iNOS在肢体IR后关节损伤中的作用及组织芯片技术的初步应用
摘要
目的：通过观察肢体IR后关节的病理变化,检测关节各组织内iNOS的表达规律和主要细胞的凋亡情况,初步探讨肢体IR后有无关节损伤及可能的机制;同时设计、使用手工组织芯片制作技术,寻找一种经济、高效的肢体IR研究方法。
方法：健康新西兰白兔54只,随机分为9组,每组6只动物。参照M. Hatoko改良方法制作兔右后肢原位IR模型；左侧肢体实施同样的手术但不阻断血运作为自身对照。按设计时点处死动物取材。观察下述指标：关节液涂片镜检；关节各组织病理变化；免疫组化、CLSM检测iNOS的表达；TUNNEL法标记凋亡细胞；苏木素-亮绿一番红三色法检测蛋白多糖含量；免疫荧光法检测软骨中胶原的变化；制作组织芯片并进行HE、免疫组化、免疫荧光检测,与常规连续切片检测结果比较,判断组织芯片技术引入肢体IR实验研究的可行性。
1.肢体IR后关节损害表现为时间依从性进行性加重；自身对照无明显变化。
2.iNOS在关节周围肌中成时间依从性动态表达,表达量与骨骼肌细胞凋亡、变性、坏死细胞计数正相关：iNOS于IR2h在毛细血管内皮细胞表达,IR4h定位胞浆,表达增加,IR8h表达最多,达高峰,24h下降,与对照侧相比具有统计学意义(p<0.01)。IR2h可见少量变性坏死细胞,IR4h、8h持续增加,24h达高峰,3d开始减少,14d可见骨骼肌萎缩,与对照组比较有统计学意义(p<0.05)。iNOS与骨骼肌细胞病理变化成正相关(r=0.682,p=0.043)。
3.iNOS在半月板中成时间依从性动态表达,表达量与半月板软骨细胞凋亡数正相关：iNOS阳性细胞在IR2h增高,8h达高峰,全层表达,定位于胞浆,24h开始减少,3d后明显下降,局限在半月板的深层细胞。与对照侧比较有统计学意义(p<0.05)凋亡细胞数IR2h增加,IR24h达高峰,3d后下降,7d后仍有表达,表现出持续高表达,与自身对照相比有显著统计学意义(p<0.01)。iNOS在半月板中的动态表达与半月板软骨细胞凋亡正相关(r=0.754,p=0.031)。
4.iNOS在关节软骨细胞中成时间依从性动态表达,表达量与软骨细胞凋亡正相关：iNOS于IR2h增高,全层表达,8h达高峰,24h开始减少,3d后明显下降,局限在同源细胞族和增殖区。与对照侧比较有显著统计学意义(p<0.01)。凋亡细胞于IR2h增加,24 h达高峰,3d稍微下降,持续到7d仍较高,表现为软骨细胞凋亡持续性异常增高。对照侧软骨细胞凋亡无明显变化,双侧比较,具有显著统计学意义(p<0.01)。iNOS在关节软骨细胞中的动态表达与软骨细胞凋亡正相关(r=0.828,p=0.006)。
5.关节软骨PG的变化：IR2h、IR4h PG开始减少(AOD值),但无统计学意义。IR24h PG继续减少,染色变浅,至IR3d组降至最低,并维持在较低水平,与对照组比较有统计学意义(p<0.01)。PG降至最低时只有对照组的56%。
6.关节软骨胶原II的变化：CollagenI I在IR2h、IR4h、IR8h组降低(AOD值)但无统计学意义。IR3d后明显降低,持续到14d仍无升高,与对照组比较有统计学意义(p<0.05)。
7.组织芯片的应用：自行设计制作组织芯片,与常规连续切片比较检测结果无统计学意义(p>0.05)。
1.肢体IR后关节损害进行性加重,存在再灌注损伤。
2.肢体IR后iNOS在关节周围肌、半月板、软骨细胞中成时间依从性动态表达,与组织细胞凋亡计数正相关。iNOS异常高表达,产生大量NO,引起细胞凋亡；胶原纤维,蛋白多糖损伤继发于细胞凋亡。iNOS的异常表达至少是肢体IR后关节损伤的主要途径之一,是软骨损伤的始动因素。
3.参照传统组织芯片制作流程,自行设计应用套针钻取、融孔植入、铝箔赋形等技术,大大简化了组织芯片制作,简单、可靠、高通量、大样本、省时快速、简便经济,易于推广,适合于相对多分组、大样本的实验研究。意义：肢体IR临床上普遍存在,但研究较少,是否存在关节IR损伤尚有争议;关节特别是关节软骨、半月板的IR机理尚无报道；多分组、多时点的肢体IR实验研究,需大量时间、人力、物力,组织芯片技术可以大幅提高效率；断指再植因关节内外粘连,功能恢复不满意,缺乏有效疗法,本实验有望为找到新的方法提供理论依据。
Objectives:To study the role of inducible nitric oxide synthase in articular tissue with limbs IR injury and introduce into tissue microarray, we designed to observe the pathologic changes, the expression of iNOS and the number of apoptotic cells in articular tissue in rabbits with limbs IR injury and apply TMA technique.
