研究背景： 大骨节病(Kashin-Beck disease，KBD)是一种局限于地方性的畸形性骨关节病，其原发性损害主要是生长发育过程中关节软骨、骺软骨以及骺板软骨出现深层凝固性坏死为特点的病理学改变。目前，大骨节病危险因子和发病机理尚不清楚，大量流行病学和分子生物学实验支持T-2毒素中毒和／或硒缺乏学说。然而，粮食中的T-2毒素和环境中的低硒如何特异性损害儿童骺板软骨和关节软骨仍然不清楚，提出的病因假说并不能够合理地阐述大骨节病病因发病机理。 目的： 1.确定大骨节病环境危险因素及其在患者关节软骨差异表达基因中的作用； 2.确定T-2毒素在大鼠体内不同组织中代谢和分布情况及其在体外培养软骨细胞中的代谢特征； 3.明确T-2毒素和HT-2毒素诱导软骨细胞凋亡的作用； 4.明确补硒对HT-2毒素诱导软骨细胞凋亡和自噬的干预作用。 方法： 1.以中英文关键词(Kashin-Beck disease or KBD or大骨节病or矮人病)和(Logistic regression or危险因素or病例对照研究)检索Medline、Web ofknowledge、 Google scholar、Springer Link、Elsevier、知网、维普和万方共8个中英文数据库。随机效应模型或固定效应模型用来合并Odds Ratio(OR)和95％Confidence Interval(CI)。利用Meta分析和系统综述鉴别大骨节病环境危险因素，进而收集大骨节病和健康人的关节软骨，软骨总RNA提取使用安捷伦总RNA提取迷你试剂盒，采用安捷伦人类全基因组表达芯片进行基因芯片杂交，使用qRT-PCR进行验证。然后基因表达谱分析技术用来鉴别大骨节病关节软骨的差异表达基因，结合大骨节病环境危险因素，检索国际环境反应基因数据库，鉴别出环境反应基因。使用DAVID、IPA和STRING对识别的环境反应基因进行生物学过程、KEGG通路富集分析、IPA经典通路、功能网络分析以及蛋白质互作网络分析。 2.选择12只健康的3w SD大鼠(雌雄各半)，按照随机数字表法分成两组。①毒素组中6只大鼠(雌雄各半)分别给予T-2毒素溶液(1 ㎎／㎏)灌胃，②对照组中6只大鼠(雌雄各半)分别给予生理盐水(0.9％NaCl溶液)灌胃。实验大鼠在灌胃8 h后被处死，并收集股骨、膝关节、肋软骨、骨骼肌、心脏、肝脏和肾脏等用于HPLC-MS／MS分析。同时给予T-2毒素孵育软骨细胞48 h，使用HPLC-MS／MS分析软骨细胞培养基和软骨细胞中T-2毒素及其代谢物的含量。最后MTT方法确定T-2毒素及其代谢物对软骨细胞活力影响，使用酶标仪在510 nm波长下测定吸光度值，计算软骨细胞活力。 3.比较T-2毒素和HT-2毒素诱导软骨细胞凋亡，软骨细胞活力检测采用MTT方法，软骨细胞凋亡采用Annexin V-FITC／PI流式细胞术双染和PI染色检测软骨细胞周期。MDA和SOD试剂盒检测软骨细胞氧化应激水平，Hoechst 33258染色检测软骨细胞凋亡荧光强度。利用qRT-PCR检测目的基因Bax的mRNA表达水平。Western Blot检测凋亡相关蛋白Bax、Caspase-9和Caspase-3的表达水平。所得到的实验数据采用均数和标准差表示，实验数据的分析使用SPSS18.0软件(t检验)，以P<0.05为差异显著性水准。 4.硒对HT-2毒素诱导软骨细胞凋亡和自噬的影响，采用MTT法检测软骨细胞活力，Annexin V-FITC／PI流式细胞术双染检测软骨细胞凋亡。Hoechst 33258染色和MDC染色分别检测软骨细胞凋亡和自噬荧光强度。利用qRT-PCR和Western Blot分别检测目的基因Bax和Beclin1的mRNA表达水平；凋亡相关蛋白Bax、Caspase-9、Caspase-3和自噬相关蛋白Beclin1、LC3-II／LC3-I比值的表达水平。采用均数和标准差表示所得到实验数据，使用SPSS18.0软件分析实验数据(t检验和方差分析)，P<0.05认为差异具有显著性。 结果： 1.纳入7项研究包括3087例大骨节病和6402例健康对照。与大骨节病发病显著相关的危险因素包括大骨节病患者年龄(OR:1.19)，父母患病率(5.16)，家庭卫生(1.68)，食物来源(3.29)，主食小麦(1.12)，小麦胚芽坏死率(6.03)，挥发性盐基氮总氮(6.85)，发硒(2.29)，蛋白质摄入(0.79)和大米(0.90)。通过Meta分析和系统综述鉴别大骨节病环境危险因素包括谷物污染(T-2毒素，DON和NIV)、微量元素(硒和碘)和维生素E缺乏。基因表达谱分析发现大骨节病关节软骨中21个上调的环境反应基因和7个下调的环境反应基因，共同参与软骨细胞的凋亡、新陈代谢、生长和发育。KEGG和IPA分析发现三条共同显著表达的信号通路PI3K-Akt Signaling Pathway，p53 Signaling Pathway和Apoptosis Pathway；基因本体分析发现，环境反应基因主要参与大骨节病的凋亡、死亡和细胞程序性死亡。 2.在股骨(173.26 μg／㎏)、膝关节(125.73μg／㎏)和肋软骨(125.35 μg／㎏)中T-2毒素和HT-2毒素总浓度显著高于心脏(60.89 μg／㎏)、肝脏(70.99μg／㎏)和肾脏(44.28μg／㎏)。T-2毒素浓度和HT-2毒素浓度在股骨和肋软骨中也显著高于心脏、肝脏和肾脏。T-2毒素进入大鼠体内8 h后，迅速代谢为HT-2毒素，其在不同组织中的代谢转化率为68.2％-90.7％。同时给予T-2毒素孵育软骨细胞48 h，培养基中T-2毒素的含量从20 ng／mL显著减少到6.67 ng／mL，HT-2毒素的浓度从0 ng／mL显著增加到6.88 ng／mL。然而，T-2毒素在软骨细胞中的相对浓度从0 ng／g增加到12.80 ng／g。T-2毒素对软骨细胞活力的影响显著高于HT-2毒素，并且具有显著剂量反应关系。 3.T-2毒素和HT-2毒素对软骨细胞活力存在显著剂量反应关系。T-2毒素和HT-2毒素显著影响软骨细胞周期(S期和G₂期)。T-2毒素诱导软骨细胞氧化应激(MDA和SOD)显著高于HT-2毒素。T-2毒素和HT-2毒素诱导软骨细胞48 h，其凋亡率分别为49.67％和36.28％。T-2毒素诱导软骨细胞凋亡相关基因和蛋白Bax、Caspase-9蛋白和Caspase-3蛋白的表达水平显著高于HT-2毒素。 4.硒能显著拮抗HT-2毒素对软骨细胞活力的影响。HT-2毒素孵育软骨细胞48 h，补硒后软骨细胞凋亡率从42.90％显著降低为33.47％。补硒显著降低HT-2毒素诱导凋亡相关基因和蛋白Bax、Caspase-9蛋白和Caspase-3蛋白的表达，同时显著降低自噬相关基因和蛋白Beclin1和LC3-II／LC3-I蛋白比值的表达。 结论： 1.结果发现谷物污染、硒缺乏和低蛋白质摄入共同作用导致大骨节病的发生。真菌毒素(T-2毒素和DON)诱导p53信号通路以及微量元素硒和维生素E缺乏诱导P13K-Akt和Ras信号通路共同诱导软骨细胞的凋亡和死亡，与大骨节病发病显著相关。 2.T-2毒素进入大鼠体内8 h后，在体内各组织器官内迅速代谢为HT-2毒素，并在骨骼系统内同时检测到T-2毒素和HT-2毒素。T-2毒素诱导软骨细胞损伤过程中主要代谢为HT-2毒素，T-2毒素对软骨细胞活力影响显著高于同等浓度的HT-2毒素。 3.T-2毒素和HT-2毒素显著降低软骨细胞活力，诱导氧化应激损伤，促进线粒体凋亡相关基因和蛋白的高表达，进而软骨细胞出现凋亡现象；T-2毒素诱导软骨细胞凋亡的作用显著高于HT-2毒素。 4.HT-2毒素诱导软骨细胞氧化应激损伤，并导致软骨细胞凋亡和自噬显著上调，具有相似的时间依赖性。补硒能够显著降低HT-2毒素对软骨细胞活力影响，同时降低软骨细胞凋亡和自噬表达水平。 关键词：大骨节病；T-2毒素；HT-2毒素；凋亡；自噬 论文类型：应用基础 *本研究得到国家自然科学基金(批准号：81472924、81620108026)和国家重点研发计划-政府间科技合作重点项目(2016YFE0119100)资助。

