大骨节病与骨性关节炎软骨细胞凋亡信号转导、差异基因及补硒干预的对比研究
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摘要
背景:
大骨节病(Kachin Beck disease,KBD)是一种以软骨坏死为主要改变的地方性变形性骨关节病,流行于我国从东北到西南的地带中,患病率和致残率很高。
自1849年俄罗斯Yurenski报道KBD以来,其发病机制至今仍不清楚。目前,对KBD病因发病机制的研究不仅局限于流行病学、病理学、环境因素(低硒、T 2毒素等)及防治等方面,且已涉及到细胞凋亡及其调控因子、差异基因和差异蛋白表达等。
骨性关节炎(Osteoarthritis, OA)是以关节软骨退变为主要特征,导致关节疼痛和功能障碍的疾病,好发于膝、髋等负重关节,常见于60岁以上的老年人群,与KBD好发于3 12岁儿童、软骨发育障碍、关节增粗畸形等继发性骨关节炎有着显著的不同。然而,这两类疾病均以关节软骨损伤为特征,严重影响成人的劳动力和生活质量。近年来,随着对OA发病机制的深入研究,证实软骨细胞凋亡、细胞因子信号转导障碍等是造成OA软骨损伤的重要原因之一。是否KBD的软骨损伤也涉及到软骨细胞凋亡及细胞因子信号转导障碍,以及两者之间有何区别,有待进一步阐明。
目前,KBD病因发病机制中存在问题很多,但急需解决的主要问题有:①发病原因是单一因素还是环境基因相互作用所致?②KBD和OA关节软骨在分子生物学水平上有何不同?③在基因水平KBD究竟发生了怎样的变化,其与OA有何区别和联系?④硒预防和治疗KBD软骨细胞的机制是什么?为此,本论文主要对比研究了KBD与OA软骨细胞凋亡信号转导、差异基因表达及补硒干预,以阐明这两类关节疾病在软骨细胞凋亡、基因表达、以及补硒对凋亡影响等方面的差异,为深入探讨KBD的病因发病机制、KBD与OA发病机制的异同,提高KBD防治效果,提供科学依据。目的:
1.通过体外培养KBD和OA的关节软骨细胞,比较软骨细胞生长增殖、形态结构特性、细胞凋亡及其信号转导通路的异同;
2.采用Agilent Human 1A基因表达谱芯片比较KBD和OA关节软骨表达基因,以筛选和确定KBD不同于OA的特征性基因;
3.通过不同浓度补硒,体外干预软骨细胞,探讨硒对KBD、OA和正常软骨细胞生长和细胞凋亡的影响。
方法:
1.将病例分为三组:KBD组,OA组和正常对照组。正常对照组和OA组选自非KBD病区,KBD组的病例选自KBD病区,年龄均在49 65岁之间。根据我国颁布的KBD病临床诊断标准,选择Ⅱ度和Ⅲ度病例;根据WOMAC诊断标准选择OA病例,排除诸如遗传性骨病,类风湿性关节炎等。在患者知情同意的情况下,软骨取材主要来自膝关节部位,并在年龄和性别等方面达到匹配。
2.在无菌条件下,体外培养KBD、OA和正常软骨细胞,运用MTT法、流式细胞仪、电镜、免疫荧光、激光共聚焦和免疫组化等实验方法,对比KBD、OA和正常软骨细胞生长特性和细胞凋亡途径的不同;对三组软骨细胞进行补硒干预,比较不同浓度硒(0.0125 g/ml～1.0 g /ml)对KBD和OA细胞生长和凋亡的影响。
3.提取KBD和OA关节软骨细胞RNA,运用Agilent Human 1A基因表达谱芯片技术,用间接标记方法,获得荧光标记的cRNA靶物,进而与基因芯片杂交,通过FeatureExtraction9.3、Spotfire 8.0和Genepix 3.0等软件,选择P<0.05的具有明显表达差异的基因,以筛选KBD不同于OA的特征性基因。
结果:
1. KBD和OA凋亡和信号转导通路的对比研究
1)KBD和OA组软骨细胞的增殖率、平均生长率显著滞后于正常对照组,其中KBD软骨细胞增殖率高于OA组。KBD软骨细胞超微结构以细胞膜形态不规则、细胞核扭曲变形、细胞膜内糖原边集、细胞膜糖原释放、高尔基体和线粒体肿胀为主,而OA组则以细胞肿胀、软骨细胞电子密度增加、细胞膜绒毛减少、胞浆内大量游离脂滴、高尔基体和内质网肿胀为主。
2)KBD和OA的软骨细胞凋亡显著高于正常组,细胞增殖周期G2/M期比例增高;Fas、Bax、Caspase 3、Caspase 9和P53表达上调;KBD软骨细胞Caspase 8和P53表达低于OA,而Caspase 9表达高于OA。
3)KBD和OA具有相似的细胞凋亡途径,与OA相比,KBD更倾向于线粒体凋亡途径。
2. KBD和OA关节软骨差异基因比较及生物通路模型的构建
在KBD与OA四组病例中筛选出显著差异的基因233个,其中共同上调的基因195个,共同下调的基因38个。根据其生物学功能分类,证实筛选出的基因中CSGALNACT、PIM2、EFNA1、SMAD 9、STK11、AQP、T cell factor/LEF、PTN,APCDD1和CAV等对KBD发生发展具有重要意义,主要涉及软骨代谢、离子通道蛋白、凋亡等多方面的基因,构建出与氧化应激、氨基酸代谢、脂代谢等相关的通路模型。
