OCCURRENCE CHARACTERISTIC OF KASHIN-BECK DISEASE BASED ON NUCLEAR FAMILY PEDIGREES

Objective The occurrence characteristic of Kashin Beck Disease (KBD) in pedigrees ascertained on the basis of one proband was estimated. Methods A total of 255 individuals in 40 pedigrees were collected from areas in the Shaanxi Province. Results ① Parents and siblings of index cases have a 3-4 times higher risk than a random unrelated individual. The odds ratio for disease is higher in mothers than in fathers of index cases; ② Prevalence in relatives of index cases (K r= 59.2% ) greatly exceeds population prevalence (K= 17.5% ); ③ K r increases with sibship size; ④ There is no significant difference of K r for male and female siblings of index cases. Also, population prevalence is not sex specific. Conclusion In conjunction with environmental agents, genetics may play an important role in KBD etiology.

In vitro effects of sodium hyaluronate on the proliferation and the apoptosis in chondrocytes from patients with Kashin-Beck disease and osteoarthritis

Objective：To identify the in vitro effects of sodium hyaluronate（HA） on the proliferation and the apoptosis of chondrocytes from patients with Kashin-Beck disease（KBD） and osteoarthritis（OA）. Methods：Samples of articular cartilages from KBD and OA patients, as well as healthy volunteers（6 subjects in each of the 3 groups） were dissected, digested with collagenase and the cells cultured in monolayers. Chondrocytes from each sample were assigned to an untreated group and two HA-treated groups： H0（no HA）, H100（HA, 0.1 g/L） and H500（HA, 0.5 g/L）. The first passage chondrocytes were used to observe proliferation using the MTT assay, and apoptosis by flow cytometry through Annexin V/PI staining. Results：HA promoted proliferation of chondrocytes in all the three groups, and.in KBD and OA groups, for cells cultured for 4 and 6 days, H500 significantly promoted the cell proliferation. The apoptotic rates of both KBD and OA group chondrocytes were in the order H500 〈 HA100 〈 H0. Conclusion：Sodium hyaluronate administration has a dosedependent in vitro effect to promote proliferation and inhibit apoptosis of chondrocytes from patients with KBD and OA.

STUDIES ON BLOOD GLUTATHIONE PEROXIDASE PROTEIN LEVEL OF CHILDREN IN KESHAN DISEASE AND KASHIN-BECK DISEASE AREAS

Objective To oberve the change in blood glutathione peroxidase (GSH-Px) protein levels of residents in the low-selenium (Se) area by contrasting the blood GSH-Px protein level of the children in the Keshan disease area with those in the Kashin-Beck disease and non-endemic areas. Methods GSH-Px protein levels were measured by enzyme-linked immunosorbent assays (ELISA). The Se content and GSH-Px activity were assayed by the 2,3-diaminonaphthalene spectrofluorimetric method and glutathione reductase-coupled method respectively. Results ①The blood Se content and GSH-Px protein level of children in Keshan disease area (Moding) were significantly lower than those in Xi’an non-endemic area, however, there was no significant difference when compared with the low-Se non-endemic area; ②The blood Se content, GSH-Px activity and GSH-Px protein level of children in the Kashin-Beck disease area (Yulin) were significantly lower than those of children in two non-endemic areas and in the Keshan disease area; ③The blood Se content and GSH-Px activity were positively correlated to the GSH-Px protein level respectively. Conclusion These results indicate that the blood GSH-Px protein level is decreased in the low-Se residents. The Se status not only affects the GSH-Px activity but also regulate the GSH-Px protein level.

EFFECT OF SELENIUM ON IMMUNE FUNCTION OF ERYTHROCYTE IN KASHIN-BECK DISEASE

Objective To investigate the relationship between erythrocyte immune function and selenium (Se) level. Methods Forty-nine Kashin-Beck patients in endemic area aged 13-16 years were divided into two groups and were orally given either selenized yeast or sodium selenite to provide 200 μ g selenium per day for 12 weeks. Erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells complement receptor typeⅠ(CR1), the immune function of red blood cells, and circulating immune complexes(CIC) were determined. Results After supplementing with selenium for 12 weeks, erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells CR1 were significantly increased. But the difference in rosette formation rates of IC and CIC content was not significant between before and after Se supplementation. Conclusion The increase of the immune function of the erythrocyte by selenium-supplement may be one of the effective mechanisms for the prevention of Kashin-Beck disease.

