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大骨节病关节软骨细胞凋亡及其相关调控因子的表达大骨节病关节软骨细胞凋亡及其相关调控因子的表达
作者:王世捷;郭雄;左弘;张银刚;许鹏;平智广;张增铁;耿冬 (西安交通大学环境与疾病基因教育部重点实验室/环境与地方病研究室,陕西 西安 710061)
摘要: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,Bax,Fas及iNos表达分布的特点.方法 收集15例大骨节病儿童和15 例正常对照儿童关节软骨.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(TUNEL)技术和Bcl-2,Bax,Fas及iNos蛋白免疫组化抗生物素蛋白-生物素-碱性磷酸酶(B-SA)法染色,观察大骨节病儿童和正常对照儿童关节软骨的凋亡细胞和Bcl-2,Bax,Fas及iNos蛋白阳性表达细胞的密度与分布.结果 (1)大骨节病儿童关节软骨中层凋亡阳性细胞数[(33.60±2.71)%]较正常关节软骨[(1.33±0.41)%]显著增多(t=11.59,P<0.01);(2)大骨节病儿童关节软骨表层和中层Bcl-2,Bax,Fas及iNos的表达显著高于正常关节软骨中的表达(t=11.75~18.65,P<0.01);大骨节病儿童关节软骨各层Bcl-2,Bax,Fas及iNos表达阳性率有显著性差异(F =73.49~114.42,P<0.01),以表层最多[分别为(41.93±12.26)%,(45.60±15.78)%,(53.60±16.49)%和(45.47±14.02)%],其次为中层[分别为(14.93±3.50)%,(13.87±4.32)%,(23.27±4.83)%,(21.67±6.82)%].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,Bax,Fas,iNos表达较正常人显著增多.
关键词:大骨节病;软骨,关节;细胞凋亡;Bcl-2;Bax;Fas;iNos
中图分类号:R684.101; R394.2 文献标识码:A 文章编号:1000-2588(2005)06-0643-04
Chondrocyte apoptosis and the expression of Bcl-2, Bax, Fas and iNos in articular cartilage in Kashin-Beck disease
WANG Shi-jie; GUO Xiong; ZUO Hong; ZHANG Yin-gang; XU Peng; PING Zhi-guang; ZHANG Zeng-tie; GENG Dong
Key Laboratory of Environment and Genes Related Diseases, Institute of Environment and Endemic Diseases of State Ministry of Education, Xi''an Jiaotong University, Xi''an 710061, China
Abstract: ObjectiveTo investigate the characteristics of chondrocyte apoptosis and distribution of Bcl-2, Bax, Fas and iNos expressions in articular cartilage in Kashin-Beck disease (KBD). MethodsSamples of articular cartilage were collected from 15 healthy children and 15 children with KBD diagnosed according to the Pathological Criteria of KBD Diagnosis in China. Chondrocyte apoptosis was detected by TUNEL method, and the articular chondrocytes positive for Bcl-2, Bax, Fas and iNos were stained by B-SA immunohistochemistry. ResultsThe percentage of apoptotic chondrocytes positively stained by TUNEL in the middle layer of articular cartilage was significantly higher in KBD children than in the control group (33.60%±2.71% vs 1.33%±0.41%, t=11.59, P<0.01). Significant difference in Bcl-2, Bax, Fas and iNos expressions was observed between the upper, middle and deep layers of the articular cartilage of KBD children (F =73.49-114.42, P<0.01), and staining for Bcl-2, Bax, Fas and iNos in KBD children was prominent in the upper layer (41.93%±12.26%, 45.60%±15.78%, 53.60%±16.49%, and 45.47%±14.02%, respectively) and the middle layer (14.93%±3.50%, 13.87%±4.32%, 23.27%±4.83%, and 21.67%±6.82%, respectively) of the articular cartilage; the percentages of chondrocytes positively stained for Bcl-2, Bax, Fas and iNos were significantly higher than those of the control group (t=11.75-18.65, P<0.01). ConclusionThe percentages of apoptotic chondrocytes and chondrocytes positive for Bcl-2, Bax, Fas and iNos in the articular cartilage of children with KBD are significantly higher than those in healthy children.
Key words: Kashin-Beck disease; cartilage, articular; apoptosis; Bcl-2; Bax; Fas; iNos
收稿日期:2004-11-22
基金项目:国家自然科学基金(39970663);西安交通大学自然科学基金(XJJ 2004133) Supported by National Natural Science Foundation of China (39970663) and Natural Science Foundation of Xi''an Jiaotong University(XJJ 2004133)
作者简介:王世捷(1971-),女,在读博士研究生,主治医师,电话:029-81906067,E-mail:sxjaner@21cn.com
大骨节病(Kashin-Beck disease,KBD)是一种慢性地方性骨关节病,主要病理特征为骺板软骨和关节软骨深层软骨细胞坏死及继发性增生和修复性改变,导致骨软骨发育障碍,继发性变形性骨关节病,是骨关节病和软骨疾患的一个较为特殊的类型,但目前其病因及发病机理仍不完全清楚.近年来国内外研究表明,软骨细胞异常凋亡是导致多种骨与软骨疾病发生(骨关节病,骨软骨发育不良)的重要环节[1].既往曾初步观察到KBD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[2],但未进行Bax,Fas,及iNos等相关调控因子的研究.因此本实验通过比较大骨节病儿童和正常儿童关节软骨细胞凋亡,相关调控因子Bcl-2,Bax,Fas及iNos表达的分布特点,分析大骨节病的软骨细胞坏死及其继发性病变是否与软骨细胞过度凋亡及其相关因子异常表达有关,以从分子水平研究大骨节病的发生机理和采取积极有效的防治措施.