3.不同浓度硒含量干预对KBD和OA软骨细胞生长和凋亡的影响
1)当Se浓度>0.05 g/ml时能显著损害正常软骨细胞的生长,但Se浓度<0.025 g/ml时仍对正常细胞的生长有损伤;尽管Se浓度>0.50 g/ml对KBD软骨细胞生长有一定的毒性作用,但在0.25 0.10 g/ml之间可促进KBD软骨细胞的生长,细胞保持增殖状态;对于OA软骨细胞,Se浓度>0.50 g/ml对细胞增殖有毒性作用,Se浓度<0.25 g/ml细胞能够保持较高增值率,硒也有一定的保护作用。
2)加硒后对正常软骨细胞生长具有毒性作用,其损害程度随加硒浓度的增高而加重;对于KBD而言,加入0.50 g/ml～1.0 g/ml硒含量对细胞生长有毒性作用,0.10 g/ml～0.25 g/ml之间为促进生长,而加入<0.05 g/ml则细胞增殖能力下降;对于OA,加入0.50 g /ml～1.0 g/ml的硒对细胞生长有毒性作用,而Se<0.25 g/ml对细胞的增殖则有促进作用。
3)补硒对软骨细胞超微结构影响的结果显示,Se<0.10 g/ml对KBD,Se<0.05 g/ml对OA的软骨细胞损伤不显著,而Se>0.025 g/ml对正常软骨细胞的亚微结构有影响。
4)加入不同浓度硒时,正常软骨细胞的凋亡率显著高于不加硒的对照组;KBD组除高浓度硒(>0.25 g/ml)产生较高的凋亡率外,补硒可降低软骨细胞的平均凋亡率;在OA组,除引起较高的软骨细胞凋亡率的硒浓度(>0.25 g/ml)外,一定浓度的补硒也可降低软骨细胞凋亡率。
5)加入一定浓度的硒对KBD和OA软骨细胞凋亡调控蛋白表达(Fas、Bax、Caspase 3、Caspase 9、P53)有抑制作用,其中补硒对KBD软骨细胞的保护作用高于OA,而对正常软骨细胞有程度不同的损伤作用;三组之间保护软骨细胞的适宜硒浓度不一。
结论:
1. KBD和OA的关节软骨细胞凋亡率显著高于正常对照组,且Fas、Bax、Caspase 3、Caspase 9、P53表达增高;其中KBD软骨细胞Caspase 8和P53表达低于OA,而Caspase 9表达则高于OA;KBD超微结构变化类似于OA,但其线粒体凋亡途径损伤较OA更明显。
2. KBD差异表达基因不同于OA,其中显著差异的233个基因中KBD下调的基因38个,上调的基因195个,主要涉及CSGALNACT、PIM2、EFNA1、SMAD 9、STK11、AQP、T cell factor/LEF、PTN、APCDD1和CAV等反映代谢、离子通道蛋白、凋亡等多方面的基因。
3.保护KBD和OA的适宜硒浓度不同,在相同补硒剂量下,对KBD保护作用高于OA,但对正常软骨细胞有不同程度的损伤作用;超出适宜的补硒浓度,对三组软骨细胞均具有毒性损伤作用。
Background:
Kashin Beck(KBD)is an endemic disease, with a high prevalence in north China. Thedisease is characterized by cartilage necrosis and local deformation of bone and joint. It wasfirst reported in 1849 by a Russian Yurenski. Since then a lot of work has been done on thisdisease but still its etiology and pathogenesis is unclear. Different aspects of Kashin Beckdisease studied so far include epidemiological as well as experimental studies includingautopsy pathology genetic interactions in the environment and the cultivation of living cells.Studies have shown that the pathogenesis of Kashin Beck disease is related to environmentalfactors (low selenium, T 2 toxin, etc.), cartilage necrosis, as well as abnormal expression ofgene and proteins.