Methods:54 adult New Zealand white rabbits(2500±100g)were divided into 9 groups randomly(Normal, Ischemia, IR2h, IR4h,IR8h, IR24h, IR3d, IR7d, IR14d,6 per group). Femoral arteries and femoral veins of right hind limbs were clamped by vessel serrefines. Tourniquets were used simultaneously (except Normal group). The left hind limbs were treated as self-control which performed same operation only. After 4 hours of ischemia, the right limbs were reperfused (except Normal and Ischemia group). Then the rabbits were killed at corresponding time. Synovia was smeared; synovial membrane, muscle, menisci, and cartilage were excised from the knee of both sides respectively. They were embedded into paraffin. Expression of iNOS was evaluated using immunohistochem-istry and immunofluorescence; Apoptotic cells were identified using the TUNNEL (terminal dUTP nick end labelling) assay; Collagen type II was evaluated using immunohistochemistry; Proteoglycan was evaluated using haematoxylin-brilliantgreen-safranine staining. Manual TMA technique was designed to simplify the experiment.
1.The articular tissue in experimental groups showed a time-dependent injury, though that in self-control groups changed insignificantly.
2. The expression of iNOS in articular muscle of right limb changed time dependently. The expression of iNOS was detected during the initial stages (IS, IR2h) and it located in capillary endothelial cells only; during the following stages, such as IR4h group and IR8h group, it located in skeleton muscle cells at large scale; Then the iNOS expression. in skeleton muscle cells declined to an undetectable level in IR3d group. Accordingly, the number of Apoptotic cells in skeleton muscle changed in the homologous way. A correlation analysis was made between the expression of iNOS and the number of apoptotic cells. The correlation (r=0.682,p=0.043) was significant at the 0.05 level (2-tailed).
3. INOS was labeled using SABC-cy-3, and the apoptotic cells were identified using the TUNNEL. Both iNOS and apoptotic cells changed time-dependently in the experimental groups. INOS which located in menisci chondrocytes cytochylema increased in IR2h group, culminated in IR8h group, and began to decline in IR24h group. The number of apoptotic cells in menisci increased from IR2h group and reach maximum in IR24h group, then decreased slightly and sustained at lower level compared with control side. It was significant at the 0.05 level (2-tailed). Correlation analysis result suggested that there was a positive correlation between the expression of iNOS and the number of apoptotic cells(r=0.754, p=0.031). It was significant at the 0.05 level (2-tailed).
4. The same methods were used to examine iNOS and apoptotic cells in cartilage. In the experimental groups iNOS located in cytochylema, which increased in IR2h group, reach maximum in IR8h group, and decreased in IR24 group. Compared with control side, it was significant at 0.01 level (two tailed). The number of apoptotic cells of cartilage appeared in IR2h group, culminated in IR24h group, then declined slightly and maintained a lower level even in IR7d group. Compared with control side, it was significant at 0.01 level(two tailed). Both iNOS and apoptotic cells changed time-dependently in the experimental groups. A correlation analysis was made between the two groups of enumeration data and its outcome (r=0.828,p=0.006) indicated that there was a positive correlation which was significant at 0.01 (at two-tailed).
