【摘要】：目的:Wnt信號通路是近年發(fā)現(xiàn)的對于細(xì)胞生物學(xué)行為具有重要調(diào)節(jié)作用的一個新的研究熱點。現(xiàn)有研究表明無論是經(jīng)典的Wnt/β-catenin/TCF(LEF1)通路,還是非經(jīng)典的Wnt通路對于具有多向分化潛能的間充質(zhì)干細(xì)胞的骨發(fā)生都具有重要的調(diào)節(jié)作用。本研究在于探索經(jīng)典Wnt/β-catenin/TCF(LEF1)通路對大鼠骨髓來源的間充質(zhì)干細(xì)胞在體外成骨分化中的調(diào)節(jié)作用。 方法:新生SD大白鼠6只,由華中科技大學(xué)同濟(jì)醫(yī)院試驗動物中心提供,用于制備骨髓間充質(zhì)干細(xì)胞。Wnt3a蛋白為PeproTech公司產(chǎn)品。細(xì)胞學(xué)體外對照觀察:采用全骨髓體外分離法培養(yǎng)骨髓間充質(zhì)干細(xì)胞,選取傳至第3代的骨髓間充質(zhì)干細(xì)胞,按5×10~7 L~(-1)接種后各分為3組,分別加入含10~8 mol/L的地塞米松、10 mol/Lβ-甘油磷酸鹽、50 mg/L維生素C的DMEM成骨誘導(dǎo)培養(yǎng)基2.0 mL。A組在誘導(dǎo)分化后的第8天起加入濃度10mg/LWnt3a,連續(xù)3天;B組在誘導(dǎo)分化后的第18天起加入濃度10mg/LWnt3a,連續(xù)3天。C組為對照組,在分化的全過程不加入Wnt3a蛋白。主要觀察指標(biāo):倒置顯微鏡觀察間充質(zhì)干細(xì)胞生長和增殖情況,流式細(xì)胞儀檢測間充質(zhì)干細(xì)胞表面標(biāo)志物的表達(dá),堿性磷酸酶染色檢測向成骨細(xì)胞分化情況,堿性磷酸酶定量分析結(jié)果。 結(jié)果:第3代骨髓間充質(zhì)干細(xì)胞的惡CD44陽性率為94.88%,CD90的陽性率為94.37%,CD45為0.95%。加入成骨誘導(dǎo)劑后7d,經(jīng)誘導(dǎo)的骨髓基質(zhì)干細(xì)胞呈堿性磷酸酶陽性。在第11天,檢測A組的堿性磷酸酶的活性較B、C組高(P0.01)。第21日,檢測B組的堿性磷酸酶活性較C組低(P0.01)。 結(jié)論:大鼠骨髓來源間充質(zhì)干細(xì)胞體的外成骨性能受Wnt3a信號通路調(diào)節(jié),并且這種調(diào)節(jié)依賴于被作用細(xì)胞的分化階段的,在間充質(zhì)干細(xì)胞進(jìn)入分化期后,Wnt3a所介導(dǎo)的經(jīng)典Wnt信號通路對于間充質(zhì)干細(xì)胞向成骨細(xì)胞分化具有促進(jìn)作用,但當(dāng)?shù)匠晒羌?xì)胞發(fā)育成熟的晚期,Wnt3a介導(dǎo)的經(jīng)典Wnt信號通路的作用由促進(jìn)變?yōu)橐种瞥晒羌?xì)胞最終發(fā)育成熟。
[Abstract]:Aim: Wnt signaling pathway is a new research hotspot in recent years, which plays an important role in the regulation of cell biological behavior. Existing studies have shown that both the classical Wnt/ 尾-catenin/TCF (LEF1) pathway and the non-classical Wnt pathway play an important role in the osteogenesis of mesenchymal stem cells with multipotential differentiation. The aim of this study was to explore the role of classical Wnt/ 尾-catenin/TCF (LEF1) pathway in the regulation of bone marrow derived mesenchymal stem cells (MSCs) in osteogenic differentiation in vitro. Methods: six newborn SD rats, provided by Experimental Animal Center of Tongji Hospital, Huazhong University of Science and Technology, were used to prepare bone marrow mesenchymal stem cells. Wnt3a protein was produced by PeproTech Company. Cytological comparative observation in vitro: bone marrow mesenchymal stem cells were cultured by whole bone marrow isolation in vitro. Bone marrow mesenchymal stem cells passed to the third generation were selected and divided into three groups according to 5 脳 10 ~ 7 L ~ (- 1) inoculation, and the cells were divided into 3 groups according to 5 脳 10 ~ 7 L ~ (- 1). The DMEM osteogenic induction medium containing 10 ~ 8 mol/L dexamethasone, 10 mol/L 尾-glycerophosphate and 50 mg/L vitamin C was added to the DMEM osteogenic induction medium 2.0 mL.A on the 8th day after differentiation, for 3 consecutive days, and the concentration was 10 mg / L / L Wnt3a on the 8th day after differentiation. The concentration of 10 mg / L Wnt3a was added to group B on the 18th day after differentiation, for 3 consecutive days. Group C was the control group, and no Wnt3a protein was added in the whole process of differentiation. Main outcome measures: the growth and proliferation of mesenchymal stem cells were observed by inverted microscope, the expression of surface markers of mesenchymal stem cells was detected by flow cytometry, and the differentiation of mesenchymal stem cells into osteoblasts was detected by alkaline phosphatase staining. Quantitative analysis of alkaline phosphatase. Results: the positive rates of malignant CD44, CD90 and CD45 were 94.88%, 94.37% and 0.95% respectively in the third generation of bone marrow mesenchymal stem cells. On the 7th day after adding osteogenic inducer, the induced bone marrow stromal stem cells showed alkaline phosphatase positive. On the 11th day, the activity of alkaline phosphatase in group A was higher than that in group B and C (P0.01). On the 21st day, the activity of alkaline phosphatase in group B was lower than that in group C (P0.01). Conclusion: the osteogenesis of rat bone marrow derived mesenchymal stem cell bodies is regulated by Wnt3a signaling pathway, and this regulation is dependent on the differentiation stage of the affected cells. After mesenchymal stem cells enter the differentiation phase, the bone forming ability of mesenchymal stem cells is regulated by the differentiation of mesenchymal stem cells. The classical Wnt signaling pathway mediated by Wnt3a promotes the differentiation of mesenchymal stem cells into osteoblasts, but at the late stage of osteoblast maturation, Wnt3a-mediated classical Wnt signaling pathway changes from promoting to inhibiting the ultimate maturation of osteoblasts.
【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】：2010
【分類號】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前5條

