Wnt信号途径对破骨细胞分化成熟的直接调控

发布时间：2018-01-10 21:00

本文关键词：Wnt信号途径对破骨细胞分化成熟的直接调控　出处：《重庆医科大学》2013年硕士论文　论文类型：学位论文

更多相关文章： β-catenin Sost Wnt3a Raw264.7 RANKL

【摘要】：目的明确Wnt信号通路是不是可以通过不依赖成骨细胞的作用直接调节破骨细胞的分化与成熟。从而可以全面的了解Wnt信号通路在骨态发育中的作用。为我们以后利用Wnt抑制剂抗体治疗骨质疏松打下坚实可靠地基础。并在这个基础上明确参与直接调节破骨细胞分化与成熟的具体Wnt信号通路。方法通过用浓度梯度的方法去诱导Raw264.7的分化与成熟并确定其最适诱导浓度，再用最佳浓度去筛选与破骨细胞分化可能相关的Wnt蛋白。根据前期实验得出的结果构建相关腺病毒及干扰病毒，再以Wnt蛋白腺病毒和干扰腺病毒去处理破骨前体细胞株Raw264.7后，检测这些Wnt通路对破骨细胞分化成熟的调控。实验时Raw264.7细胞分成四组分别为：空Raw264.7（阴性对照组）、Ad-GFP or Ad-RFP(实验对照组)、Ad-Wnt3a,Ad-β-catenin,Ad-Sost(实验组)和50ng/mLRANKL诱导Raw264.7(阳性对照组)检测它对Raw264.7分化成熟的影响。结果确定RANKL诱导Raw264.7的最佳浓度为50ng/mL，用50ng/mL的RANKL筛选相关蛋白。选出Wnt3、Wnt3a、Wnt5b、Wnt7a、Wnt7b、β-catenin、Sost可能与Raw264.7的分化成熟有关，并构建了这几种腺病毒（由美国芝加哥大学肿瘤研究中心何通川教授实验室赠送并保存）分别用这些腺病毒感染Raw64.7细胞并按空白组、实验对照组、实验组和阳性对照组共四组。分析结果得：β-catenin诱导的Raw264.7对比空白组对照组都呈单核细胞；Wnt3a诱导的Raw264.7对比空白组和对照组也呈单核细胞；而Sost诱导的Raw264.7对比空白组和对照组呈多核细胞。结论Wnt信号通路可以不通过成骨细胞间接地调节破骨细胞分化成熟，，并且直接参与其调控。并且β-catenin可以抑制Raw264.7细胞分化成熟；Wnt3a也可以抑制Raw264.7分化成成熟的破骨细胞；而Sost则可以促进Raw264.7的分化成熟。
[Abstract]:Objective to clarify the Wnt signaling pathway is not available through does not depend on the role of osteoblastic cells directly regulate osteoclast differentiation and maturation. Thus a comprehensive understanding of the Wnt signaling pathway in bone development in state. For our future use of a Wnt inhibitor antibodies for the treatment of osteoporosis and lay a solid foundation. Reliable and clearly involved in this based on the direct regulation of osteoclast differentiation and maturation of the Wnt signaling pathway.
By using the method of concentration gradient to induce Raw264.7 differentiation and maturation and determine the optimal induction concentration, and the optimal concentration to screening and differentiation of osteoclast may be related to Wnt protein. According to the preliminary experimental results to construct adeno-associated virus and virus interference, and then to Wnt protein of adenovirus and adenovirus to deal with interference the osteoclast precursor cell line Raw264.7, detect the Wnt pathway on the differentiation of osteoclast regulation. The Raw264.7 cells were divided into four groups respectively: Raw264.7 (negative control group), Ad-GFP or Ad-RFP (experimental group), Ad-Wnt3a, Ad- beta -catenin, Ad-Sost (experimental group) and 50ng/mLRANKL induced by Raw264.7 (positive control group) to detect its effect on Raw264.7 differentiation and maturation.
The optimal concentration of RANKL induced by Raw264.7 50ng/mL, 50ng/mL RANKL related protein screening. The selected Wnt3, Wnt3a, Wnt5b, Wnt7a, Wnt7b, beta -catenin, Sost and Raw264.7 could be differentiated mature, and constructed the several adenovirus (presented by the University of Chicago cancer research center Professor He Tongchuan laboratory and preservation) respectively. The adenovirus infected Raw64.7 cells in blank control group, experimental control group, experimental group and positive control group of four groups. Results: -catenin beta induced Raw264.7 compared with blank control group showed mononuclear cells induced by Wnt3a Raw264.7; compared with the blank group and the control group also showed mononuclear cells; Sost by contrast Raw264.7 blank group and control group were multinucleated cells.
Conclusion Wnt signaling pathway may not by osteoblasts indirectly regulate osteoclast differentiation and maturation, and directly involved in the regulation of beta. And -catenin can inhibit the differentiation of Raw264.7 cells; Wnt3a can also inhibit Raw264.7 differentiation into mature osteoclasts; while Sost can promote the differentiation of Raw264.7 into maturity.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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