 Backgrounds: Kashin-Beck disease (KBD) is an endemic and deformity osteoarthropathy. The pathological changes of primary damage are deeper coagulation necrosis in the epiphyseal cartilage, epiphyseal plate cartilage and articular cartilage during the growth and development process. At present, the etiology and pathogenesis of KBD are still unclear, a large number of epidemiological and molecular biological experiments supported the theory of T-2 toxin poisoning and selenium deficiency. However, it is still unclear that how the T-2 toxin and low selenium specifically damage children's epiphyseal plate cartilage and articular cartilage, it is not one hypothesis can fully explain the pathogenesis of KBD. Objective: 1.To determine the environmental risk factors for KBD, and the role of environmental risk factors on the differential expression genes of KBD patient's articular cartilage. 2.To explore the concentration of T-2 toxin and HT-2 toxin in different tissues of rats administrated of T-2 toxin, and the metabolism of T-2 toxin in cultured human chondrocytes. 3.Comparaison of apoptosis of chondrocytes induced by T-2 toxin and HT-2 toxin. 4.Investigate supplementation selenium play an important role on the apoptosis and autophagy of chondrocyte induced by HT-2 toxin. Methods: 1.The search strategy used keywords “Kashin-Beck disease” or “KBD” and “logistic regression” or “risk factor” or “case-control study”, and we searched five English and three Chinese databases (Medline, Web of knowledge, Google scholar, Springer Link, Elsevier, CNKI, WANFANG, CQVIP) to identify environmental risk factors for KBD. Dersimonian and Laird randomized or fixed effect model were applied in processing data using pooled odds ratios (OR) and 95% confidence intervals (CI). We have identified environmental risk factors for KBD using meta-analysis and systematic review. Total RNA was isolated from articular cartilage of KBD and healthy controls. Agilent human whole genome microarray chip was used to analyze the amplified, labeled and hybridized total RNA, and the validated microarray data was partially verified using qRT-PCR. Gene expression profile analysis technology was used to identify differentially expressed genes of articular cartilage in KBD.These environmental response genes (ERGs) were derived from the comparative toxicogenomics database combined with environmental risk factors of KBD. The identified ERGs were subjected to KEGG pathway enrichment, biological process (BP), ingenuity canonical pathways, genes network analysis and interaction network analyses using the database for annotation, visualization and integrated discovery (DAVID), ingenuity pathway analysis (IPA) and STRING. 2.Total 12 healthy SD rats (half males and half females) aged 3 weeks, which were divided into two groups using the random number table. (1) T-2 toxin group included six rats (half males and half females), which received a gavage of T-2 toxin with a dosage of 1 ㎎/㎏, and (2)control group included six rats (half males and half females), which received a gavage of normal saline (0.9% NaCl solution). These rats were sacrificed after 8 h, and the thighbone, knee joints, costal cartilage, skeletal muscle, heart, liver, and kidneys were collected for HPLC-MS/MS analyses. The chondrocytes were incubated with T-2 toxin for 48 h, the medium supematants and chondrocytes were analyzed by HPLC-MS/MS separately, and identified metabolites of T-2 toxin in cultured chondrocytes. For determination of viability of chondrocytes induced by T-2 toxin and its metabolites using the MTT assay, optical density values were read with an automatic enzyme-linked immunosorbent assay reader at 510 nm. Cell survival rate was calculated and estimated. 