Osteoarthritis:Osteoarthritis (OA) is characterized by articular cartilage degeneration,causing joint pain and dysfunction. It occurs in knee, hip and other weight bearing joints. Itusually occurs in late years of life, mainly at the age of 60 years or older, while KBD startsin 3 12 year old child. They significantly differ in terms of abnormal thickening of thecartilage in joint’s development disorders. Recent studies on OA show that the disease isrelated to apoptosis, disorder in cytokine and signal transduction mechanism. Tissue sectionexperiments have proved that KBD cartilage damage is also related to abnormal expressionof apoptotic factors. Thus both KBD and OA shares some mechanism of the diseasedevelopment but it needs further clarifications about differences in development of both thediseases.
At present there are many unanswered questions about the pathogenesis and etiology ofKBD. Some of the questions which need further research include:①The etiology of KBD.Is it a single factor or an interaction between genes and environment?②What is thedifference between the pathological changes of the articular cartilage in KBD and OA?③What is the genetic basis of KBD and OA?④What is the mechanism of the KBD prevention and treatment by selenium .Keeping in view the aim of differentiating these twodiseases the etiology and pathogenesis of KBD and OA was further which provided ascientific basis for KBD prevention and treatment. This aim in this study we focused on thecomparative study of KBD and OA chondrocyte apoptosis signal transduction, genome widescan screening differential gene profiles and selenium intervening on chondrocyte apoptosis;
Objectives:
1. To compare the differences between cartilage cells of KBD and OA in terms ofchondrocyte growth and proliferation, morphological structure, apoptosis and signaltransduction pathway by culturing the cartilage cells of KBD and OAin vitro;
2. To identify the characteristic gene expression of KBD, which is different from the OA byAgilent Human 1AcDNAmicroarray;
3. To investigate the effect of selenium on cell growth characters and apoptosis among KBD,OAand normal cartilage groups by exposing them to different levels of selenium.
Method:
1. The patients were divided into three groups: KBD, OAand the normal group. The patientsof control and OA groups were selected from the non KBD endemic area, while patients ofKBD were selected from KBD endemic area, aged 49 65 years old. The diagnostic criteriafor KBD included assessment on fingers, joints and immunohistochemistry analysis. Patientswith StageⅡandⅢwere included in this study. OA patients were diagnosed according toWOMAC, patients with genetic bone disease and rheumatoid arthritis were excluded fromthe study. All cartilage were taken from the same location in the joint as far as possible toachieve all aspects to be matched.
2. In aseptic conditions, KBD, OA and normal cartilage cells were cultured in vitro. Wecontrast KBD, OA and normal chondrocytes of different growth characteristics and cellapoptosis pathway by MTT, flow cytometry, electron microscopy, immunofluorescence,laser confocal and immunohistochemistry; Selenium intervention on three groups ofcartilage cells was done to find the impact of different concentrations of selenium on cellgrowth and apoptosis in KBD and OA.
3. We compared difference in gene expression between KBD and OA articular cartilage bygenome wide scan, and checked them by real time PCR; Microarray were scanned byGene Axon 4000B and data was analyzed by Genepix 3.0 software package. Special KBDgenes which were different to OAwere selected.
Results:
I. Comparision of cell apoptosis and signal transduction pathways between KBD and OA
1) The average cell proliferation of KBD and OA cartilage group were significantly lowerthan the normal group, while OA growth rate was less than KBD cartilage cell. Theultrastructure in KBD cartilage cell were as follows: irregular cell shape, distorted nuclei,glycogen gathered within the cell membrane, glycogen release, Golgi apparatus andsignifican mitochondrial swelling; OAgroup: cell swelling, cartilage cells, increased electrondensity, reduced membrane villus in cytoplasm with a large number of free lipid droplets,Golgi apparatus and endoplasmic reticulum swelling.
2) The apoptosis in KBD and OA cartilage was significantly higher than normal group, cellcycle G1 / M phase was increased; The expression of Fas, Bax, Caspase 3, Caspase 9 andP53 increased; Caspase 8, P53 expression in KBD cartilage cells is relatively lower than theOA, while the expression of caspase 9 is relatively higher than OA.
3) The comparision of apoptosis genes in KBD and OA cartilage by gene chips, indicatedthat KBD and OA have similar apoptosis pathway, KBD was prone to mitochondriaapoptosis signal pathway.