1 材料和方法
1.1 组织来源和处理
取死于其他疾病的3~15岁大骨节病儿童指骨关节软骨共15例(男5例,女10例),同时选择死于其他疾病的3~12岁非大骨节病儿童指骨关节软骨15例(男7例,女8例).切取0.5~1 cm软骨样品,4%多聚甲醛固定,3% EDTA脱钙,石蜡包埋.用10%多聚赖氨酸处理载玻片,软骨组织切片为6 μm,分别做HE,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)和免疫组织化学染色.
1.2 方法
1.2.1 TUNEL法检测细胞凋亡 3%H2O2处理样品切片10 min后,用蛋白酶K于37 ℃消化5 min;取TDT和DIG-d-UTP各1 μl,加入18 μl标记缓冲液于软骨组织片,37 ℃杂交2 h;封闭液孵育30 min,生物素化抗地高辛抗体反应30 min;SABC稀释液反应30 min,DAB显色,苏木素复染.阳性对照:在切片置TDT反应前,先在200 ng/ml DNase I溶液中37 ℃下反应15 min;阴性对照:不加TDT酶,其余步骤相同.
1.2.2 免疫组化染色 软骨切片二甲苯脱蜡,梯度酒精脱水,0.3%H2O2的甲醇液冲洗,磷酸盐缓冲液冲洗.加入1%牛血清白蛋白,20 ℃孵育15 min.第一抗体在4 ℃下孵育16 h,第二抗体孵育20 min;用3-hydroxy-2-naphthy-locid2,4-dimethylaniline进行呈色反应.细胞核用苏木素染色.阴性对照:用PBS代替一抗,其余步骤相同.兔抗人Bcl-2,Fas,Bax,iNos多克隆抗体及TUNEL细胞凋亡试剂盒均购自于美国Santa Cruz公司.
1.2.3 关节软骨的细胞分层 根据Robert等[3]报道,将关节软骨大致分为3层.表层(a):细胞扁平,体积较小,长轴与关节面平行;中层(b):细胞排列稍紧密,圆形或椭圆形,细胞稍大;深层(c):细胞胞体浑圆,胞质丰富,呈柱状或散在整齐排列.
1.3 统计学处理
在光学显微镜10×10倍视野下,在每个样本关节软骨各个细胞层中随机选取4个视野,分别计100个细胞内平均凋亡阳性细胞数和Bcl-2,Bax,Fas及iNos阳性细胞数.采用t检验检验大骨节病与正常组间的显著性水平,α=0.05,双侧检验;方差分析检验不同软骨细胞层间的显著性水平.
2 结果
2.1 关节软骨HE染色特点
大骨节病患者关节软骨HE染色显示各细胞层次分界清楚,各层细胞形态特征明显(图1A).即表层细胞扁平,体积较小,胞核蓝染;中层细胞排列稍紧密,圆形或椭圆形,胞核蓝染;深层可见片状坏死细胞,胞核红染,胶原显现.正常儿童关节软骨HE染色特点见图2A.
2.2 关节软骨凋亡细胞的分布
凋亡阳性细胞为散在分布,胞核或胞核和胞质同时呈棕黄色.凋亡阴性细胞胞核呈蓝色,胞质无色,胞膜完整.大骨节病儿童关节软骨凋亡阳性细胞以中层出现最为明显,延续到深层仍然可见,表层未见凋亡阳性细胞(图1B).正常儿童关节软骨凋亡阳性细胞散在分布于深层,表层未见凋亡阳性细胞(图2B).统计分析显示,患病儿童关节软骨中层凋亡阳性细胞数与正常软骨中层相比差异有显著性(P<0.01),大骨节病组较正常组平均增多32%(表1);患病儿童关节软骨中层凋亡阳性细胞数最多,与表层和深层相比有显著性差异(P<0.01,表1).
2.3 关节软骨Bcl-2表达的分布
Bcl-2表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.患病儿童关节软骨Bcl-2表达的阳性细胞主要分布在关节软骨的表层和中层上部,少数见于深层(图1C);正常儿童在关节软骨的表层和深层有少量的Bcl-2阳性表达(图2C).患病儿童关节软骨表层和中层Bcl-2阳性细胞数明显多于其在正常儿童关节软骨中的表达(t=12.44-16.55,P<0.01,表1);患病儿童关节软骨各层Bcl-2表达阳性率有显著性差异(F=114.42,P<0.01),表层最高,深层最低(表1).
2.4 关节软骨Bax表达的分布
Bax表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.正常儿童在关节软骨的表层有少量Bax阳性表达(1D),患病儿童关节软骨Bax表达的阳性细胞主要分布在关节软骨的表层和中层上部(图2D);患病儿童关节软骨表层和中层Bax阳性细胞数明显多于其在正常儿童关节软骨中的表达(P<0.01),大骨节病组较正常组表层平均增多44%,中层平均增多14%,见表1.患病儿童关节软骨各层Bax表达阳性率有显著性差异(F=91.87,P<0.01),表层最高,深层最低,见表1.