5. Proteoglycan was evaluated using haematoxylin-brilliantgreen-safranine staining and collagen typell was evaluated using immunohistochemistry. There was no difference of PG and collagen typell between the control and experimental group during the initial time. Both PG and collagen typell decreased significantly after 3d and sustained a lower level to 14d in the experimental group.. It was significant at 0.05 level (at two-tailed) compared with the control side.
6. Designed an manual method to prepare TMA and use them to examine the expression of iNOS, apoptotic cells in both experimental and control group. The results were no significant compared with that examined by usual paraffin section.
1. The injury of articular tissue aggravated time-dependently after reperfusion, as obviously suggests that there was a IR injury in knee joint in rabbits with hind limbs IR.
2. Both the expression of iNOS and the number of apoptotic cells in muscle, menisci and cartilage changed time-dependently in the experimental group. There was positive correlation between the two signs. PG and collagen typeⅡdecreased significantly after 3d and sustained a lower level to 14d in the experimental group. It was significant compared with those in control side (p<0.05). INOS is considered to be one of the mechanisms responsible for the death of cells during reperfusion time. Go further and say; it may be one of the mechanisms responsible for the articular tissue IR injury in rabbits.
3. Self-designed TMA preparation method possess the following properties:①imple;②practical;③economical;④reliable;⑤efficient;⑥easy to be popularized.
方法：健康新西兰白兔54只,随机分为9组,每组6只动物。参照M. Hatoko改良方法制作兔右后肢原位IR模型；左侧肢体实施同样的手术但不阻断血运作为自身对照。按设计时点处死动物取材。观察下述指标：关节液涂片镜检；关节各组织病理变化；免疫组化、CLSM检测iNOS的表达；TUNNEL法标记凋亡细胞；苏木素-亮绿一番红三色法检测蛋白多糖含量；免疫荧光法检测软骨中胶原的变化；制作组织芯片并进行HE、免疫组化、免疫荧光检测,与常规连续切片检测结果比较,判断组织芯片技术引入肢体IR实验研究的可行性。
1.肢体IR后关节损害表现为时间依从性进行性加重；自身对照无明显变化。
2.iNOS在关节周围肌中成时间依从性动态表达,表达量与骨骼肌细胞凋亡、变性、坏死细胞计数正相关：iNOS于IR2h在毛细血管内皮细胞表达,IR4h定位胞浆,表达增加,IR8h表达最多,达高峰,24h下降,与对照侧相比具有统计学意义(p<0.01)。IR2h可见少量变性坏死细胞,IR4h、8h持续增加,24h达高峰,3d开始减少,14d可见骨骼肌萎缩,与对照组比较有统计学意义(p<0.05)。iNOS与骨骼肌细胞病理变化成正相关(r=0.682,p=0.043)。
3.iNOS在半月板中成时间依从性动态表达,表达量与半月板软骨细胞凋亡数正相关：iNOS阳性细胞在IR2h增高,8h达高峰,全层表达,定位于胞浆,24h开始减少,3d后明显下降,局限在半月板的深层细胞。与对照侧比较有统计学意义(p<0.05)凋亡细胞数IR2h增加,IR24h达高峰,3d后下降,7d后仍有表达,表现出持续高表达,与自身对照相比有显著统计学意义(p<0.01)。iNOS在半月板中的动态表达与半月板软骨细胞凋亡正相关(r=0.754,p=0.031)。
4.iNOS在关节软骨细胞中成时间依从性动态表达,表达量与软骨细胞凋亡正相关：iNOS于IR2h增高,全层表达,8h达高峰,24h开始减少,3d后明显下降,局限在同源细胞族和增殖区。与对照侧比较有显著统计学意义(p<0.01)。凋亡细胞于IR2h增加,24 h达高峰,3d稍微下降,持续到7d仍较高,表现为软骨细胞凋亡持续性异常增高。对照侧软骨细胞凋亡无明显变化,双侧比较,具有显著统计学意义(p<0.01)。iNOS在关节软骨细胞中的动态表达与软骨细胞凋亡正相关(r=0.828,p=0.006)。
5.关节软骨PG的变化：IR2h、IR4h PG开始减少(AOD值),但无统计学意义。IR24h PG继续减少,染色变浅,至IR3d组降至最低,并维持在较低水平,与对照组比较有统计学意义(p<0.01)。PG降至最低时只有对照组的56%。
6.关节软骨胶原II的变化：CollagenI I在IR2h、IR4h、IR8h组降低(AOD值)但无统计学意义。IR3d后明显降低,持续到14d仍无升高,与对照组比较有统计学意义(p<0.05)。
7.组织芯片的应用：自行设计制作组织芯片,与常规连续切片比较检测结果无统计学意义(p>0.05)。
1.肢体IR后关节损害进行性加重,存在再灌注损伤。
2.肢体IR后iNOS在关节周围肌、半月板、软骨细胞中成时间依从性动态表达,与组织细胞凋亡计数正相关。iNOS异常高表达,产生大量NO,引起细胞凋亡；胶原纤维,蛋白多糖损伤继发于细胞凋亡。iNOS的异常表达至少是肢体IR后关节损伤的主要途径之一,是软骨损伤的始动因素。