II. The comparative study of genome wide scan and biological modeling in KBD and OAcartilage.
In the comparative study of KBD and OA cartilage, there were 233 same array of genesexpression, in which 195 genes up regulated and 38 genes down regulated in the four pairgroups. They were divided according to their biological function into categories such ascellular metabolism, ion channels and transport proteins, signal transduction, cytokines, cellreceptors and the cytoskeleton .It was confirmed before gene selction that the genes selectedCSGALNACT, PIM2, EFNA1, SMAD 9, STK11, AQP, T cell factor / LEF, PTN, APCDD1,CAV and others in KBD were of great significance. Based on the results, KBD is mainlyrelated to metabolism, ion channel proteins, apoptosis and other aspects of gene expressionabnormalities. We build oxidative stress, amino acid metabolism, lipid metabolism andrelated pathways model.
III. The effect of various concentrations of selenium (Se) on growth and apoptosis in KBDand OA.
1) Selenium in high concentrations i.e Se>0.05 g/ml produced toxic damage to normalcartilage cells while the low concentration of selenium also have an impact on the growthrate and the proliferation rate became negative.Studies on KBD cartilage have shown that acertain concentration of selenium can promote the KBD cartilage cells growth, but the highconcentrations of selenium have some of its toxic effects. The concentration of seleniumbetween 0.25 g/ml～0.10 g/ml significantly promote cell proliferation, but the low environment selenium can reduce the proliferation rate. Studies on OA have showedtolerance to high concentrations of selenium and toxic effects of selenium on cellproliferation, but still the cells will be able to maintain growth and proliferation; inSe<0.25 g/ml cell proliferation and growth rate were also high.
2) Normal cartilage after exposure to higher concentration of selenium showed toxic effectson the growth with more damage to cell growth; in terms of KBD, 1.0 g/ml～0.50 g/ml ofselenium produces toxic effect on cell growth, and a concentration of 0.25 g/ml～0.10 g/mlpromoted cell growth; while environmental selenium with a concentration of <0.05 g/mlwas the safety range; for OA, 1.0 g/ml～0.50 g/ml of selenium produced toxic effects oncell growth, Se<0.25 g/ml promoted cell proliferation.
3) The tolerance of KBD and OA to selinum was higher than the normal group, and amongthem KBD had higher tolerance than OA group and the tolerance of OA group is higher thanthe normal group. Microstructure indicated that cartilage cells were tolerant to Se<0.10 g/ml in KBD group and to Se<0.05 g/ml in OA group. While in the normal group thesub structure was by a concentration of Se>0.025 g/ml.
4) Effect of selenium on apoptosis: In normal cartilage cells the the apoptosis rate was higherwith different concentrations of selenium as compared to apoptosis rates without selenium.In the KBD group, the higher concentrations of selenium produced higher apoptotic rate butthe average apoptotic rate declined with selenium. In the OA group, the higher concentrationof selenium resulted in high apoptosis rate but the certain concentration of selenium mayreduce the apoptosis rate.
5) Immunohistochemistry of Fas, Bax, Caspase 3, Caspase 9, P53 and other apoptosisindicators also showed that a certain concentration of selenium added to the KBD and OAchondrocyte in culture can inhibit apoptosis protein expression. The tolerance to selenium ofKBD was higher than OA, and OA was higher than normal cartilage cells. Beyond a certainconcentration selenium can induce apoptosis.
Conclusion:
1. The rate of apoptosis in KBD and OA articular cartilage was significantly higher than thenormal group, and expression of Fas, Bax, Caspase 3, Caspase 9 and P53 increased. In thecomparison of KBD and OA, Caspase 8 and P53 expression in KBD were relatively lowerthan OA, while the caspase 9 expression is relatively higher than OA. Micro structuralchanges in KBD by electron microscopy confirmed that KBD apoptosis pathway is similarto OA, and may be more prone to mitochondrial apoptosis pathway.
2. There is significant difference in 233 gene expressions in KBD and OA, out of these 38genes were down regulated and 195 genes were up regulated in KBD. CSGALNACT, PIM2, EFNA1, SMAD 9, STK11, AQP, T cell factor / LEF, PTN, APCDD1, CAV and other geneswere confirmed to be of greater significance in KBD, and that is mainly related tometabolism, ion channel proteins, apoptosis and other abnormal genes expression.
3. A certain concentration of selenium has protective effect on the KBD and OAchondrocytes. Under the same dose of selenium, the tolerance of KBD is higer than OA andOA tolerance is higher than normal group.Higher concentrations of selenium have damagingand toxic effects on the normal cells and the three groups of chondrocytes have differentconcentrations of selenium tolerance.