STUDIES ON LYMPHOCYTE SUBSETS, HUMORAL IMMUNITY AND THEIR RELATIONSHIP WITH SELENIUM IN PATIENTS WITH KASHIN-BECK DISEASE

Objective To study the humoral immunity status and distribution pattern of lymphocyte subgroups of peripheral blood mononuclear cell (PBMC) in patients with Kashin-Beck Disease (KBD), and their relationship with erythrocyte selenium. Methods 23 X-ray diagnosed patients, 22 age- and sex- matched healthy children in KBD affected area (KAA), and 25 in KBD non-affected area (KNAA) were randomly selected. Immunohistochemistry with monoclonal antibodies anti-CD4, anti-CD8, anti-CD20 was conducted to analyze the lymphocyte subsets. Serum IgM, IgA, IgG, Complement C3 and C4 were assayed using rate nephelometry (Array 360 System, USA). The contents of erythrocyte selenium was determined by 2,3-diaminonaphthalene fluorescence assay. Results CD4+ and CD8+ cells percentage in PBMCs and serum IgA were significantly lower in KAA than those in KNAA(P< 0.05). CD20+ percentage in KAA displayed a decreasing trend compared to KNAA, although not statistically significantly. No statistical differences were found in CD4/CD8 ratio, serum IgG, IgM, C3 and C4 levels. Erythrocyte selenium level in KAA still showed a pronounced decrease compared to that in KNAA. Correlation analysis showed that erythrocyte selenium contents had a strong association with the CD4 cell percentage (r= 0.625, P< 0.05), as well as serum IgA (r= 0.462, P< 0.05). In addition, a moderate correlation between the serum IgA and CD4+ percentage (r= 0.130, P> 0.05) was found. Conclusion These results suggested that children in KAA had a comparably low cellular immunity level and their humoral immunity status was also in a state of moderate immune suppression. Of this immune disorder in Kashin-Beck disease patients, selenium deficiency probably played a critical role via affecting the distribution pattern of peripheral blood lymphocyte. Selenium-deficiency and immune impairment maybe both have something to do with the cause-effect chain of KBD.

POSSIBLE HYDROGEOCHEMICAL PRECONDITIONS FOR KASHIN－BECK DISEASE IN TRANSBAIKALIA

In the basin of the Amur River at the territory of Russia, in the northern regions of China and Korea the osteal-articulate disease of people and animals, well known as Kashin-Beck disease is spread. About 20 hypotheses had been suggested for explaining the disease causes. some of them are briefly introduced. The hydrogeochemical researches in Transbaikalia. Russia, showed that natural water of the endemic areas have several features in chemical composition.some of them are considered by existing hypotheses as the causes of the disease.others do not concern the possible etiologic factors of the disease for the present. The author examines these features respectively.

A STUDY ON RELATIONSHIP BETWEEN ORGANIC SUBSTANCES IN WATER AND PREVALENCE OF KASHIN BECK DISEASE

This study is to acquire the relationship between organic substances in water and prevalence of Kashin Beck disease (KBD). We sampled in three endemic areas and their corresponding neighboring non endemic areas, determined the contents of organic substances and examined the children. The results showed that:①humus type B exited only in endemic areas; ②the content of humus type B is proportional to the state of the illness. KBD may be related to the humus type B in drinking water.

EFFECTS OF CEREALS FROM KASHIN－BECK DISEASE ENDEMIC AREA ON FIBRILLOGENESIS IN VITRO OF CARTILAGE COLLAGEN TYPE Ⅱ IN RATS

In this feeding trial rats were fed on diets of cereals from Kashin-Beck Disease (KBD) endemic area, Se-supplemented cereals from the above area, and cereals from Non-KBD endemic area. The purpose of this paper was to investigate the effects of cereals from KBD endemic area and Se on the formation kinetics of cartilage type Ⅱ collagen fibril, the stability and ultrastructure of fibrils formed in vitro. The results indicated that low-selenium cereals from KBD endemic area resulted in a decelerated rate and extant of forming the type Ⅱ collagen fibril, the fibril stability reduced, fibril diameters diminished, and fibril banding periods increased or decreased in vitro. Sesupplemented cereals from KBD endemic area partially rectified the pathologic changes mentioned above. These data are important for further studying the etiology and pathology of KBD.

