【摘要】：目的觀察經(jīng)典Wnt/β-catenin以及骨形態(tài)發(fā)生蛋白(BMP)信號通路對大鼠骨髓來源的間充質(zhì)干細(xì)胞體外增殖以及向成骨方向分化的影響。 方法應(yīng)用密度梯度離心聯(lián)合貼壁篩選法分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞并分成四組:對照組,Wnt組,BMP組,Wnt3a和BMP-2聯(lián)合組,在不同時間點用細(xì)胞計數(shù)試劑盒-8(CCK-8)法檢測細(xì)胞增殖活性,堿性磷酸酶(ALP)活性定量測定、Von Kossa染色觀察細(xì)胞向成骨分化情況和基質(zhì)礦化程度。反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測成骨特異性標(biāo)志物表達(dá)。 結(jié)果培養(yǎng)第7d,CKK-8測吸光度值聯(lián)合組為0.845,Wnt組為0.738,明顯高于對照組0.409(P0.05)。培養(yǎng)第6d和第12d,ALP活性吸光度值聯(lián)合組為63.8和144.3,BMP-2組為40.8和104.1,均明顯高于對照組7.3和18.9(P0.05)。培養(yǎng)14d后,聯(lián)合組骨鈣素mRNA表達(dá)量高于對照組,而Runx2及Osterix表達(dá)量與其他組相比增高不顯著。培養(yǎng)3w后,Von Kossa染色顯示聯(lián)合組鈣結(jié)節(jié)數(shù)量及大小均高于對照組。 結(jié)論Wnt3a因子和骨形態(tài)發(fā)生蛋白2聯(lián)合誘導(dǎo)能有效地促進(jìn)骨髓間充質(zhì)干細(xì)胞增殖及向成骨方向分化,兩者具有協(xié)同作用。
[Abstract]:Objective to investigate the effects of classical Wnt/ 尾 -catenin and bone morphogenetic protein (BMP) signaling pathway on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Methods Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation combined with adherent screening and were divided into four groups: control group, Wnt group, BMP group, Wnt3a group and BMP-2 group. Cell proliferation activity was detected by cell count kit 8 (CCK-8) at different time points. Alkaline phosphatase (ALP) activity was measured quantitatively by Von Kossa staining to observe the osteogenic differentiation and the degree of matrix mineralization. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of osteogenic specific markers. Results on the 7th day of culture, the absorbance value of CKK-8 in the combined group was 0.738, which was significantly higher than that in the control group (0.409) (P0.05). The absorbance values of ALP activity in the combined group were 40.8 and 104.1, which were significantly higher than those in the control group (7.3 and 18.9, P0.05). After 14 days of culture, the expression of osteocalcin mRNA in the combined group was higher than that in the control group, while the expression of Runx2 and Osterix in the combined group was not significantly higher than that in the other groups. After 3 weeks of culture, Von Kossa staining showed that the number and size of calcium nodules in the combined group were higher than those in the control group. Conclusion combined induction of Wnt3a factor and bone morphogenetic protein 2 can effectively promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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本文編號：2201944