3.Comparison of apoptosis of chondrocytes induced by T-2 toxin and HT-2 toxin. The viability of chondrocytes was measured by MTT method. The apoptosis of chontrodytes was measured by Annexin V-FITC/PI flow cytometry, and PI was used to measure the chondrocyte cycle. The MDA and SOD test kit were used to measure the oxidative stress levels of chontrodytes. Hoechst 33258 staining agent was used to measure the immune strength of apoptosis of chondrocytes respectively. qRT-PCR was used to measure the mRNA expression levels of Bax. Western Blot was used to measure the protein expression level of Bax, Caspase-9 and Caspase-3. All the obtained data were represented by mean and standard deviation, and SPSS18.0 software was used to analyze the test data (t test), and P<0.05 was considered to be significant. 4.Effect of selenium on the apoptosis and autophagy of chondrocytes induced by HT-2 toxin. MTT method was used to measure the viability of chondrocytes. Annexin V-FITC/PI flow cytometry was used to measure the apoptosis of chontrodytes. Hoechst 33258 and MDC staining agent were used to measure the immune strength of apoptosis and autophagy of chondrocytes respectively. qRT-PCR was used to measure the mRNA expression levels of Bax and Beclinl. Western Blot was used to measure the protein expression level of Bax, Caspase-9 and Caspase-3, Beclinl, ratio of LC3-II/ LC3-1 . All the obtained data were represented by mean and standard deviation, and SPSS18.0 software was used to analyze the test data (t test and analysis of variance), and p<0.05 was considered to be significant. Results: 1.We identified seven studies covered 3087 KBD and 6402 controls. The onset of KBD were significantly associated with risk factors including age (OR: 1.19), parents prevalence (5.16), family hygiene (1.68), food source (3.29), wheat (1.12), wheat germ necrosis rate (6.03), total volatile basic nitrogen (6.85) and low selenium in hair (2.29), protein intake (0.79) and rice (0.90). The polluted grains (T-2 toxin, deoxynivalenol and nivalenol), trace element (selenium and iodine) and vitamin E deficiency were identified as the ERFs for KBD using meta-analysis and systematic review. Gene expression profiling analysis identified 21 up-regulated ERGs and 7 down-regulated ERGs in cartilage of KBD, which involved with apoptosis, metabolism and growth and development. DAVID and IPA analysis found out three significant common pathways involved with PI3K-Akt signaling pathway, p53 signaling pathway and apoptosis pathway, and gene ontology function analysis found out 28 ERGs were involved with apoptosis, death and cell death in KBD. 2.Total concentrations of T-2 toxin and HT-2 toxin in the thighbone (173.26 μg/㎏), knee joints (125.73 μg/㎏), and costal cartilage (125.35 μa/㎏) were significantly higher than heart (60.89 μg/㎏), liver (70.99 μg/㎏) and kidney (44.28 μg/㎏). The concentrations of T-2 toxin and HT-2 toxin in the thighbone and costal cartilage were also significantly higher than heart, liver, and kidney. T-2 toxin was rapidly metabolized into HT-2 toxin in 8 h, and the metabolic conversion rates in the different tissues were range from 68.20% to 90.70%. The concentration of T-2 toxin in the cell medium decreased from 20 ng/mL to 6.67 ng/mL, and the concentration of HT-2 toxin increased from 0 ng/mL to 6.88 ng/mL during the 48 h incubation, whereas the concentration of T-2 toxin in chondrocytes increased from 0 ng/g to 12.80 ng/g. The viability of chondrocytes induced by T-2 toxin was was significantly much higher than HT-2 toxin, with a significant dose-response relationship. 