不同层次软骨细胞亚群SIRT1基因特异性敲除对骨关节炎的影响及其机制研究

Osteoarthritis (OA) is a multifactorial disease,especially aging. Prevention of chondral degeneration and consequent cartilage loss is the key center in the process of AO. There are increasing evidence suggesting that the disequilibrium between anabolism and catabolism, the changes of chondral matrix and subchondral bone result in the aforementioned phenomena, and aging accelerate these pathological process. According to amount of publised articles, the gene, SIRT1 might play a crucial role in OA onset. In our apllication,we are going to establish three kinds of the OA mouse model in which SIRT1 gene in the diverse subgroups of chondrocytes will be specifically knockouted using Cre/LoxP system (superficial and middle chondrocytes in cartilage expresses type II collagen whereas deep chongdrocytes expresses type X collagen. when DNA fragment encoding the Cre recombinase,a site-specific recombinase,is inserted downstream to the promotors of type II,X the recombinase will clip off the LoxP site flanked gene, and the specific gene in the subgroup chondrocytes in diverse levels of catilage is knockout, such as SIRT1. Namely there will be three kinds of SIRT1 gene knockout mouse model: type II promotor or type X promotor mediated SIRT1 knockout and the SIRT1 knockout in whole cartilage.),the classical OA model established in the wildtype mice is used as the control. Next, in such SIRT1 knockout OA model, we would detect the pathology, apoptosis of chondrocytes, morphological structure changes of subchondral bone, bone density and the angiogenesis between the bottom of cartilage and the underlying subchondral bone plate in various indicated time point,as well as the SIRT1-FOXO and SIRT1-NF-κB signalling pathway and the phosphorylation, acetylation and activities of the related proteins. Moreover, in the cellular level, we will elucidate the concrete and detailed machanism of SIRT1 using SIRT1 overexpression and SIRT1 siRNA vector transfection into the subgroups of chondrocytes.

骨关节炎(OA)是与年龄因素密切相关的多因素疾病，延缓软骨进行性的退变和丢失是防治的中心环节。国内外研究显示软骨的退变丢失受软骨细胞分解合成代谢紊乱、软骨基质结构性质的改变以及软骨下骨向软骨内侵蚀等共同影响所致，而衰老带来的一系列内环境的改变加速了这些过程，其中长寿基因SIRT1可能在OA的发生发展中起着重要的作用。本课题拟建立野生型和不同亚群软骨细胞的SIRT1基因条件性敲除小鼠的OA模型，检测不同时期的软骨病理性损伤，软骨细胞凋亡，软骨下骨形态结构、骨密度和两者交界处血管生成等改变，以及SIRT1-FOXO和SIRT1-NF-κB信号转导途径相关蛋白磷酸化、乙酰化水平和活性变化，并结合离体实验：将SIRT1表达载体和siRNA转染不同亚群的软骨细胞，检测上述信号通路中相关蛋白在不同时间点的磷酸化、乙酰化水平以及细胞凋亡程度，明确SIRT1基因在OA中的作用及其分子机制。

目的：通过改变软骨组织及软骨相关细胞中SIRT1基因的表达情况，在体及离体条件下研究SIRT1基因改变对骨关节炎的影响及其可能的调控机制。方法：建立软骨组织特异性SIRT1基因敲除小鼠并体外培养原代软骨细胞，通过相关手术及物质刺激建立小鼠及软骨细胞骨关节炎模型，通过特殊染色、基因芯片、RT-PCR、Western Blot、Elisa、免疫组化等方法观察小鼠膝关节软骨组织及软骨细胞mRNA、蛋白质的变化情况及软骨组织的改变情况和软骨细胞的增殖衰老情况。结果：他莫昔芬诱导后小鼠软骨组织中SIRT1基因敲除成功，SIRT1基因敲除后骨关节炎发生时，膝关节软骨组织退变明显，Mankin评分升高，SIRT1、AKT、II型胶原蛋白表达量降低，SREBP2、VEGF、HMGCR蛋白表达量明显升高；在软骨细胞中，SIRT1基因表达升高时，II型胶原蛋白、聚集蛋白聚糖mRNA的表达升高，IL-1β、TNF-α mRNA的表达降低，PGC-1α蛋白的表达升高，而p53、NF-κB、bax、SREBP-2、MMP-13的表达降低，细胞外基质减少，而SIRT1基因表达量降低时则出现相反的情况。 结论：SIRT1基因改变可能通过调控PI3K/AKT、NF-κB等信号通路中相关因子的表达情况影响软骨细胞的衰老、凋亡并调节骨关节炎的发生发展。