X-ray image characteristics and related measurements in the ankles of 118 adult patients with Kashin-Beck disease

Background Kashin-Beck disease （KBD） is a chronic,degenerative osteoarthropathy that causes severe skeletal deformation.Although many researchers have proven that almost all KBD patients who showed an increaseing proximo-distal gradient had radiographic abnormalities of the ankle,few detailed description of radiographic changes in the ankles of patients with KBD has been reported,especially for variable measurements of ankle changes.The purpose of this study was to demonstrate the radiographic characteristics of the ankles of adult KBD patients.Methods One hundred and eighteen adult KBD patients from september to October 2010 in Rongtang county in China were examined with lateral radiographs of the right ankle.The morphological abnormalities in the talus,calcaneus,navicular bone,distal tibia,and joint space were analyzed,and the calcaneus length,height,length-height ratio,tuber angle,front angle,plantar angle,and distal tibia anteroposterior （AP） length were measured using Riepert＇s method.Results Eighty-one patients （68.6％） had abnormal ankle radiographs; 72 （88.9％） patients had talus changes,69 （85.2％） patients had calcaneus changes,28 （34.6％） patients had navicular bone changes,and 48 （59.2％） patients had distal tibia changes.For 118 KBD patients,the average calcaneus length was 7.4 cm,height was 4.3 cm,and the length-height ratio was 1.7.The calcaneus tuber angle was 28.2°,front angle was 38.0° and the plantar angle was 74.2°.The distal tibia anteroposterior length was 4.05 cm.Compared with 50 normal adults （control group）,significant differences were found for the calcaneus length,the calcaneus length-height ratio,and the distal tibia AP length.Conclusions Patients with KBD have characteristic abnormalities on ankle radiographs; talus depression and deformity,calcaneus shortening deformity,and distal tibia deformity with AP length widening were the most typical changes.

西藏土壤-植物-动物(人)系统中硒含量与大骨节病的关系

通过对西藏大骨节病区与非病区中土壤、粮食和儿童发样匹配采样以及对样品中硒含量的分析,探讨了硒元素在土壤-植物-动物生态系统中的含量特征及其与大骨节病分布流行的关系。结果表明,西藏地区整体处于低硒水平,所有病区样品的硒含量显著低于非病区(P<0.05)。土壤低硒是导致西藏大骨节病区粮食、人体硒水平偏低及病情流行的一个主要原因。改善土壤硒环境,提高土壤硒含量是西藏地区大骨节病防治的主要措施之一;补硒能够显著提高人体发硒水平,并有效控制大骨节病,但是需要长期坚持。

大骨节病患者关节软骨细胞凋亡及相关调控因子Bcl-2表达分布的特点

目的 探讨大骨节病患者关节软骨细胞凋亡及相关调控因子 Bcl- 2表达分布的特点。方法 采用脱氧核糖核酸末端转移酶介导的脱氧核糖核酸缺口标记法 (TUNEL氏法 )检测凋亡阳性细胞 ,利用单克隆抗体免疫组化检测 Bcl- 2在关节软骨中的表达。结果 1TU NEL法染色的大骨节病儿童关节软骨凋亡阳性细胞数较正常关节软骨增多 ,且主要分布在中层。 2大骨节病儿童关节软骨 Bcl- 2的表达显著高于正常关节软骨中的表达 ,以表层最多 ,其次为中层 ;成人晚期大骨节病患者剥蚀的关节软骨表层 Bcl- 2的表达聚集在软骨细胞团中。结论 大骨节病患者关节软骨细胞凋亡及相关调控因子 Bcl- 2表达较正常人显著增多。

大骨节病患者血清NO、NOS、sFas/APO-1检测分析

目的探讨一氧化氮(NO)、一氧化氮合酶(NOS)、诱导型一氧化氮合酶(iNOS)、可溶性Fas(sFas)诱导大骨节病发生发展的作用与特点,为大骨节病防治研究提供新的思路。方法选择陕西省永寿县、榆林市榆阳区大骨节病区与咸阳市渭城区非大骨节病区120例调查对象,分为成人、儿童大骨节病组;成人、儿童病区对照组;成人、儿童非病区对照组,每组20例,平均年龄、性别无差别。用酶还原法检测了血清NO,化学比色法检测了血清NOS、iNOS,ELISA法检测了血清sFas水平。结果①大骨节病成人组血清NO、iNOS显著高于病区和非病区成人对照组(P< 0.05);血清NOS、sFas/APO鄄1水平高于非病区成人对照组(P< 0.005)。②大骨节病儿童组血清NO高于非病区儿童对照组(P< 0.05);血清NOS、iNOS均高于病区和非病区儿童对照组(P< 0.005)。结论儿童大骨节病发生发展中NO诱导途径起一定的作用,而Fas途径作用不明显。当疾病发展到成人大骨节病时,二者均起一定作用。

