Wnt信号通路在压力刺激下大鼠髁突软骨病理变化过程中的作用及其机制研究

发布时间：2018-01-06 04:41

  本文关键词：Wnt信号通路在压力刺激下大鼠髁突软骨病理变化过程中的作用及其机制研究　出处：《南京大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 大鼠髁突软骨 骨关节炎 软骨细胞 细胞增殖 Wnt信号通路 骨关节炎样变 细胞外基质 炎症反应 Wnt

【摘要】：第一部分Wnt信号通路在压力刺激下大鼠髁突软骨细胞增殖过程中的作用[目的]研究软骨细胞增殖活力受到抑制的原因及其在髁突软骨病理性变薄过程中的作用,本研究拟探讨Wnt信号通路对压力刺激下的大鼠髁突软骨病理性变薄,软骨细胞增殖水平受到抑制的影响,明确其具体的作用和机制。[方法]选择6周龄SD雄性大鼠应用本课题组自主设计的颞下颌关节压应力加载动物模型装置(专利号：201120210396.4),对大鼠髁突软骨施加向上、向后的80g/侧超负荷压应力刺激诱导大鼠髁突软骨发生病理性变薄,将所有动物设为早期组和晚期组,每组分为四个亚组：空白对照组,单纯加药组,单纯加力组和加力加药组。苏木素-伊红(HE)染色观察大鼠髁突软骨层厚度及形态学的变化;免疫组化(immunohistochemistry, IHC)检测Wnt信号通路下游标记分子β-catenin和GSK3β,以及细胞增殖标记分子cyclin D1, PCNA, Histone H3的表达改变。采用RT-PCR检测Wnt下游标志分子Axin2在mRNA水平的表达改变。[结果]1.髁突软骨受到机械力刺激的晚期,Wnt信号通路受损。2.机械力刺激髁突软骨的晚期,应用GSK3β抑制剂可以一定程度恢复Wnt信号通路的活力,并显著提高髁突软骨细胞的增殖活力。3.机械力刺激髁突软骨的晚期,GSK3β抑制剂通过上调cyclin D1, PCNA和Histone H3的表达,促进细胞的DNA合成,恢复软骨细胞的增殖水平。[结论]1.机械力刺激髁突软骨导致Wnt信号通路受损,软骨细胞的增殖活力受到抑制及髁突软骨病理性变薄。2.应用GSK3β抑制剂恢复Wnt信号通路的活力可以上调软骨细胞的增殖水平并缓解髁突软骨的病理性变薄。第二部分Wnt信号通路在压力刺激下大鼠髁突软骨骨关节炎样病理变化过程中的作用[目的]为了研究Wnt信号通路对大鼠髁突软骨在持续机械应力作用下病理性改变的作用,我们对已经证实调节软骨细胞增殖活力的Wnt信号通路进一步研究其在维持细胞外基质内环境稳态和炎症反应中的作用。探讨Wnt信号通路在大鼠髁突软骨骨关节炎样病理变化过程中的作用及其机制。[方法]选择6周龄SD雄性大鼠建立髁突软骨骨关节炎动物模型,同样分为早期组和晚期组,每组分为四个亚组：空白对照组,单纯加药组,单纯加力组合加力加药组。采用阿辛蓝(Ab)染色观察大鼠髁突软骨蛋白多糖含量的变化；免疫组化(immunohistochemistry, IHC)检测Col-Ⅱ和NF-α的蛋白水平变化,RT-PCR检测Col-Ⅱ和Col-X型胶原蛋白,蛋白酶MMPs,炎症因子TNF-α和IL-1的mRNA水平的表达改变,研究胶原蛋白和多糖表达水平,以及炎症因子表达水平的变化。[结果]1.GSK3β抑制剂恢复机械应力加载大鼠髁突软骨晚期的Wnt信号通路活力,抑制MMPs的活性并上调胶原蛋白合蛋白多糖的表达。2.GSK3β抑制剂还显著下调了机械力刺激晚期的软骨细胞中炎症因子TNF-α和IL-1的高表达。[结论]机械力刺激髁突软骨抑制了Wnt信号通路,导致软骨细胞外基质受损和炎症应答的激活。GSK3β抑制剂恢复受损的Wnt信号通路活力,并维持软骨细胞外基质内环境的稳态,抑制一系列炎症反应。
[Abstract]:The first part of the Wnt signaling pathway in the process of pressure stimulated proliferation of rat condylar cartilage cells [Objective] to study the proliferation of cartilage cells inhibited and the reasons in the condylar cartilage pathological thinning process, this paper intends to discuss the Wnt pathway thorn Condylar Cartilage Pathological rats induced by the pressure becomes thin, the proliferation level of cartilage cells inhibited the effect, clear its function and mechanism. The specific methods of selection of 6 week old male SD rats using the research group designed the temporomandibular joint stress loading animal model device (Patent No.: 201120210396.4), the rat condylar cartilage is applied to on the back side, 80g/ overload stress induced rat condylar Cartilage Pathological thinning, all animal for early and late groups, each group was divided into four subgroups: blank control group, simple dosing group, simple Afterburner afterburner group and drug group. Hematoxylin and eosin (HE) staining was used to observe the changes of condylar cartilage thickness and morphology of rats; immunohistochemistry (immunohistochemistry, IHC) detection of Wnt signaling pathways downstream molecular markers of beta -catenin and GSK3 beta cell proliferation marker cyclin, D1, PCNA, Histone H3 expression the detection of Wnt by RT-PCR. Expression of Axin2 in mRNA downstream molecular level changes. Results the]1. condylar cartilage by mechanical stimulation of the late Wnt pathway impaired.2. mechanical