3.参照传统组织芯片制作流程,自行设计应用套针钻取、融孔植入、铝箔赋形等技术,大大简化了组织芯片制作,简单、可靠、高通量、大样本、省时快速、简便经济,易于推广,适合于相对多分组、大样本的实验研究。意义：肢体IR临床上普遍存在,但研究较少,是否存在关节IR损伤尚有争议;关节特别是关节软骨、半月板的IR机理尚无报道；多分组、多时点的肢体IR实验研究,需大量时间、人力、物力,组织芯片技术可以大幅提高效率；断指再植因关节内外粘连,功能恢复不满意,缺乏有效疗法,本实验有望为找到新的方法提供理论依据。
Objectives:To study the role of inducible nitric oxide synthase in articular tissue with limbs IR injury and introduce into tissue microarray, we designed to observe the pathologic changes, the expression of iNOS and the number of apoptotic cells in articular tissue in rabbits with limbs IR injury and apply TMA technique.
Methods:54 adult New Zealand white rabbits(2500±100g)were divided into 9 groups randomly(Normal, Ischemia, IR2h, IR4h,IR8h, IR24h, IR3d, IR7d, IR14d,6 per group). Femoral arteries and femoral veins of right hind limbs were clamped by vessel serrefines. Tourniquets were used simultaneously (except Normal group). The left hind limbs were treated as self-control which performed same operation only. After 4 hours of ischemia, the right limbs were reperfused (except Normal and Ischemia group). Then the rabbits were killed at corresponding time. Synovia was smeared; synovial membrane, muscle, menisci, and cartilage were excised from the knee of both sides respectively. They were embedded into paraffin. Expression of iNOS was evaluated using immunohistochem-istry and immunofluorescence; Apoptotic cells were identified using the TUNNEL (terminal dUTP nick end labelling) assay; Collagen type II was evaluated using immunohistochemistry; Proteoglycan was evaluated using haematoxylin-brilliantgreen-safranine staining. Manual TMA technique was designed to simplify the experiment.
1.The articular tissue in experimental groups showed a time-dependent injury, though that in self-control groups changed insignificantly.
2. The expression of iNOS in articular muscle of right limb changed time dependently. The expression of iNOS was detected during the initial stages (IS, IR2h) and it located in capillary endothelial cells only; during the following stages, such as IR4h group and IR8h group, it located in skeleton muscle cells at large scale; Then the iNOS expression. in skeleton muscle cells declined to an undetectable level in IR3d group. Accordingly, the number of Apoptotic cells in skeleton muscle changed in the homologous way. A correlation analysis was made between the expression of iNOS and the number of apoptotic cells. The correlation (r=0.682,p=0.043) was significant at the 0.05 level (2-tailed).
3. INOS was labeled using SABC-cy-3, and the apoptotic cells were identified using the TUNNEL. Both iNOS and apoptotic cells changed time-dependently in the experimental groups. INOS which located in menisci chondrocytes cytochylema increased in IR2h group, culminated in IR8h group, and began to decline in IR24h group. The number of apoptotic cells in menisci increased from IR2h group and reach maximum in IR24h group, then decreased slightly and sustained at lower level compared with control side. It was significant at the 0.05 level (2-tailed). Correlation analysis result suggested that there was a positive correlation between the expression of iNOS and the number of apoptotic cells(r=0.754, p=0.031). It was significant at the 0.05 level (2-tailed).