1 于晶晶;王紅;劉曦;賀曾;劉嘉馨;;2種方法制備的血小板凝膠釋放PGFs含量及生物活性的初步研究[J];中國輸血雜志;2012年02期

2 胡振宇;;TGF-β1促進(jìn)rhBMP-2對成骨前體細(xì)胞增殖分化的觀察[J];口腔頜面外科雜志;2012年01期

3 陳學(xué)鵬;段銀鐘;錢紅;金作林;;PKC、PKA信號通路在調(diào)控體外培養(yǎng)人牙囊細(xì)胞VEGF表達(dá)中的作用[J];中國細(xì)胞生物學(xué)學(xué)報;2012年05期

4 黃興;曹烈虎;李海航;蘇佳燦;;復(fù)合骨移植替代物的臨床應(yīng)用[J];中國組織工程研究;2013年34期

5 楊陽;李瑪琳;;促骨修復(fù)的重組生長因子的研究開發(fā)進(jìn)展[J];云南中醫(yī)學(xué)院學(xué)報;2013年04期

相關(guān)博士學(xué)位論文 前2條

1 崔福愛;BMP-6和VEGF表達(dá)載體的構(gòu)建及其促進(jìn)成骨作用的實驗研究[D];山東大學(xué);2010年

2 閆香珍;重組人骨形成蛋白2和重組人神經(jīng)生長因子β在三度根分叉缺損修復(fù)過程的作用研究[D];山東大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 程永帥;富含血小板血漿協(xié)同BMP-4促成骨分化研究[D];青島大學(xué);2009年

，

本文編號：2446156