ICP-AES法测定西藏大骨节病区及非病区饮用水中的微量元素

运用ICP-AES法准确测定了西藏地区大骨节病区和非病区中沟水、河水、泉水和溪水等四种饮用水中Cu，Zn，Fe，Ca，Mg，Mn，A1，K，Ba，P，Sr，Cd，V，Cr，Ni，PB和Co等多种微量元素的含量。该方法的相对标准偏差在0．80G～2．83％之间，标准物质的测定结果满意，方法简单可靠。文章通过对测定结果的分析，得出西藏不同地区、不同饮用水类型中微量元素的含量差异，为改善西藏大骨节病区居民的生活饮用水提供了科学理论依据。

大骨节病和骨性关节炎患者滑膜白介素-1β和肿瘤坏死因子-α的比较

目的比较大骨节病(KBD)和骨性关节炎(OA)关节滑膜和滑液中白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达,探讨两种病的发病机制。方法分别收集大骨节病和骨性关节炎患者关节滑膜和滑液20例和18例,以单纯半月板损伤患者的关节滑膜和滑液19例为对照;采用免疫组织化学染色法,观察大骨节病、骨性关节和半月板患者关节滑膜IL-1β和TNF-α阳性表达强度;采用ELISA法检测三种滑液标本IL-1β和TNF-α的水平。结果TNF-α在KBD和OA滑膜组织中的表达与对照组相比有显著性差异(P<0.05);IL-1β在KBD和OA滑膜组织中的表达与对照组相比也有显著性差异(P<0.01);IL-1β和TNF-α在KBD滑膜组织中的表达与OA相比无显著性差异(P>0.05)。TNF-α和IL-1β在KBD和OA滑液中的水平与对照组相比均有显著性差异(P<0.05);IL-1β和TNF-α在KBD滑液中的水平与OA相比均无显著性差异(P>0.05)。结论IL-1β和TNF-α可能在KBD和OA发生发展中扮演着重要的角色。

硫酸软骨素纳米硒可抑制T-2毒素诱导的大骨节病软骨细胞凋亡

目的确定硫酸软骨素纳米硒对T-2毒素干预体外大骨节病软骨细胞生长的影响。方法合成并表征硫酸软骨素纳米硒粒子,依据《大骨节病临床诊断标准》(WS/T 207-2010),选择Ⅱ/Ⅲ度KBD患者6例关节软骨进行体外分离、培养。分别给予硫酸软骨素纳米硒联合T-2毒素进行干预,采用MTT、HE染色和流式细胞仪观察细胞生长和凋亡的变化。结果合成的硫酸软骨素纳米硒中硒的含量为10.1%,可在蒸馏水中自组装成粒径为30-200 nm纳米粒子,红外图谱提示纳米硒与硫酸软骨素可能以共价键的方式结合;硫酸软骨素纳米硒在浓度为50-200 ng/ml可有效抑制20 ng/ml T-2毒素诱导大骨节病软骨细胞的凋亡作用,降低T-2毒素诱导软骨细胞的早期凋亡率[(8.64±1.57)%,P<0.05]。结论硫酸软骨素纳米硒具有抗T-2毒素诱导软骨细胞凋亡的作用,可作为一种潜在的治疗大骨节病的药物制剂。

线粒体细胞色素c介导的caspase-9激活通路参与大骨节病关节软骨细胞凋亡的发生机制

目的通过评价线粒体功能研究线粒体介导的caspase-9激活通路参与成人大骨节病关节软骨细胞凋亡的机制。方法关节软骨细胞培养,Hoechst 33258染色检测软骨细胞核形态,流式细胞仪检测软骨细胞凋亡率和细胞内活性氧含量,Western bloting检测细胞色素c的表达,荧光法检测caspase-9和caspase-3活性。结果大骨节病患者关节软骨细胞较正常软骨细胞凋亡百分率增加,线粒体细胞色素c释放,caspase-9和caspase-3活性增高,且活性氧含量增加。结论线粒体氧化应激参与了大骨节病软骨细胞的病理生理变化,线粒体功能障碍可能在大骨节病关节软骨细胞凋亡过程发挥了重要作用。