2.5 关节软骨Fas表达的分布
Fas表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.患病儿童关节软骨Fas表达的阳性细胞主要分布在关节软骨的表层和中层上部,少数见于深层(1E),正常儿童在关节软骨的表层有少量的Fas阳性表达(图2E);患病儿童关节软骨各层Fas阳性细胞数明显多于其在正常儿童关节软骨中的表达(P<0.01),其中大骨节病组较正常组表层平均增多52%,中层平均增多24%.患病儿童关节软骨各层Fas表达阳性率有显著性差异(F=88.78,P<0.01),表层最高,深层最低,见表1.
2.6 关节软骨iNos表达的分布
iNos表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.患病儿童关节软骨iNos表达的阳性细胞主要分布在关节软骨的表层和中层上部,少数见于深层(1F),正常儿童在关节软骨的表层有少量的iNos阳性表达(图2F);患病儿童关节软骨各层iNos阳性细胞数明显多于其在正常儿童关节软骨中的表达(t=11.75-13.41,P<0.01,表1).患病儿童关节软骨各层iNos表达阳性率有显著性差异(F=73.49,P<0.01),表层最高,深层最低,表1.
图1 大骨节病儿童(男,4岁,大骨节病早期)指骨关节软骨的HE,TUNEL和Bcl-2,Bax,Fas及iNos免疫组织化学染色
Fig.1 HE, TUNEL and immunohistochemical staining of the finger articular cartilage from a child with Kashin-Beck disease (KBD) (4 years of age with clinical manifestations of the first-degree KBD)
A: Large chondrocyte necrotic areas without cells in the deep layer (Original magnification: ×100); B: Occasional TUNEL-positive chondrocytes in the middle layer (Original magnification: ×100); C: Bcl-2 (Original magnification: ×200); D: Bax (Original magnification: ×200); E: Fas (Original magnification: ×200); F: iNos (Original magnification: ×200)
图2 正常儿童(男,5岁)指骨关节软骨HE,TUNEL和Bcl-2,Bax,Fas及iNos免疫组织化学染色(原放大倍数:×200)
Fig.2 HE, TUNEL and immunohistochemical staining of finger articular cartilage of a healthy 5-year-old boy (Original magnification: ×200)
A: Absence of chondrocyte necrosis in the deep layer; B: Absence of TUNEL-positive chondrocytes in the middle layer; C: Bcl-2; D: Bax; E: Fas; F: iNos
3 讨论
本实验结果表明:(1)大骨节病儿童关节软骨出现以软骨中层为主,深层极少的软骨细胞过度凋亡特征,提示细胞凋亡信号已在大骨节病儿童关节软骨中层发生;(2)与对照组比较,大骨节病关节软骨表,中层中Bcl-2,Bax,Fas和软骨表,中,深层iNos表达明显增多,提示大骨节病软骨细胞凋亡相关调控因子表达异常.
近年研究表明软骨细胞凋亡及其基因调控在软骨细胞增殖,生长,分化及疾病的发生中有重要作用.软骨细胞凋亡的异常,不仅影响骨和软骨的正常发育,而且也与软骨发育障碍,骨关节病的发生发展有关.在正常生理状况下,关节软骨细胞凋亡发生在肥大层,凋亡的软骨细胞约占5%~10%,关节软骨可发生10%左右的程序性细胞凋亡[4],但大骨节病在发生深层软骨细胞坏死同时又出现中层软骨细胞凋亡.大骨节病早期儿童关节和骺板软骨的DNA含量减少约50%,软骨细胞明显减少[5] .然而,软骨细胞的减少是软骨细胞过度凋亡还是软骨细胞坏死的结果,尚未见文献报道.大骨节病表,中层关节软骨基质中,尚未发生坏死的软骨细胞Ⅱ型胶原合成减少[6],而软骨细胞Ⅱ型胶原的减少被认为是一种诱导凋亡的信号.因此推测大骨节病患者存在软骨细胞过度凋亡,而且可能与大骨节病软骨细胞的坏死有联系.
大骨节病关节软骨表层和中层中Bcl-2的表达明显多于正常关节软骨,与Kim等[7]观察到的骨关节炎患者软骨细胞Bcl-2在表层损伤区表达增多的特点相似.这可能是大骨节病患者关节软骨细胞仍具有能力通过上调Bcl-2的表达来补偿进行性的细胞凋亡.最近还有报道Bcl-2的转化产物可导致细胞凋亡[8].大骨节病关节软骨Bax的表达分布,与Kim等[7]报道Bax在正常与骨关节炎患者软骨细胞中表达无明显差异不同,大骨节病关节软骨表层和中层中Bax的表达明显多于正常关节软骨(P<0.05),因此可以认为Bax和Bcl-2共同参与了大骨节病儿童软骨细胞的凋亡.Bax的作用与Bcl-2相反,具有促进细胞凋亡的作用,Bcl-2与Bax的比例决定细胞的生死.本研究显示二者的阳性数之间无显著性差异,可能是大骨节病的病程缓慢的原因之一.