大骨节病(Kachin Beck disease,KBD)是一种以软骨坏死为主要改变的地方性变形性骨关节病,流行于我国从东北到西南的地带中,患病率和致残率很高。
自1849年俄罗斯Yurenski报道KBD以来,其发病机制至今仍不清楚。目前,对KBD病因发病机制的研究不仅局限于流行病学、病理学、环境因素(低硒、T 2毒素等)及防治等方面,且已涉及到细胞凋亡及其调控因子、差异基因和差异蛋白表达等。
骨性关节炎(Osteoarthritis, OA)是以关节软骨退变为主要特征,导致关节疼痛和功能障碍的疾病,好发于膝、髋等负重关节,常见于60岁以上的老年人群,与KBD好发于3 12岁儿童、软骨发育障碍、关节增粗畸形等继发性骨关节炎有着显著的不同。然而,这两类疾病均以关节软骨损伤为特征,严重影响成人的劳动力和生活质量。近年来,随着对OA发病机制的深入研究,证实软骨细胞凋亡、细胞因子信号转导障碍等是造成OA软骨损伤的重要原因之一。是否KBD的软骨损伤也涉及到软骨细胞凋亡及细胞因子信号转导障碍,以及两者之间有何区别,有待进一步阐明。
目前,KBD病因发病机制中存在问题很多,但急需解决的主要问题有:①发病原因是单一因素还是环境基因相互作用所致?②KBD和OA关节软骨在分子生物学水平上有何不同?③在基因水平KBD究竟发生了怎样的变化,其与OA有何区别和联系?④硒预防和治疗KBD软骨细胞的机制是什么?为此,本论文主要对比研究了KBD与OA软骨细胞凋亡信号转导、差异基因表达及补硒干预,以阐明这两类关节疾病在软骨细胞凋亡、基因表达、以及补硒对凋亡影响等方面的差异,为深入探讨KBD的病因发病机制、KBD与OA发病机制的异同,提高KBD防治效果,提供科学依据。目的:
1.通过体外培养KBD和OA的关节软骨细胞,比较软骨细胞生长增殖、形态结构特性、细胞凋亡及其信号转导通路的异同;
2.采用Agilent Human 1A基因表达谱芯片比较KBD和OA关节软骨表达基因,以筛选和确定KBD不同于OA的特征性基因;
3.通过不同浓度补硒,体外干预软骨细胞,探讨硒对KBD、OA和正常软骨细胞生长和细胞凋亡的影响。
方法:
1.将病例分为三组:KBD组,OA组和正常对照组。正常对照组和OA组选自非KBD病区,KBD组的病例选自KBD病区,年龄均在49 65岁之间。根据我国颁布的KBD病临床诊断标准,选择Ⅱ度和Ⅲ度病例;根据WOMAC诊断标准选择OA病例,排除诸如遗传性骨病,类风湿性关节炎等。在患者知情同意的情况下,软骨取材主要来自膝关节部位,并在年龄和性别等方面达到匹配。
2.在无菌条件下,体外培养KBD、OA和正常软骨细胞,运用MTT法、流式细胞仪、电镜、免疫荧光、激光共聚焦和免疫组化等实验方法,对比KBD、OA和正常软骨细胞生长特性和细胞凋亡途径的不同;对三组软骨细胞进行补硒干预,比较不同浓度硒(0.0125 g/ml～1.0 g /ml)对KBD和OA细胞生长和凋亡的影响。
3.提取KBD和OA关节软骨细胞RNA,运用Agilent Human 1A基因表达谱芯片技术,用间接标记方法,获得荧光标记的cRNA靶物,进而与基因芯片杂交,通过FeatureExtraction9.3、Spotfire 8.0和Genepix 3.0等软件,选择P<0.05的具有明显表达差异的基因,以筛选KBD不同于OA的特征性基因。
结果:
1. KBD和OA凋亡和信号转导通路的对比研究
1)KBD和OA组软骨细胞的增殖率、平均生长率显著滞后于正常对照组,其中KBD软骨细胞增殖率高于OA组。KBD软骨细胞超微结构以细胞膜形态不规则、细胞核扭曲变形、细胞膜内糖原边集、细胞膜糖原释放、高尔基体和线粒体肿胀为主,而OA组则以细胞肿胀、软骨细胞电子密度增加、细胞膜绒毛减少、胞浆内大量游离脂滴、高尔基体和内质网肿胀为主。
2)KBD和OA的软骨细胞凋亡显著高于正常组,细胞增殖周期G2/M期比例增高;Fas、Bax、Caspase 3、Caspase 9和P53表达上调;KBD软骨细胞Caspase 8和P53表达低于OA,而Caspase 9表达高于OA。
3)KBD和OA具有相似的细胞凋亡途径,与OA相比,KBD更倾向于线粒体凋亡途径。
2. KBD和OA关节软骨差异基因比较及生物通路模型的构建
在KBD与OA四组病例中筛选出显著差异的基因233个,其中共同上调的基因195个,共同下调的基因38个。根据其生物学功能分类,证实筛选出的基因中CSGALNACT、PIM2、EFNA1、SMAD 9、STK11、AQP、T cell factor/LEF、PTN,APCDD1和CAV等对KBD发生发展具有重要意义,主要涉及软骨代谢、离子通道蛋白、凋亡等多方面的基因,构建出与氧化应激、氨基酸代谢、脂代谢等相关的通路模型。