4. The same methods were used to examine iNOS and apoptotic cells in cartilage. In the experimental groups iNOS located in cytochylema, which increased in IR2h group, reach maximum in IR8h group, and decreased in IR24 group. Compared with control side, it was significant at 0.01 level (two tailed). The number of apoptotic cells of cartilage appeared in IR2h group, culminated in IR24h group, then declined slightly and maintained a lower level even in IR7d group. Compared with control side, it was significant at 0.01 level(two tailed). Both iNOS and apoptotic cells changed time-dependently in the experimental groups. A correlation analysis was made between the two groups of enumeration data and its outcome (r=0.828,p=0.006) indicated that there was a positive correlation which was significant at 0.01 (at two-tailed).
5. Proteoglycan was evaluated using haematoxylin-brilliantgreen-safranine staining and collagen typell was evaluated using immunohistochemistry. There was no difference of PG and collagen typell between the control and experimental group during the initial time. Both PG and collagen typell decreased significantly after 3d and sustained a lower level to 14d in the experimental group.. It was significant at 0.05 level (at two-tailed) compared with the control side.
6. Designed an manual method to prepare TMA and use them to examine the expression of iNOS, apoptotic cells in both experimental and control group. The results were no significant compared with that examined by usual paraffin section.
1. The injury of articular tissue aggravated time-dependently after reperfusion, as obviously suggests that there was a IR injury in knee joint in rabbits with hind limbs IR.
2. Both the expression of iNOS and the number of apoptotic cells in muscle, menisci and cartilage changed time-dependently in the experimental group. There was positive correlation between the two signs. PG and collagen typeⅡdecreased significantly after 3d and sustained a lower level to 14d in the experimental group. It was significant compared with those in control side (p<0.05). INOS is considered to be one of the mechanisms responsible for the death of cells during reperfusion time. Go further and say; it may be one of the mechanisms responsible for the articular tissue IR injury in rabbits.
3. Self-designed TMA preparation method possess the following properties:①imple;②practical;③economical;④reliable;⑤efficient;⑥easy to be popularized.
引文
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[13]陈建常,史振满,王乐农等.肢体缺血再灌注后关节软骨及滑膜损伤的组织学观察[J].中国矫形外科杂志,2001,8(2)：156-157.
[14]宋一平,黄耀添,王钢,等,肢体缺血再灌注损伤兔膝关节软骨细胞核DNA含量的图像分析[J].骨与关节损伤杂志,1995；10(6)：355-357.
[15]陈建常,史振满,王乐农,等.丹参对缺血肢体关节软骨及滑膜损伤的影响[J].骨与关节损伤杂志,2000；15(2)：121-122.
[16]Blaisdell FW,et al. The pathophysiology of skeletal muscle ischemia and the reperfusion sndrome [J] Cardiovasc Surg J,2002,10(6):620-630.
[17]陈瑗,周玫.自由基医学基础与病理生理[M]第一版,上海：人民卫生出版社,2002：265.
[18]倪江东,付荫宇,李贺君.骨骼肌缺血再灌注损伤及其保护的实验研究[J].湖南医学,1998,15(2)：92-93.
[19]Bhendel M,Volk T,Kox WJ. The influence of cellular hypoxia and reactive oxygen species on the development of endothelial cell edema, [J] Microcirc Clin Exp,1997,17:67-74.
[20]Beetsch JW, Park TS, Dugan LL, et al. Xanthine oxides-derived superoxide causes reoxygenation injury of ischemia cerebral endothelial cells[J] Brain Res, 1998,786:89-95.
[21]周君琳,凌亦凌,李陈利等.一氧化氮和过氧亚硝基阴离子在肢体缺血再灌注致肺损伤中的作用,200l,17(2)：158-160.
[22]孙宇一,韩竹,吕松岑等.一氧化氮在骨骼肌缺血再灌注损伤中的作用[J].哈尔滨医科大学学报,2002,36(6)：440-443.
[23]Lepore DA. Nitric oxide synthase-independent generation of nitric oxide in muscle ischemia-reperfusion injury[J].Nitric Oxide,2000,4 (6):541-565.
[24]郑庆丰,郭平凡.陈福真等.肿瘤坏死因子α介导骨骼肌缺血再灌注损伤的实验研究[J].中华普通外科杂志,2002,17(8)：486-488.
[25]王亦璁主编.膝关节外科的基础与临床[M].第一版,北京：人民卫生出版社,2000：2-23.
