋势，镜下发现BMP9+Wnt3a组的钙盐结节要明显多于BMP9组、Wnt3a组。 3、在第9天应用Western blotting和Q-PCR检测OC、OPN的表达，结果发现OC和OPN的表达在BMP9+Wnt3a组高于BMP9组和Wnt3a组。 4、在第2天应用Western blotting和Q-PCR检测Runx2的表达，结果发现Runx2在BMP9+Wnt3a组中的表达要高于BMP9组和Wnt3a组 5、动物体内接种4W后取出异位成骨的骨块，观测大体标本以及进行HE、Masson染色，结果显示BMP9+Wnt3a组的骨块大于BMP9组和Wnt3a组（P＜0.05），通过HE、Masson染色发现BMP9+Wnt3a组的骨块比BMP9组和Wnt3a组的成熟度高。结论：Wnt3a促进了BMP9诱导的小鼠间充质干细胞C3H10T1/2的骨向分化，其中成骨关键基因Runx2可能作为二者的交汇点起了重要作用。
[Abstract]:Aim: to investigate the effects of classical Wnt signaling pathway and bone morphogenetic protein 9 (bone morphogenetic protein9,BMP9 (BMP 9) on bone differentiation of mouse mesenchymal stem cells (C3H10T1/2). Methods: 1. Murine mesenchymal stem cells (C3H10T1/2) were divided into four groups according to different treatment factors: BMP9 group (Wnt3a) and BMP9 Wnt3a group. (2) the activity of alkaline phosphatase (ALP) was detected by staining and quantitative analysis, and the expression of osteopontin (OPN) (OPN), osteopontin (OC),) was detected by Western blotting,Q-PCR. (4) calcium deposition was detected by alizarin red staining, and the expression of osteogenic transcription factor (Runx2) was detected by Western blotting,Q-PCR. 6. The effects of ectopic osteoblasts on osteogenic differentiation of stem cells were further verified by the animal model of ectopic osteogenesis. Results: 1. ALP staining and activity determination were performed on the 7th day after adding the treatment factors. It was found that the ALP activity of the Wnt3a BMP9 group was significantly higher than that of the BMP9 group (P < 0. 01). 2. The calcium deposition was observed by alizarin red staining on the 14th day. The results showed that the color of BMP9 Wnt3a group increased gradually from GFP group Wnt3a group. Under microscope, the calcium nodule of BMP9 Wnt3a group was significantly higher than that of BMP9 group Wnt3a group. On the 9th day, the expression of OC,OPN was detected by Western blotting and Q-PCR. The results showed that the expression of OC and OPN in BMP9 Wnt3a group was higher than that in BMP9 group and Wnt3a group. On the second day, Western blotting and Q-PCR were used to detect the expression of Runx2. The results showed that the expression of Runx2 in BMP9 Wnt3a group was higher than that in BMP9 group and Wnt3a group 5. After 4 weeks of inoculation in vivo, ectopic osteoblasts were taken out, gross specimens were observed and HE,Masson staining was performed. The results showed that the bone mass of BMP9 Wnt3a group was larger than that of BMP9 group and Wnt3a group (P < 0. 05), and the maturity of BMP9 Wnt3a group was higher than that of BMP9 group and Wnt3a group by HE,Masson staining. Conclusion the bone differentiation of mouse mesenchymal stem cells (C3H10T1/2) induced by BMP9 was promoted by Wnt3a, and the osteogenic key gene Runx2 may play an important role in the intersection of the two.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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