stimulation of the condylar cartilage in the late application of GSK3 beta inhibitors can be recovered to a certain degree of Wnt signaling pathway activity, and significantly improve the condylar cartilage the cell proliferation activity of.3. mechanical stimulation of the condylar cartilage in the late GSK3 beta inhibitors through upregulation of cyclin expression of D1, PCNA and Histone H3, promote the synthesis of DNA cells, restore the proliferation of chondrocytes. [conclusion]1. mechanical stimulation of condylar cartilage impairs Wnt signaling, cartilage cell proliferation activity was inhibited and the condylar cartilage pathological thinning of.2. application of GSK3 beta Wnt signaling pathway inhibitor to restore vitality can change the pathological increase of cartilage cell proliferation level and ease of condylar cartilage is thin. The second part of the Wnt signaling pathway in the process of pressure under the stimulation of condylar cartilage in osteoarthritis pathological changes of rats [Objective] to study the signal transduction of Wnt in rat condylar cartilage under continuous mechanical stress change of force under the action of Wnt signaling pathway we have proven to regulate cartilage cell proliferation activity further its role in maintaining cell homeostasis and inflammation in stroma. To investigate the Wnt signal pathway in the process of condylar cartilage of osteoarthritis like pathological changes in rats. Method: effect and mechanism To establish the condylar cartilage of osteoarthritis animal model of 6 week old male SD rats were equally divided into early and late groups, each group was divided into four subgroups: blank control group, simple dosing group, single drug group. The combination of afterburner afterburner alcian blue (Ab) staining to observe the changes of rat condylar cartilage bone proteoglycan content; immunohistochemistry (immunohistochemistry, IHC) changes in protein levels of Col- II and NF- alpha detection, RT-PCR detection and Col- II type Col-X collagen, protease MMPs, change the expression of inflammatory factor TNF- alpha and IL-1 mRNA levels of collagen, protein and polysaccharide expression levels and inflammatory factors the expression level of]1.GSK3. The beta Wnt signaling pathway inhibitor restored the mechanical dynamic loading of condylar cartilage in rats with the expression of.2.GSK3 beta inhibitors inhibit MMPs activity and increase of collagen and proteoglycan also significantly reduced Mechanical stimulation of cartilage cells in the late inflammatory factor TNF- alpha and IL-1 expression. Conclusion] mechanical stimulation of condylar cartilage inhibits the Wnt signaling pathway, Wnt signaling pathway leading to the recovery of activity impaired activation of.GSK3 beta inhibitors of extracellular matrix of cartilage damage and inflammatory response, and maintain the chondrocyte extracellular matrix homeostasis of internal environment a series of inflammatory reaction, inhibition.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R782
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本文编号：1386355