低硒与胶原基因多态性及DNA甲基化在大骨节病中联合作用研究

Based on Kashin-Beck disease(KBD) is characteristic by chondrocyte necrosis, and seriously endanger the health of the population, however the cause remains unclear，In accordance with the studied conclusions " abnormal collagen gene expression will cause cartilage metabolic disorders and lead to bone and joint disease", but there is no research to explore the relationship between cartilage collagens (Ⅰ, II, III, VI, Ⅸ, Ⅹ, Ⅺ type collagens) and KBD at home and abroad; refered to the deeply studies in environment - gene interaction. In this study, cartilage collagen should be selscted as the studied target, refer to its research results in osteoarthritis, TaqMan gene SNP genotyping technology shoule be used to get the polrmorphism loci and the high risk gentypes of KBD; Methylation changes of cartilage collagen gene in KBD should be cleared by Methylation specific PCR technique, collagen protein and mRNA expression in cartilage also be detected. So the role of cartilage collagen in the pathogenesis of KBD disease and its possible mechanism should be understood. Advanced environment-gene interaction analysis technology shoule be used to quantitative estimate and test the combined effects of low selenium and suspicious gene in the further study. To clarify the role and nature of cartilage collagen in Kashin-Beck disease; To promote the studies of environment- gene interaction role in the pathogenic pathway and blocking measures for Kashin-Beck disease;To enhance the prevention and treatment of Kashin-Beck disease in China.

针对以软骨细胞坏死为特点、病因不清而严重危害人口健康的大骨节病；依据"胶原基因的异常表达导致软骨代谢障碍与骨关节病有关"的研究结论，且在国内外尚无软骨胶原基因（Ⅰ、Ⅱ、Ⅲ、Ⅵ﹑Ⅸ﹑Ⅹ﹑Ⅺ型胶原）与大骨节病的相关研究；基于国际上环境-基因联合作用的研究进展。本研究将以软骨胶原作为研究基质物，参照国际上对其在骨关节病中的研究结果，用TaqMan基因SNP分型技术获得大骨病多态性基因位点和高风险基因型；用甲基化特异性PCR技术明确大骨节病胶原基因甲基化现象，同时检测胶原蛋白及其mRNA在软骨中的表达，得到软骨胶原在大骨节病发病中的作用及可能的机制；进一步采用先进的环境-基因交互作用的分析技术，定量估算并检验低硒与可疑基因的联合作用。以阐明软骨胶原在大骨节病发病中的作用与本质；推进大骨节病环境-基因联合致病作用的致病通路及阻断措施的研究；以应用于大骨节病的疾病控制、监测和预防。

首次探讨软骨胶原及金属基质蛋白多态性及其蛋白表达在大骨节病（KBD）发病中的作用，结果发现：（1）COL9A1基因的rs6910140位点上的野生型等位基因“C”是KBD的保护因素；(2) 在COL11A1基因的rs2229783位点中，纯合子“G” 的基因型分布是KBD的危险因素；(3)MMP-3基因中的rs679620 和rs639752的多态性和KBD的易感性相关，（矫正P值均小于0.05）;（4）未发现研究位点与疾病的分期（严重度）相关（P值均大于0.05）。(5) KBD 患者中，MMP-1和MMP-3蛋白表达增多，而Ⅸ、Ⅺ型胶原，TIMP-3蛋白表达降低。以上结果提示KBD易感性与胶原基因多态性有关，可能与其对细胞外基质（胶原和金属基质蛋白酶）蛋白表达水平调控有关，使软骨细胞外基质降解能力大于合成能力，最终造成关节软骨退变坏死，与KBD的发病有关。首次在KBD患者中进行全基因甲基化分析，对差异化基因进行基因本体论（GO）和 KEGG通路分析，结果发现了97个差异基因，包括36个高甲基化和61个低甲基化位点，在这些位点中有14个有意义的信号通路，且这些生物学途径支持KBD的自身免疫疾病和炎症疾病假说。进一步用bisulfite sequencing polymerase chain reaction （BSP）方法在软骨中验证，其结果与高分辨率阵列的结果一致。表明KBD和对照之间存在DNA甲基化的差异。总之，本研究结果提示细胞外基质（胶原和金属基质蛋白酶）蛋白表达水平与KBD患病有关，细胞外基质蛋白的表达差异与对应基因的多态性有关，表现出相同环境不同的发病情况。而DNA甲基化改变参与了KBD发病，可能与自身免疫疾病途径相关。