Hashimoto[9]研究认为至少有两种相互独立的途径参与了骨关节炎软骨细胞的凋亡:一种是与滑膜炎症无关的途径,由NO介导;另一种是同滑膜炎症相关的途径,由Fas介导,针对NO合成的抑制剂不能抑制Fas途径诱导的凋亡.大骨节病儿童患者关节软骨各层的iNOS表达明显多于正常关节软骨,与张宝弟等[10]检测大骨节病儿童血液中NO含量增高相一致.同时,大骨节病关节软骨表层和中层中Fas的表达较正常关节软骨明显增多,说明大骨节病儿童关节软骨细胞的凋亡同时由NO和Fas介导.
综上所述,大骨节病儿童除了表现为深层的软骨细胞坏死之外,还存在异常的软骨细胞凋亡.软骨细胞的凋亡增多主要发生在关节软骨的中层,Bcl-2,Bax,Fas及iNos在其发病机理中发挥着重要作用.大量的软骨细胞凋亡可导致继发性软骨细胞坏死,因此是否过度的软骨细胞凋亡与软骨细胞坏死有关,还需要进一步寻找证据.
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[7] Kim HA, Lee YJ, Seong SC, et al. Apoptotic chondrocyte death in human osteoarthritis[J]. J Rheumatol, 2000, 10(10): 455-62.
[8] Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspase[J]. Science, 1997, 278(5345): 1966-8.
[9] Hashimoto S, Takahashi H, Aiel D, et al. Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis[J]. Arthritis Rheum, 1998, 41(9): 1226-34.
[10] 张宝弟, 郭 雄, 白广禄, 等. 大骨节病患者血清NO, NOS, sFas/ APO-1检测分析[J]. 中国地方病学杂志, 2004, 31(4): 172-5.
Zhang BD, Guo X, Bai GL, et al. The changes of nitric oxide, NO synthase and sFas/Apo-1 in serum among the patients with Kashin-Beck disease[J]. Chin J Endemiol, 2004, 31(4): 172-5.
参考文献:
[1] Aigner T, McKenna L. Molecular pathology and pathobiology of osteoarthritic cartilage[J]. Cell Mol Life Sci, 2002, 59(1): 5-18.
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Yang J, Guo X, Zhang ZT, et al. The distributed characteristics of chondrocyte apoptosis and the expression of Bcl-2 in articular cartilage with Kashin-Beck disease[J]. Chin J Endemiol, 2002, 21(5): 341-3.
[3] Robert K, Schen K, Peter SE, et al. Articular cartilage biochemistry[M]. New York: Raven Press, 1986. 3-22.
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[6] 郭 雄, Thomas A, Pirkko L, 等. 大骨节病关节软骨胶原表型表达和软骨细胞异常分化的研究[J]. 中华病理学杂志, 1998, 27(1): 19-22.
Guo X, Thomas A, Pirkko L, et al. A study on abnormal chon- drocyte differentiation and abnormal expression of collagen types in AC from patients with Kashin-Beck disease[J]. Chin J Pathol, 1998, 27(1): 19-22.
[7] Kim HA, Lee YJ, Seong SC, et al. Apoptotic chondrocyte death in human osteoarthritis[J]. J Rheumatol, 2000, 10(10): 455-62.
[8] Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspase[J]. Science, 1997, 278(5345): 1966-8.
[9] Hashimoto S, Takahashi H, Aiel D, et al. Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis[J]. Arthritis Rheum, 1998, 41(9): 1226-34.
[10] 张宝弟, 郭 雄, 白广禄, 等. 大骨节病患者血清NO, NOS, sFas/ APO-1检测分析[J]. 中国地方病学杂志, 2004, 31(4): 172-5.
Zhang BD, Guo X, Bai GL, et al. The changes of nitric oxide, NO synthase and sFas/Apo-1 in serum among the patients with Kashin-Beck disease[J]. Chin J Endemiol, 2004, 31(4): 172-5.
391789
·上一篇：2006年中央补助地方大骨节病项目技术方案
·下一篇：大骨节病(KBD)病因尚未定论至今仅根据三种主要病因假说...
作者:王世捷;郭雄;左弘;张银刚;许鹏;平智广;张增铁;耿冬 (西安交通大学环境与疾病基因教育部重点实验室/环境与地方病研究室,陕西 西安 710061)
摘要: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,Bax,Fas及iNos表达分布的特点.方法 收集15例大骨节病儿童和15 例正常对照儿童关节软骨.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(TUNEL)技术和Bcl-2,Bax,Fas及iNos蛋白免疫组化抗生物素蛋白-生物素-碱性磷酸酶(B-SA)法染色,观察大骨节病儿童和正常对照儿童关节软骨的凋亡细胞和Bcl-2,Bax,Fas及iNos蛋白阳性表达细胞的密度与分布.结果 (1)大骨节病儿童关节软骨中层凋亡阳性细胞数[(33.60±2.71)%]较正常关节软骨[(1.33±0.41)%]显著增多(t=11.59,P<0.01);(2)大骨节病儿童关节软骨表层和中层Bcl-2,Bax,Fas及iNos的表达显著高于正常关节软骨中的表达(t=11.75~18.65,P<0.01);大骨节病儿童关节软骨各层Bcl-2,Bax,Fas及iNos表达阳性率有显著性差异(F =73.49~114.42,P<0.01),以表层最多[分别为(41.93±12.26)%,(45.60±15.78)%,(53.60±16.49)%和(45.47±14.02)%],其次为中层[分别为(14.93±3.50)%,(13.87±4.32)%,(23.27±4.83)%,(21.67±6.82)%].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,Bax,Fas,iNos表达较正常人显著增多.
