AMPK及Sclerostin在骨关节炎发病机制中的作用研究

发布时间：2018-02-02 13:57

本文关键词： 骨关节炎 AMPK 骨硬化蛋白合成代谢分解代谢和凋亡　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：骨关节炎(Osteoarthritis,OA)是一种退行性骨关节病,该病的发生率随着年龄增高而增多。由于目前没有很好的预防和治疗骨关节炎的药物,所以进一步的阐明该疾病的发生发展的机制,对于开发新的药物有重要意义。第一部分:软骨特异性敲除AMPK加重成年小鼠关节不稳和老年自发性的骨关节炎AMPK是一种进化上保守的丝氨酸/苏氨酸蛋白激酶,包含一个催化亚基(α1和α2)和两个调节亚基。正常的关节软骨细胞表达AMPK。已有研究表明软骨细胞中AMPK活性下降与OA和衰老相关。在小鼠膝关节不稳OA模型和人OA膝关节软骨中均发现AMPKα的磷酸化水平降低。在老年的小鼠膝关节软骨中AMPKα 的磷酸化水平也降低。Interleukin-1β(IL-1β)和 tumor necrosis factor αα(TNFα)都可以下调软骨细胞中AMPK活性。体外上调AMPK活性会减轻IL-1β或TNFα介导的分解代谢。此外,研究也发现了 AMPK激活剂在体内具有软骨保护作用。但是最近的一项研究却发现软骨特异性敲除AMPKα1在手术诱导的小鼠OA模型中不会影响骨关节炎进程。在本项研究中,我们利用Cre-Loxp技术,获得了软骨特异性敲除AMPK的小鼠(AMPKα1 cKO,AMPKα2 cKO和AMPKα cDKO小鼠)来研究其在骨关节炎发生发展中的作用。结果发现,和同窝野生小鼠相比,AMPK敲除小鼠创伤后关节炎加重,特别是AMPKα cDKO小鼠。此外,雄性的AMPKα cDKO小鼠在老年自发性骨关节炎模型中关节损伤也加重。非常有意思的是,雌性老年AMPKαcDKO小鼠关节损伤不严重。进一步的体外研究发现小鼠原代软骨细胞中敲除AMPK会增强IL-1β刺激后的分解代谢。体内研究表明AMPKα cDKO小鼠关节面上MMP-3,MMP-13,phospho-NF-KB和凋亡标记物的表达增高。综上所述,我们的结果发现软骨细胞中的AMPK活性对于维持关节软骨稳态具有重要作用。第二部分:小鼠高表达SOST蛋白会加重内侧半月板不稳定造成的骨关节炎Wnt/β-catenin信号通路在维持骨和软骨稳态中扮演重要角色。骨关节炎患者的软骨损伤与Wnt/β-catenin信号通路异常有关。Sclerostin(SOST)是一种可溶的Wnt信号通路的抑制剂,可以调节骨稳态。SOST最初被认为只是骨细胞分泌的多肽,但是最近的研究表明肥大软骨细胞也可以分泌SOST蛋白。小鼠和绵羊手术诱导的OA关节软骨和人晚期OA关节软骨中SOST的表达也增多。此外,Interleukin-1α(IL-1α)会上调SOST的mRNA水平。最近的研究表明上调的SOST在关节损伤中具有保护作用。体外实验表明SOST可以剂量依赖性的抑制IL-1α介导的分解代谢,体内实验表明Sost敲除会加速手术介导的关节损伤。在本项研究中,我们利用SOST转基因(SOST Tg)小鼠来研究它在维持关节稳态中的作用。结果发现,与对照组相比,SOST Tg小鼠创伤后关节损伤没有减轻,反而加重。此外,SOST Tg小鼠非手术侧的软骨下骨厚度均比对照组小,但是术后8周,SOST Tg小鼠的手术组的软骨下骨厚度比非手术组有显著性增厚。SOST Tg小鼠骨关节炎加重可能与β-catenin信号通路下调和软骨细胞凋亡增多有关。高表达SOST会加重关节不稳诱导的OA。我们的结果进一步表明关节软骨的稳态需要精密调控的Wnt信号通路。
[Abstract]:Osteoarthritis (Osteoarthritis, OA) is a degenerative joint disease, the incidence of the disease increases with age increased. Because the drugs are not currently well the prevention and treatment of osteoarthritis, the occurrence and development mechanism so that further clarify the disease, have important significance for the development of new drugs. The first part: the cartilage specific knockout AMPK mice increased joint instability and senile spontaneous osteoarthritis AMPK is an evolutionarily conserved serine / threonine protein kinase that contains a catalytic subunit (alpha 1 and alpha 2) and two regulatory subunits. Articular cartilage cells expression of AMPK. has been studied the activity of AMPK in cartilage cells decreased with OA and aging related normal. In the mouse knee instability model of OA and OA in articular cartilage of knee joint were found in AMPK alpha decreased phosphorylation levels in mice. Articular cartilage in elderly AMPK alpha phosphate The level of.Interleukin-1 decreased beta (IL-1 beta) and tumor necrosis factor alpha alpha (TNF alpha) can reduce the activity of AMPK in cartilage cells. In vitro upregulation of AMPK activity will reduce the IL-1 or TNF alpha beta mediated catabolism. In addition, the study also found that the AMPK activator with cartilage protective effect in vivo but. A recent study found that cartilage specific knockdown of AMPK alpha 1 not in mouse OA model