3.不同浓度硒含量干预对KBD和OA软骨细胞生长和凋亡的影响
1)当Se浓度>0.05 g/ml时能显著损害正常软骨细胞的生长,但Se浓度<0.025 g/ml时仍对正常细胞的生长有损伤;尽管Se浓度>0.50 g/ml对KBD软骨细胞生长有一定的毒性作用,但在0.25 0.10 g/ml之间可促进KBD软骨细胞的生长,细胞保持增殖状态;对于OA软骨细胞,Se浓度>0.50 g/ml对细胞增殖有毒性作用,Se浓度<0.25 g/ml细胞能够保持较高增值率,硒也有一定的保护作用。
2)加硒后对正常软骨细胞生长具有毒性作用,其损害程度随加硒浓度的增高而加重;对于KBD而言,加入0.50 g/ml～1.0 g/ml硒含量对细胞生长有毒性作用,0.10 g/ml～0.25 g/ml之间为促进生长,而加入<0.05 g/ml则细胞增殖能力下降;对于OA,加入0.50 g /ml～1.0 g/ml的硒对细胞生长有毒性作用,而Se<0.25 g/ml对细胞的增殖则有促进作用。
3)补硒对软骨细胞超微结构影响的结果显示,Se<0.10 g/ml对KBD,Se<0.05 g/ml对OA的软骨细胞损伤不显著,而Se>0.025 g/ml对正常软骨细胞的亚微结构有影响。
4)加入不同浓度硒时,正常软骨细胞的凋亡率显著高于不加硒的对照组;KBD组除高浓度硒(>0.25 g/ml)产生较高的凋亡率外,补硒可降低软骨细胞的平均凋亡率;在OA组,除引起较高的软骨细胞凋亡率的硒浓度(>0.25 g/ml)外,一定浓度的补硒也可降低软骨细胞凋亡率。
5)加入一定浓度的硒对KBD和OA软骨细胞凋亡调控蛋白表达(Fas、Bax、Caspase 3、Caspase 9、P53)有抑制作用,其中补硒对KBD软骨细胞的保护作用高于OA,而对正常软骨细胞有程度不同的损伤作用;三组之间保护软骨细胞的适宜硒浓度不一。
结论:
1. KBD和OA的关节软骨细胞凋亡率显著高于正常对照组,且Fas、Bax、Caspase 3、Caspase 9、P53表达增高;其中KBD软骨细胞Caspase 8和P53表达低于OA,而Caspase 9表达则高于OA;KBD超微结构变化类似于OA,但其线粒体凋亡途径损伤较OA更明显。
2. KBD差异表达基因不同于OA,其中显著差异的233个基因中KBD下调的基因38个,上调的基因195个,主要涉及CSGALNACT、PIM2、EFNA1、SMAD 9、STK11、AQP、T cell factor/LEF、PTN、APCDD1和CAV等反映代谢、离子通道蛋白、凋亡等多方面的基因。
3.保护KBD和OA的适宜硒浓度不同,在相同补硒剂量下,对KBD保护作用高于OA,但对正常软骨细胞有不同程度的损伤作用;超出适宜的补硒浓度,对三组软骨细胞均具有毒性损伤作用。
Background:
Kashin Beck(KBD)is an endemic disease, with a high prevalence in north China. Thedisease is characterized by cartilage necrosis and local deformation of bone and joint. It wasfirst reported in 1849 by a Russian Yurenski. Since then a lot of work has been done on thisdisease but still its etiology and pathogenesis is unclear. Different aspects of Kashin Beckdisease studied so far include epidemiological as well as experimental studies includingautopsy pathology genetic interactions in the environment and the cultivation of living cells.Studies have shown that the pathogenesis of Kashin Beck disease is related to environmentalfactors (low selenium, T 2 toxin, etc.), cartilage necrosis, as well as abnormal expression ofgene and proteins.