关键词:大骨节病;软骨,关节;细胞凋亡;Bcl-2;Bax;Fas;iNos
中图分类号:R684.101; R394.2 文献标识码:A 文章编号:1000-2588(2005)06-0643-04
Chondrocyte apoptosis and the expression of Bcl-2, Bax, Fas and iNos in articular cartilage in Kashin-Beck disease
WANG Shi-jie; GUO Xiong; ZUO Hong; ZHANG Yin-gang; XU Peng; PING Zhi-guang; ZHANG Zeng-tie; GENG Dong
Key Laboratory of Environment and Genes Related Diseases, Institute of Environment and Endemic Diseases of State Ministry of Education, Xi''an Jiaotong University, Xi''an 710061, China
Abstract: ObjectiveTo investigate the characteristics of chondrocyte apoptosis and distribution of Bcl-2, Bax, Fas and iNos expressions in articular cartilage in Kashin-Beck disease (KBD). MethodsSamples of articular cartilage were collected from 15 healthy children and 15 children with KBD diagnosed according to the Pathological Criteria of KBD Diagnosis in China. Chondrocyte apoptosis was detected by TUNEL method, and the articular chondrocytes positive for Bcl-2, Bax, Fas and iNos were stained by B-SA immunohistochemistry. ResultsThe percentage of apoptotic chondrocytes positively stained by TUNEL in the middle layer of articular cartilage was significantly higher in KBD children than in the control group (33.60%±2.71% vs 1.33%±0.41%, t=11.59, P<0.01). Significant difference in Bcl-2, Bax, Fas and iNos expressions was observed between the upper, middle and deep layers of the articular cartilage of KBD children (F =73.49-114.42, P<0.01), and staining for Bcl-2, Bax, Fas and iNos in KBD children was prominent in the upper layer (41.93%±12.26%, 45.60%±15.78%, 53.60%±16.49%, and 45.47%±14.02%, respectively) and the middle layer (14.93%±3.50%, 13.87%±4.32%, 23.27%±4.83%, and 21.67%±6.82%, respectively) of the articular cartilage; the percentages of chondrocytes positively stained for Bcl-2, Bax, Fas and iNos were significantly higher than those of the control group (t=11.75-18.65, P<0.01). ConclusionThe percentages of apoptotic chondrocytes and chondrocytes positive for Bcl-2, Bax, Fas and iNos in the articular cartilage of children with KBD are significantly higher than those in healthy children.
Key words: Kashin-Beck disease; cartilage, articular; apoptosis; Bcl-2; Bax; Fas; iNos
收稿日期:2004-11-22
基金项目:国家自然科学基金(39970663);西安交通大学自然科学基金(XJJ 2004133) Supported by National Natural Science Foundation of China (39970663) and Natural Science Foundation of Xi''an Jiaotong University(XJJ 2004133)
作者简介:王世捷(1971-),女,在读博士研究生,主治医师,电话:029-81906067,E-mail:sxjaner@21cn.com
大骨节病(Kashin-Beck disease,KBD)是一种慢性地方性骨关节病,主要病理特征为骺板软骨和关节软骨深层软骨细胞坏死及继发性增生和修复性改变,导致骨软骨发育障碍,继发性变形性骨关节病,是骨关节病和软骨疾患的一个较为特殊的类型,但目前其病因及发病机理仍不完全清楚.近年来国内外研究表明,软骨细胞异常凋亡是导致多种骨与软骨疾病发生(骨关节病,骨软骨发育不良)的重要环节[1].既往曾初步观察到KBD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[2],但未进行Bax,Fas,及iNos等相关调控因子的研究.因此本实验通过比较大骨节病儿童和正常儿童关节软骨细胞凋亡,相关调控因子Bcl-2,Bax,Fas及iNos表达的分布特点,分析大骨节病的软骨细胞坏死及其继发性病变是否与软骨细胞过度凋亡及其相关因子异常表达有关,以从分子水平研究大骨节病的发生机理和采取积极有效的防治措施.
1 材料和方法
1.1 组织来源和处理
取死于其他疾病的3~15岁大骨节病儿童指骨关节软骨共15例(男5例,女10例),同时选择死于其他疾病的3~12岁非大骨节病儿童指骨关节软骨15例(男7例,女8例).切取0.5~1 cm软骨样品,4%多聚甲醛固定,3% EDTA脱钙,石蜡包埋.用10%多聚赖氨酸处理载玻片,软骨组织切片为6 μm,分别做HE,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)和免疫组织化学染色.