[26]孙新军,王成琪,周继红等.缺血再灌注损伤对再植肢体关节滑膜的早期影响[J].第三军医大学学报,2001,23(10)：1191-1193.
[27]Joseph A. Buckwalter,MD,MS Thomas A. Einhom, MD Sheldon R. Simon, MD主 编.陈启明,梁国穗,秦岭主译.骨科基础科学.[M].第一版,北京：人民卫生出版社,2001：459-464.
[28]钟慈声,孙安阳主编.一氧化氮的生物医学[M]第2版.上海：上海医科大学出版社,1997,281-5.
[29]Stangel M, Zettl UK, Mix E, et al.H2O2 and nitric oxide-mediated oxidative stress induce apoptosis in rat skeletal muscle myoblaster. [J] Exp Neurol,1996,55:36-43.
[30]陈瑗,周玫.自由基医学基础与病理生理.[M].第一版,上海：人民卫生出版社,2002,110-118.
[31]N.J. Goodstone, T.E. Hardingham. Tumour necrosis factor astimulates nitric oxide production more potently than interleukin-1βin porcine articular chondrocytes. Rheumatology 2002,883-891.
[32]Mel S Lee,Takashi Ikenoue, Michael C D Trindade, et al. Protective effects of intermittent hydrostatic pressure on osteoarthritic chondrocytes activated by bacterial endotoxin in vitro[J]. Journal of Orthopaedic Research,2003,21(1): 117-122.
[33]Clancy RM, Gomez PF, Abramson SB. Nitric oxide sustains nuclear factor kappa B activation in cytokine-stimulated chondrocytes[J].Osteoarthritis Cartilage,2004,12 (7):552-558.
[34]Mendes AF, Carvalho AP, Caramona MM, et al. Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS Ⅱ expression in articular chondrocytes. [J] Inflamm Res 2002,51(7):369-375.
[35]王传家,李宇,霍霞,等.一氧化氮对兔关节软骨细胞凋亡的影响[J].实用美容整形外科杂志,2002,13(6)：324-326.
[36]Ayse Karan, Mehmet Akif Karan, Pervin Vural, et al. Synovial fluid nitric oxide levels in patients with knee osteoarthritis[J]. Clin Rheumatol 2003,22:397-399.
[37]彭丹,孙材江,周江南.一氧化氮在实验性骨关节炎软骨细胞凋亡中的作用[J].中华风湿病学杂志,2000,4(4)：232-4.
[38]Studer RK, Levicoff E, Georgescu H, et al. Nitric oxide inhibits chondrocyte response to IGF-1:inhibition of IGF-IRbgr; tyrosine phosphorylation. [J] Am J Physiol 2000,279:C961-969.
[39]Taskiran D, Stefanovic-Racic M, Georgescu. et al. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1 [J]. Biochem Biophys Res Commun 1994,200:142-148.
[40]孙炜,王吉兴,秦立斌,等.一氧化氮合酶抑制剂在实验性兔关节软骨修复中的作用[J].中华风湿病学杂志,2002,6(4)：227-30.
[41]王吉兴,孙炜,金大地,等.一氧化氮合酶抑制剂对关节软骨修复的影响[J].中华骨科杂志,2003,23(2)：108-113.
[42]A Scharstuhl,HM van Beuningen,ELVitters,et al. Loss of transforming growth factor counteraction on interleukin 1 mediated effects in cartilage of old mice [J].Annals of the Rheumatic Diseases,2002,21(5):914.
[43]Pelletier JP,Lascar CV,Jovanovic D,et al. Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors. [J] Rheumatol 1999,26:2002-2014.
[44]张梅,李平,陈朝晖,等.马钱子碱对一氧化氮诱导软骨细胞凋亡的影响[J].中国临床康复,2003,7(26)：3554.
[45]R K Studer,K Decker,S Melhem,et al. Nitric oxide inhibition of IGF-1 stimulated proteoglycan synthesis:Role of cGMP [J].Journal of Orthopaedic Research, 2003,002040:468-475.
[46]van't Hof RJ, Hocking L,Wright PK,et al. Nitric oxide is a mediator of apoptosis in the rheumatoid joint [J]. Rheumatology (Oxford),2000,30(9):1004-1008.