Osteoarthritis:Osteoarthritis (OA) is characterized by articular cartilage degeneration,causing joint pain and dysfunction. It occurs in knee, hip and other weight bearing joints. Itusually occurs in late years of life, mainly at the age of 60 years or older, while KBD startsin 3 12 year old child. They significantly differ in terms of abnormal thickening of thecartilage in joint’s development disorders. Recent studies on OA show that the disease isrelated to apoptosis, disorder in cytokine and signal transduction mechanism. Tissue sectionexperiments have proved that KBD cartilage damage is also related to abnormal expressionof apoptotic factors. Thus both KBD and OA shares some mechanism of the diseasedevelopment but it needs further clarifications about differences in development of both thediseases.
At present there are many unanswered questions about the pathogenesis and etiology ofKBD. Some of the questions which need further research include:①The etiology of KBD.Is it a single factor or an interaction between genes and environment?②What is thedifference between the pathological changes of the articular cartilage in KBD and OA?③What is the genetic basis of KBD and OA?④What is the mechanism of the KBD prevention and treatment by selenium .Keeping in view the aim of differentiating these twodiseases the etiology and pathogenesis of KBD and OA was further which provided ascientific basis for KBD prevention and treatment. This aim in this study we focused on thecomparative study of KBD and OA chondrocyte apoptosis signal transduction, genome widescan screening differential gene profiles and selenium intervening on chondrocyte apoptosis;
Objectives:
1. To compare the differences between cartilage cells of KBD and OA in terms ofchondrocyte growth and proliferation, morphological structure, apoptosis and signaltransduction pathway by culturing the cartilage cells of KBD and OAin vitro;
2. To identify the characteristic gene expression of KBD, which is different from the OA byAgilent Human 1AcDNAmicroarray;
3. To investigate the effect of selenium on cell growth characters and apoptosis among KBD,OAand normal cartilage groups by exposing them to different levels of selenium.
Method:
1. The patients were divided into three groups: KBD, OAand the normal group. The patientsof control and OA groups were selected from the non KBD endemic area, while patients ofKBD were selected from KBD endemic area, aged 49 65 years old. The diagnostic criteriafor KBD included assessment on fingers, joints and immunohistochemistry analysis. Patientswith StageⅡandⅢwere included in this study. OA patients were diagnosed according toWOMAC, patients with genetic bone disease and rheumatoid arthritis were excluded fromthe study. All cartilage were taken from the same location in the joint as far as possible toachieve all aspects to be matched.
2. In aseptic conditions, KBD, OA and normal cartilage cells were cultured in vitro. Wecontrast KBD, OA and normal chondrocytes of different growth characteristics and cellapoptosis pathway by MTT, flow cytometry, electron microscopy, immunofluorescence,laser confocal and immunohistochemistry; Selenium intervention on three groups ofcartilage cells was done to find the impact of different concentrations of selenium on cellgrowth and apoptosis in KBD and OA.
3. We compared difference in gene expression between KBD and OA articular cartilage bygenome wide scan, and checked them by real time PCR; Microarray were scanned byGene Axon 4000B and data was analyzed by Genepix 3.0 software package. Special KBDgenes which were different to OAwere selected.
Results:
I. Comparision of cell apoptosis and signal transduction pathways between KBD and OA
1) The average cell proliferation of KBD and OA cartilage group were significantly lowerthan the normal group, while OA growth rate was less than KBD cartilage cell. Theultrastructure in KBD cartilage cell were as follows: irregular cell shape, distorted nuclei,glycogen gathered within the cell membrane, glycogen release, Golgi apparatus andsignifican mitochondrial swelling; OAgroup: cell swelling, cartilage cells, increased electrondensity, reduced membrane villus in cytoplasm with a large number of free lipid droplets,Golgi apparatus and endoplasmic reticulum swelling.
2) The apoptosis in KBD and OA cartilage was significantly higher than normal group, cellcycle G1 / M phase was increased; The expression of Fas, Bax, Caspase 3, Caspase 9 andP53 increased; Caspase 8, P53 expression in KBD cartilage cells is relatively lower than theOA, while the expression of caspase 9 is relatively higher than OA.
3) The comparision of apoptosis genes in KBD and OA cartilage by gene chips, indicatedthat KBD and OA have similar apoptosis pathway, KBD was prone to mitochondriaapoptosis signal pathway.