1.2 方法
1.2.1 TUNEL法检测细胞凋亡 3%H2O2处理样品切片10 min后,用蛋白酶K于37 ℃消化5 min;取TDT和DIG-d-UTP各1 μl,加入18 μl标记缓冲液于软骨组织片,37 ℃杂交2 h;封闭液孵育30 min,生物素化抗地高辛抗体反应30 min;SABC稀释液反应30 min,DAB显色,苏木素复染.阳性对照:在切片置TDT反应前,先在200 ng/ml DNase I溶液中37 ℃下反应15 min;阴性对照:不加TDT酶,其余步骤相同.
1.2.2 免疫组化染色 软骨切片二甲苯脱蜡,梯度酒精脱水,0.3%H2O2的甲醇液冲洗,磷酸盐缓冲液冲洗.加入1%牛血清白蛋白,20 ℃孵育15 min.第一抗体在4 ℃下孵育16 h,第二抗体孵育20 min;用3-hydroxy-2-naphthy-locid2,4-dimethylaniline进行呈色反应.细胞核用苏木素染色.阴性对照:用PBS代替一抗,其余步骤相同.兔抗人Bcl-2,Fas,Bax,iNos多克隆抗体及TUNEL细胞凋亡试剂盒均购自于美国Santa Cruz公司.
1.2.3 关节软骨的细胞分层 根据Robert等[3]报道,将关节软骨大致分为3层.表层(a):细胞扁平,体积较小,长轴与关节面平行;中层(b):细胞排列稍紧密,圆形或椭圆形,细胞稍大;深层(c):细胞胞体浑圆,胞质丰富,呈柱状或散在整齐排列.
1.3 统计学处理
在光学显微镜10×10倍视野下,在每个样本关节软骨各个细胞层中随机选取4个视野,分别计100个细胞内平均凋亡阳性细胞数和Bcl-2,Bax,Fas及iNos阳性细胞数.采用t检验检验大骨节病与正常组间的显著性水平,α=0.05,双侧检验;方差分析检验不同软骨细胞层间的显著性水平.
2 结果
2.1 关节软骨HE染色特点
大骨节病患者关节软骨HE染色显示各细胞层次分界清楚,各层细胞形态特征明显(图1A).即表层细胞扁平,体积较小,胞核蓝染;中层细胞排列稍紧密,圆形或椭圆形,胞核蓝染;深层可见片状坏死细胞,胞核红染,胶原显现.正常儿童关节软骨HE染色特点见图2A.
2.2 关节软骨凋亡细胞的分布
凋亡阳性细胞为散在分布,胞核或胞核和胞质同时呈棕黄色.凋亡阴性细胞胞核呈蓝色,胞质无色,胞膜完整.大骨节病儿童关节软骨凋亡阳性细胞以中层出现最为明显,延续到深层仍然可见,表层未见凋亡阳性细胞(图1B).正常儿童关节软骨凋亡阳性细胞散在分布于深层,表层未见凋亡阳性细胞(图2B).统计分析显示,患病儿童关节软骨中层凋亡阳性细胞数与正常软骨中层相比差异有显著性(P<0.01),大骨节病组较正常组平均增多32%(表1);患病儿童关节软骨中层凋亡阳性细胞数最多,与表层和深层相比有显著性差异(P<0.01,表1).
2.3 关节软骨Bcl-2表达的分布
Bcl-2表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.患病儿童关节软骨Bcl-2表达的阳性细胞主要分布在关节软骨的表层和中层上部,少数见于深层(图1C);正常儿童在关节软骨的表层和深层有少量的Bcl-2阳性表达(图2C).患病儿童关节软骨表层和中层Bcl-2阳性细胞数明显多于其在正常儿童关节软骨中的表达(t=12.44-16.55,P<0.01,表1);患病儿童关节软骨各层Bcl-2表达阳性率有显著性差异(F=114.42,P<0.01),表层最高,深层最低(表1).
2.4 关节软骨Bax表达的分布
Bax表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.正常儿童在关节软骨的表层有少量Bax阳性表达(1D),患病儿童关节软骨Bax表达的阳性细胞主要分布在关节软骨的表层和中层上部(图2D);患病儿童关节软骨表层和中层Bax阳性细胞数明显多于其在正常儿童关节软骨中的表达(P<0.01),大骨节病组较正常组表层平均增多44%,中层平均增多14%,见表1.患病儿童关节软骨各层Bax表达阳性率有显著性差异(F=91.87,P<0.01),表层最高,深层最低,见表1.
2.5 关节软骨Fas表达的分布
Fas表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.患病儿童关节软骨Fas表达的阳性细胞主要分布在关节软骨的表层和中层上部,少数见于深层(1E),正常儿童在关节软骨的表层有少量的Fas阳性表达(图2E);患病儿童关节软骨各层Fas阳性细胞数明显多于其在正常儿童关节软骨中的表达(P<0.01),其中大骨节病组较正常组表层平均增多52%,中层平均增多24%.患病儿童关节软骨各层Fas表达阳性率有显著性差异(F=88.78,P<0.01),表层最高,深层最低,见表1.
2.6 关节软骨iNos表达的分布
iNos表达的阳性细胞胞质呈均匀红染颗粒,胞核蓝染,胞膜完整.患病儿童关节软骨iNos表达的阳性细胞主要分布在关节软骨的表层和中层上部,少数见于深层(1F),正常儿童在关节软骨的表层有少量的iNos阳性表达(图2F);患病儿童关节软骨各层iNos阳性细胞数明显多于其在正常儿童关节软骨中的表达(t=11.75-13.41,P<0.01,表1).患病儿童关节软骨各层iNos表达阳性率有显著性差异(F=73.49,P<0.01),表层最高,深层最低,表1.
