【摘要】：目的：探討經(jīng)典Wnt信號(hào)通路與骨形態(tài)發(fā)生蛋白9（bone morphogenetic protein9，BMP9）誘導(dǎo)小鼠間充質(zhì)干細(xì)胞C3H10T1/2細(xì)胞骨向分化的相互影響及分子機(jī)制。 方法： 1、以小鼠間充質(zhì)干細(xì)胞C3H10T1/2為目的細(xì)胞，，根據(jù)不同的處理因素將其分為4個(gè)組：GFP組、BMP9組、Wnt3a組、BMP9+Wnt3a組； 2、通過(guò)染色和定量測(cè)定早期成骨標(biāo)志堿性磷酸酶（ALP）的活性； 3、應(yīng)用Western blotting、Q-PCR檢測(cè)晚期成骨標(biāo)志骨鈣蛋白（OC）、骨橋蛋白（OPN）的表達(dá)； 4、應(yīng)用茜素紅染色檢測(cè)鈣鹽沉積的情況； 5、應(yīng)用Western blotting、Q-PCR檢測(cè)成骨轉(zhuǎn)錄因子Runx2的表達(dá)； 6、應(yīng)用異位成骨動(dòng)物模型進(jìn)行進(jìn)一步驗(yàn)證二者對(duì)干細(xì)胞成骨分化的相互影響。 結(jié)果： 1、在加入處理因素后第7天進(jìn)行ALP染色和活性測(cè)定，發(fā)現(xiàn)Wnt3a+BMP9組的ALP活性比BMP9組、Wnt3a組明顯增高（P＜0.01）。 2、第14天進(jìn)行茜素紅染色觀測(cè)鈣鹽沉積，結(jié)果發(fā)現(xiàn)從大體標(biāo)本可以看到從GFP組、Wnt3a組、BMP9組、BMP9+Wnt3a組顏色有遞增的趨勢(shì)，鏡下發(fā)現(xiàn)BMP9+Wnt3a組的鈣鹽結(jié)節(jié)要明顯多于BMP9組、Wnt3a組。 3、在第9天應(yīng)用Western blotting和Q-PCR檢測(cè)OC、OPN的表達(dá)，結(jié)果發(fā)現(xiàn)OC和OPN的表達(dá)在BMP9+Wnt3a組高于BMP9組和Wnt3a組。 4、在第2天應(yīng)用Western blotting和Q-PCR檢測(cè)Runx2的表達(dá)，結(jié)果發(fā)現(xiàn)Runx2在BMP9+Wnt3a組中的表達(dá)要高于BMP9組和Wnt3a組 5、動(dòng)物體內(nèi)接種4W后取出異位成骨的骨塊，觀測(cè)大體標(biāo)本以及進(jìn)行HE、Masson染色，結(jié)果顯示BMP9+Wnt3a組的骨塊大于BMP9組和Wnt3a組（P＜0.05），通過(guò)HE、Masson染色發(fā)現(xiàn)BMP9+Wnt3a組的骨塊比BMP9組和Wnt3a組的成熟度高。 結(jié)論：Wnt3a促進(jìn)了BMP9誘導(dǎo)的小鼠間充質(zhì)干細(xì)胞C3H10T1/2的骨向分化，其中成骨關(guān)鍵基因Runx2可能作為二者的交匯點(diǎn)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329

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