T-2毒素致软骨细胞损伤临界值和硒保护实验研究

目的：主要探索Ｔ－２毒素对体外培养软骨细胞致损临界值以及硒保护作用。方法：应用单层培养传代软骨细胞，用生化方法检测软骨细胞ＤＮＡ量和蛋白量，透射电子显微镜观察软骨细胞超微结构。结果：（１）Ｔ－２毒素在０．００１～０．００５ｍｇ／Ｌ培养液中（以下相同）剂量范围内，随浓度增加，Ｔ－２毒素对软骨细胞ＤＮＡ合成和细胞分裂增殖影响逐渐增大，加硒后该影响有所减弱，但仍不能阻止损伤的发生；（２）Ｔ－２毒素在０．０００５ｍｇ／Ｌ时，对软骨细胞的影响甚微；Ｔ－２毒素在０．００１ｍｇ／Ｌ和０．００５ｍｇ／Ｌ时，细胞损伤明显，与对照组比较有显著差别（Ｐ＜０．００１）。

大骨节病、骨关节炎软骨细胞分泌IL-1β、TNF-α及透明质酸对其影响实验研究

目的:探讨在不同剂量透明质酸作用下,人大骨节病(KBD)软骨细胞和骨关节炎(OA)软骨细胞对IL-1β、TNF-α合成分泌的影响,为临床上透明质酸钠治疗OA及KBD提供实验依据。方法:将符合临床诊断标准的KBD及OA患者与从遭遇意外事故的病人作为正常对照,分离、体外培养三组患者关节软骨细胞,分别给予100μg/ml(H100组)、500μg/ml(H500组)不同剂量的透明质酸钠(HA)干预及阴性对照(0.00μg/ml,H0组),通过放免测定细胞培养上清液中IL-1β、TNF-α的含量变化。结果:与正常组比较,KBD和OA组软骨细胞IL-1β、TNF-α明显增高;HA干预随其剂量增高抑制IL-1β、TNF-α合成增强,对TNF-α的抑制有统计学意义。结论:骨关节炎和大骨节病患者软骨细胞合成分泌IL-1β、TNF-α明显增加;应用透明质酸后,可降低两种炎性因子的分泌。

大骨节病患者膝关节置换术后IL-1β、TNF-α表达变化及其与疼痛指数的相关性

目的探讨大骨节病患者膝关节置换术后IL-1β及TNF-α表达变化及其与膝关节疼痛指数的相关性。方法大骨节病组与骨性关节炎组患者各30例,单纯半月板损伤患者20例作为对照组。采用ELISA法检测3组患者血液及滑液标本IL-1β和TNF-α的表达水平,流式细胞术检测关节软骨细胞凋亡率,疼痛指数变化采用VAS评分,分析VAS与IL-1β、TNF-α变化的相关性。结果大骨节病组与骨性关节炎组血液标本、滑液标本IL-1β和TNF-α的水平均显著高于对照组(P<0.05);大骨节病组及骨性关节炎组细胞凋亡率显著高于对照组(P<0.05);Pearson相关分析表明,患者手术前后血液中IL-1β和TNF-α表达水平变化与VAS呈显著正相关。结论大骨节病患者IL-1β和TNF-α的水平较高,且其表达变化与VAS密切相关。

Difference in apoptosis-associated genes expression profiling and immunohistology analysis between Kashin-Beck disease and primary osteoarthritis

Kashin-Beck disease(KBD)is a chronic and deformed endemic osteoarthritis,without fully known etiology.As compared to primary osteoarthritis(POA),its pathogenesis still exists controversial.We performed this study to discriminate the difference in genes expression involved in apoptosis from KBD cartilages and POA cartilages.Microarray analysis and Ingenuity Pathway Analysis(IPA)were used to identify the molecular mechanisms/canonical pathways implicated in KBD arthritis.Immunohistochemistry staining was carried out to detect tissue distribution of PDCD5 and EGR1 in the knee cartilages from KBD and POA.The 23 up-regulated apoptosis-related genes with[2-fold change were identified.‘‘Role of Macrophages,Fibroblasts,and Endothelial Cells in Rheumatoid Arthritis’’signaling was screened as the most relevant pathway through IPA.The 8 differentially regulated genes were verified by qRT-PCR analysis.Programmed cell death 5 in middle and deeper zones and early growth response 1 in superficial and middle zones showed protein overexpressed in KBD cartilage samples comparing to those in POA cartilage samples.Data indicates a higher expression level of apoptosis-related functional genes in KBD articular cartilage compared with POA articular cartilage.