图1 大骨节病儿童(男,4岁,大骨节病早期)指骨关节软骨的HE,TUNEL和Bcl-2,Bax,Fas及iNos免疫组织化学染色
Fig.1 HE, TUNEL and immunohistochemical staining of the finger articular cartilage from a child with Kashin-Beck disease (KBD) (4 years of age with clinical manifestations of the first-degree KBD)
A: Large chondrocyte necrotic areas without cells in the deep layer (Original magnification: ×100); B: Occasional TUNEL-positive chondrocytes in the middle layer (Original magnification: ×100); C: Bcl-2 (Original magnification: ×200); D: Bax (Original magnification: ×200); E: Fas (Original magnification: ×200); F: iNos (Original magnification: ×200)
图2 正常儿童(男,5岁)指骨关节软骨HE,TUNEL和Bcl-2,Bax,Fas及iNos免疫组织化学染色(原放大倍数:×200)
Fig.2 HE, TUNEL and immunohistochemical staining of finger articular cartilage of a healthy 5-year-old boy (Original magnification: ×200)
A: Absence of chondrocyte necrosis in the deep layer; B: Absence of TUNEL-positive chondrocytes in the middle layer; C: Bcl-2; D: Bax; E: Fas; F: iNos
3 讨论
本实验结果表明:(1)大骨节病儿童关节软骨出现以软骨中层为主,深层极少的软骨细胞过度凋亡特征,提示细胞凋亡信号已在大骨节病儿童关节软骨中层发生;(2)与对照组比较,大骨节病关节软骨表,中层中Bcl-2,Bax,Fas和软骨表,中,深层iNos表达明显增多,提示大骨节病软骨细胞凋亡相关调控因子表达异常.
近年研究表明软骨细胞凋亡及其基因调控在软骨细胞增殖,生长,分化及疾病的发生中有重要作用.软骨细胞凋亡的异常,不仅影响骨和软骨的正常发育,而且也与软骨发育障碍,骨关节病的发生发展有关.在正常生理状况下,关节软骨细胞凋亡发生在肥大层,凋亡的软骨细胞约占5%~10%,关节软骨可发生10%左右的程序性细胞凋亡[4],但大骨节病在发生深层软骨细胞坏死同时又出现中层软骨细胞凋亡.大骨节病早期儿童关节和骺板软骨的DNA含量减少约50%,软骨细胞明显减少[5] .然而,软骨细胞的减少是软骨细胞过度凋亡还是软骨细胞坏死的结果,尚未见文献报道.大骨节病表,中层关节软骨基质中,尚未发生坏死的软骨细胞Ⅱ型胶原合成减少[6],而软骨细胞Ⅱ型胶原的减少被认为是一种诱导凋亡的信号.因此推测大骨节病患者存在软骨细胞过度凋亡,而且可能与大骨节病软骨细胞的坏死有联系.
大骨节病关节软骨表层和中层中Bcl-2的表达明显多于正常关节软骨,与Kim等[7]观察到的骨关节炎患者软骨细胞Bcl-2在表层损伤区表达增多的特点相似.这可能是大骨节病患者关节软骨细胞仍具有能力通过上调Bcl-2的表达来补偿进行性的细胞凋亡.最近还有报道Bcl-2的转化产物可导致细胞凋亡[8].大骨节病关节软骨Bax的表达分布,与Kim等[7]报道Bax在正常与骨关节炎患者软骨细胞中表达无明显差异不同,大骨节病关节软骨表层和中层中Bax的表达明显多于正常关节软骨(P<0.05),因此可以认为Bax和Bcl-2共同参与了大骨节病儿童软骨细胞的凋亡.Bax的作用与Bcl-2相反,具有促进细胞凋亡的作用,Bcl-2与Bax的比例决定细胞的生死.本研究显示二者的阳性数之间无显著性差异,可能是大骨节病的病程缓慢的原因之一.
Hashimoto[9]研究认为至少有两种相互独立的途径参与了骨关节炎软骨细胞的凋亡:一种是与滑膜炎症无关的途径,由NO介导;另一种是同滑膜炎症相关的途径,由Fas介导,针对NO合成的抑制剂不能抑制Fas途径诱导的凋亡.大骨节病儿童患者关节软骨各层的iNOS表达明显多于正常关节软骨,与张宝弟等[10]检测大骨节病儿童血液中NO含量增高相一致.同时,大骨节病关节软骨表层和中层中Fas的表达较正常关节软骨明显增多,说明大骨节病儿童关节软骨细胞的凋亡同时由NO和Fas介导.
综上所述,大骨节病儿童除了表现为深层的软骨细胞坏死之外,还存在异常的软骨细胞凋亡.软骨细胞的凋亡增多主要发生在关节软骨的中层,Bcl-2,Bax,Fas及iNos在其发病机理中发挥着重要作用.大量的软骨细胞凋亡可导致继发性软骨细胞坏死,因此是否过度的软骨细胞凋亡与软骨细胞坏死有关,还需要进一步寻找证据.