3.T-2 toxin and HT-2 toxin were decreased the viability of chondrocytes with a significant dose-response relationship. T-2 toxin and HT-2 toxin can influence the chondrocyte cycle (S and G₂). The oxidative stress (MDA and SOD) of chondrocytes induced by T-2 toxin was significantly higher than HT-2 toxin. The apoptosis rate of chondrocytes induced by T-2 and HT-2 toxin increased 49.67% and 36.28% in 48 h respectively. The apoptosis-relatived gene and protein Bax, Caspase-9 and Caspase-3 proteins in chondrocytes induced by T-2 toxin were significantly higher expression than HT-2 toxin. 4.Selenium could significantly antagonise the viability of chondrocytes induced by HT-2 toxin. Chondrocytes were incubated with HT-2 toxin for 48 h, and the apoptosis rate of chondrocytes was significantly reduced from 42.9% to 33.47% after supplementation selenium. Supplementation selenium significantly reduced the expression level of Bax and Beclinl genes of chondrocytes, and significantly reduced the proteins expression level of Bax, Caspase-9, Caspase-3, Beclinl, ratio of LC3-II/ LC3-I proteins of chondrocytes induced by HT-2 toxin. Conclusion: 1.Meta-analysis found out the polluted grain, selenium deficiency and low protein intake may contribute to be onset of KBD together. The mycotoxins (T-2 toxin and DON) induce p53 signaling pathway and the trace element selenium and vitamin E deficiency induce PI3K-Akt and Ras signaling pathway contributing to the apoptosis and cell death of chondrocyte, and result into the development of KBD. 2.The rats were admination of T-2 toxin for 8 h, it was rapidly metabolized into HT-2 toxin in various tissues, and T-2 toxin and HT-2 toxin were detected in the skeletal system simultaneously. T-2 toxin in the cultured chondrocytes can be metabolized into HT-2 toxin, and T-2 toxin was more toxic to chondrocyte than HT-2 toxin at equivalent concentrations. 3.T-2 toxin and HT-2 toxin significantly reduced viability of chondrocytes, induced oxidative stress injury, promoted the high expression of apoptosis-related gene and proteins in mitochondria, ultimately induced the apoptosis of chondrocytes. Meanwhile, the apoptosis of chondrocytes induced by T-2 toxin were much higher than HT-2 toxin significantly. 4.HT-2 toxin induced oxidative stress injury of chondrocytes, and result into the apoptosis and autophagy of chondrocytes with similar time-dependent manner. Selenium supplementation can significantly reduce the viability of chondrocytes induced by HT-2 toxin, and reduce the apoptosis and autophagy of chondrocytes. KEY WORDS: Kashin-Beck disease, T-2 toxin, HT-2 toxin, Apoptosis, Autophagy TYPE OF THESIS: Application Fundamentals *This research was supported by the national natural science fund (no: 81472924, 81620108026) and national key research and development program project - China-South Korea international cooperation project funded (2016YFE0119100).