induced by surgery of osteoarthritis process. In this study, we used Cre-Loxp technology to obtain the cartilage specific AMPK knockout mice (AMPK alpha 1 cKO, AMPK cKO and AMPK alpha cDKO alpha 2 mouse) to study its role in the development of osteoarthritis. The results showed that compared with littermate wild-type mice, AMPK knockout mice and posttraumatic arthritis increased, especially AMPK alpha cDKO mice. In addition, the male AMPK cDKO mice alpha in elderly osteoarthritis In the model of joint injury also increased. Interestingly, female cDKO mice aged AMPK a joint injury is not serious. In vitro studies further show that knockdown of AMPK can enhance the catabolism of IL-1 beta stimulated mouse primary cartilage cells. In vivo studies showed that MMP-3, AMPK alpha cDKO mouse articular surface MMP-13 expression increased. Phospho-NF-KB and apoptosis markers. In summary, our results suggest that the cartilage cells in AMPK activity has an important role in maintaining the homeostasis of articular cartilage. The second part: the high expression of SOST protein in mice will aggravate the medial meniscus instability caused by osteoarthritis Wnt/ beta -catenin signaling pathway plays an important role in maintaining the homeostasis of bone and cartilage in patients. Osteoarthritis cartilage damage and Wnt/ beta -catenin signal pathway on.Sclerostin (SOST) is a soluble inhibitor of Wnt signaling pathway can regulate the bone Steady state.SOST initially considered only polypeptide osteoblasts, but recent studies have shown that hypertrophic chondrocytes can secrete SOST protein. The expression of SOST induced mouse and sheep surgery OA articular cartilage and articular cartilage in late OA also increased. In addition, Interleukin-1 alpha (IL-1 alpha) can up regulate SOST mRNA levels. The study shows that upregulation of SOST has protective effect on the joint damage. In vitro experiments showed that SOST can inhibit the catabolism of IL-1 alpha induced dose-dependent manner, in vivo experiments show that knockdown of Sost could accelerate joint damage mediated by surgery. In this study, we use SOST (SOST Tg) transgenic mice to study it is in the maintenance of homeostasis in the role of joint. The results showed that compared with the control group, SOST Tg mice after trauma joint injury not reduced, but increased. In addition, SOST Tg mice non operative side of the thickness of subchondral bone Were lower than control group, but after 8 weeks, SOST Tg mice group thickness of subchondral bone non surgical group were significantly thickened.SOST Tg mice osteoarthritis may aggravate down-regulation and beta -catenin signaling pathway and apoptosis of cartilage cells increased. The high expression of SOST will increase the joint instability induced by our OA. the results further showed that Wnt signaling pathway requires precise regulation of articular cartilage homeostasis.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R684.3

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