参考文献:
[1] Aigner T, McKenna L. Molecular pathology and pathobiology of osteoarthritic cartilage[J]. Cell Mol Life Sci, 2002, 59(1): 5-18.
[2] 杨 珺, 郭 雄, 张增铁, 等. 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[J].中国地方病学杂志, 2002, 21(5): 341-3.
Yang J, Guo X, Zhang ZT, et al. The distributed characteristics of chondrocyte apoptosis and the expression of Bcl-2 in articular cartilage with Kashin-Beck disease[J]. Chin J Endemiol, 2002, 21(5): 341-3.
[3] Robert K, Schen K, Peter SE, et al. Articular cartilage biochemistry[M]. New York: Raven Press, 1986. 3-22.
[4] Roach HI, Aigner T, Erenpeisa J. Ostegenic differentiation of hyper- trophic chondrocytes involves asymmetric cell divisions and apoptosis[J]. J Cell Biol, 1995, 131(3): 483-91.
[5] Yang TS, Hou LZ, Li FS. Biochemical characteristics of Kashin- Beck disease[A]. In: Kashin-Beck disease and non-communicable diseases[M]. Beijing: Chinese Academy of Preventive Medicine, 1990. 116-28.
[6] 郭 雄, Thomas A, Pirkko L, 等. 大骨节病关节软骨胶原表型表达和软骨细胞异常分化的研究[J]. 中华病理学杂志, 1998, 27(1): 19-22.
Guo X, Thomas A, Pirkko L, et al. A study on abnormal chon- drocyte differentiation and abnormal expression of collagen types in AC from patients with Kashin-Beck disease[J]. Chin J Pathol, 1998, 27(1): 19-22.
[7] Kim HA, Lee YJ, Seong SC, et al. Apoptotic chondrocyte death in human osteoarthritis[J]. J Rheumatol, 2000, 10(10): 455-62.
[8] Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspase[J]. Science, 1997, 278(5345): 1966-8.
[9] Hashimoto S, Takahashi H, Aiel D, et al. Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis[J]. Arthritis Rheum, 1998, 41(9): 1226-34.
[10] 张宝弟, 郭 雄, 白广禄, 等. 大骨节病患者血清NO, NOS, sFas/ APO-1检测分析[J]. 中国地方病学杂志, 2004, 31(4): 172-5.
Zhang BD, Guo X, Bai GL, et al. The changes of nitric oxide, NO synthase and sFas/Apo-1 in serum among the patients with Kashin-Beck disease[J]. Chin J Endemiol, 2004, 31(4): 172-5.
参考文献:
[1] Aigner T, McKenna L. Molecular pathology and pathobiology of osteoarthritic cartilage[J]. Cell Mol Life Sci, 2002, 59(1): 5-18.
[2] 杨 珺, 郭 雄, 张增铁, 等. 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[J].中国地方病学杂志, 2002, 21(5): 341-3.
Yang J, Guo X, Zhang ZT, et al. The distributed characteristics of chondrocyte apoptosis and the expression of Bcl-2 in articular cartilage with Kashin-Beck disease[J]. Chin J Endemiol, 2002, 21(5): 341-3.
[3] Robert K, Schen K, Peter SE, et al. Articular cartilage biochemistry[M]. New York: Raven Press, 1986. 3-22.
[4] Roach HI, Aigner T, Erenpeisa J. Ostegenic differentiation of hyper- trophic chondrocytes involves asymmetric cell divisions and apoptosis[J]. J Cell Biol, 1995, 131(3): 483-91.
[5] Yang TS, Hou LZ, Li FS. Biochemical characteristics of Kashin- Beck disease[A]. In: Kashin-Beck disease and non-communicable diseases[M]. Beijing: Chinese Academy of Preventive Medicine, 1990. 116-28.
[6] 郭 雄, Thomas A, Pirkko L, 等. 大骨节病关节软骨胶原表型表达和软骨细胞异常分化的研究[J]. 中华病理学杂志, 1998, 27(1): 19-22.
Guo X, Thomas A, Pirkko L, et al. A study on abnormal chon- drocyte differentiation and abnormal expression of collagen types in AC from patients with Kashin-Beck disease[J]. Chin J Pathol, 1998, 27(1): 19-22.
[7] Kim HA, Lee YJ, Seong SC, et al. Apoptotic chondrocyte death in human osteoarthritis[J]. J Rheumatol, 2000, 10(10): 455-62.
[8] Cheng EH, Kirsch DG, Clem RJ, et al. Conversion of Bcl-2 to a Bax-like death effector by caspase[J]. Science, 1997, 278(5345): 1966-8.
[9] Hashimoto S, Takahashi H, Aiel D, et al. Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis[J]. Arthritis Rheum, 1998, 41(9): 1226-34.
[10] 张宝弟, 郭 雄, 白广禄, 等. 大骨节病患者血清NO, NOS, sFas/ APO-1检测分析[J]. 中国地方病学杂志, 2004, 31(4): 172-5.
Zhang BD, Guo X, Bai GL, et al. The changes of nitric oxide, NO synthase and sFas/Apo-1 in serum among the patients with Kashin-Beck disease[J]. Chin J Endemiol, 2004, 31(4): 172-5.
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