II. The comparative study of genome wide scan and biological modeling in KBD and OAcartilage.
In the comparative study of KBD and OA cartilage, there were 233 same array of genesexpression, in which 195 genes up regulated and 38 genes down regulated in the four pairgroups. They were divided according to their biological function into categories such ascellular metabolism, ion channels and transport proteins, signal transduction, cytokines, cellreceptors and the cytoskeleton .It was confirmed before gene selction that the genes selectedCSGALNACT, PIM2, EFNA1, SMAD 9, STK11, AQP, T cell factor / LEF, PTN, APCDD1,CAV and others in KBD were of great significance. Based on the results, KBD is mainlyrelated to metabolism, ion channel proteins, apoptosis and other aspects of gene expressionabnormalities. We build oxidative stress, amino acid metabolism, lipid metabolism andrelated pathways model.
III. The effect of various concentrations of selenium (Se) on growth and apoptosis in KBDand OA.
1) Selenium in high concentrations i.e Se>0.05 g/ml produced toxic damage to normalcartilage cells while the low concentration of selenium also have an impact on the growthrate and the proliferation rate became negative.Studies on KBD cartilage have shown that acertain concentration of selenium can promote the KBD cartilage cells growth, but the highconcentrations of selenium have some of its toxic effects. The concentration of seleniumbetween 0.25 g/ml～0.10 g/ml significantly promote cell proliferation, but the low environment selenium can reduce the proliferation rate. Studies on OA have showedtolerance to high concentrations of selenium and toxic effects of selenium on cellproliferation, but still the cells will be able to maintain growth and proliferation; inSe<0.25 g/ml cell proliferation and growth rate were also high.
2) Normal cartilage after exposure to higher concentration of selenium showed toxic effectson the growth with more damage to cell growth; in terms of KBD, 1.0 g/ml～0.50 g/ml ofselenium produces toxic effect on cell growth, and a concentration of 0.25 g/ml～0.10 g/mlpromoted cell growth; while environmental selenium with a concentration of <0.05 g/mlwas the safety range; for OA, 1.0 g/ml～0.50 g/ml of selenium produced toxic effects oncell growth, Se<0.25 g/ml promoted cell proliferation.
3) The tolerance of KBD and OA to selinum was higher than the normal group, and amongthem KBD had higher tolerance than OA group and the tolerance of OA group is higher thanthe normal group. Microstructure indicated that cartilage cells were tolerant to Se<0.10 g/ml in KBD group and to Se<0.05 g/ml in OA group. While in the normal group thesub structure was by a concentration of Se>0.025 g/ml.
4) Effect of selenium on apoptosis: In normal cartilage cells the the apoptosis rate was higherwith different concentrations of selenium as compared to apoptosis rates without selenium.In the KBD group, the higher concentrations of selenium produced higher apoptotic rate butthe average apoptotic rate declined with selenium. In the OA group, the higher concentrationof selenium resulted in high apoptosis rate but the certain concentration of selenium mayreduce the apoptosis rate.
5) Immunohistochemistry of Fas, Bax, Caspase 3, Caspase 9, P53 and other apoptosisindicators also showed that a certain concentration of selenium added to the KBD and OAchondrocyte in culture can inhibit apoptosis protein expression. The tolerance to selenium ofKBD was higher than OA, and OA was higher than normal cartilage cells. Beyond a certainconcentration selenium can induce apoptosis.
Conclusion:
1. The rate of apoptosis in KBD and OA articular cartilage was significantly higher than thenormal group, and expression of Fas, Bax, Caspase 3, Caspase 9 and P53 increased. In thecomparison of KBD and OA, Caspase 8 and P53 expression in KBD were relatively lowerthan OA, while the caspase 9 expression is relatively higher than OA. Micro structuralchanges in KBD by electron microscopy confirmed that KBD apoptosis pathway is similarto OA, and may be more prone to mitochondrial apoptosis pathway.
2. There is significant difference in 233 gene expressions in KBD and OA, out of these 38genes were down regulated and 195 genes were up regulated in KBD. CSGALNACT, PIM2, EFNA1, SMAD 9, STK11, AQP, T cell factor / LEF, PTN, APCDD1, CAV and other geneswere confirmed to be of greater significance in KBD, and that is mainly related tometabolism, ion channel proteins, apoptosis and other abnormal genes expression.
3. A certain concentration of selenium has protective effect on the KBD and OAchondrocytes. Under the same dose of selenium, the tolerance of KBD is higer than OA andOA tolerance is higher than normal group.Higher concentrations of selenium have damagingand toxic effects on the normal cells and the three groups of chondrocytes have differentconcentrations of selenium tolerance.
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