[47]Bezerra MM,Bezerra MM,Greenacre S,et al. Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan-induced arthritis.[J] Br J Pharmacol,2004,141(1):172-182.
[1]师建国,林雨冬,闫庆国等.乳腺癌组织芯片中凋亡相关基因BAG-l的表达及其意义[J].中国普通外科杂志,2004,13(5)：369-371.
[2]徐志秀,李新功,董艳光.组织芯片的Feulgen反应和免疫组化染色[J].临床与实验病理学杂志,2003,19(2)：214-215.
[3]张彦宁,周小鸽,张长淮等.应用组织芯片技术检测抗体特异性和敏感性[J].诊断病理学杂志,2003,l0(5)：313.
[4]张建中,韩瑞刚,郑艳华等.多肿瘤组织芯片在评价肿瘤细胞增殖与凋亡状态研究中的作用[J].肿瘤防治杂志,2004,11(1)：25-28.
[5]向正华,吕军,蒋平等. 组织芯片技术及其应用[J].解剖学杂志,2002,25(4)403-407.
[6]Schrml P, Kononen J, Bubendorf L, tal.Tissue microarrays for sene amplificationsurveys in many different tumor types [J].Clin Canceer Res,1999,5(8): 1966-1975.
[7]韩瑞刚,张建中,黄英武等.肺癌石蜡包埋组织及新鲜标本组织芯片制备方法学探讨.[J].临床与实验病理学杂志,2002,18(4)：404-407.
[2]胥少汀,葛宝丰,徐印坎主编.实用骨科学[M].第二版,北京：人民军医出版社,2004：985,99l-992.
[3]R K Studer,K Decker,S Melhem,et al. Nitric oxide inhibition of IGF-1 stimulated proteoglycan synthesis:Role of cGMP [J].Journal of Orthopaedic Research,2003, 21(5):914.
[4]A R Poole,M Kobayashi,et al. Type Ⅱ collagen degradation and its regulation in articular cartilage in osteoarthritis [J]. Annals of the Rheumatic Diseases,2002, 61:1178-1181.
[5]崔国庆,田得祥,陈启明等.一氧化氮合成酶在骨关节炎关节软骨及骨刺中的表达[J] 中华骨科杂志,1998,18(10)：619-621.
[6]孙炜,王吉兴,金大地等.一诱导型一氧化氮合酶抑制剂与软骨修复的研究进展[J].中华骨科杂志,2001,21(2)：119-120.
[7]王传家,李宇,霍霞,等.一氧化氮对兔关节软骨细胞凋亡的影响[J].实用美容整形外科杂志,2002,13(6)：324-326
[8]邵欣欣,田得祥,于长隆等.一氧化氮抑制兔关节软骨细胞II型胶原的合成[J].中国运动医学杂志,200l,20(1)：31-34.
[9]Lisa A Fortier,Molly M Deak,Stacy A Semevolos,et al. Insulin-like growth factor-I diminishes the activation status and expression of the small GTPase Cdc42 in articular chondrocytes [J] Journal of Orthopaedic Research,2004,22(2):436-445.
[10]裴明,曲绵域,于长隆等.诱导型一氧化氮合酶和白介素1受体拮抗剂在骨关节炎滑膜和软骨中的表达及意义[J].中国运动医学杂志,2000,19(3)：246-248.
[11]张俐,LE Chen高效选择性iNOS抑制剂1400W对骨骼肌缺血再灌注损伤的实验研究[J].中国中医骨伤科杂志,2000,8(6)：10-15.
[12]M. Hatoko A, Tanaka M, Kuwahara S.Yurugi, et al. stress response of systemic organs to ischemia-reperfusion of rat hind limb-study of induction of 72-kD stress protein (HSP72) [J]. Eur J Plast Surg 2002,25:143-78.
[13]陈建常,史振满,王乐农等.肢体缺血再灌注后关节软骨及滑膜损伤的组织学观察[J].中国矫形外科杂志,2001,8(2)：156-157.
[14]宋一平,黄耀添,王钢,等,肢体缺血再灌注损伤兔膝关节软骨细胞核DNA含量的图像分析[J].骨与关节损伤杂志,1995；10(6)：355-357.
[15]陈建常,史振满,王乐农,等.丹参对缺血肢体关节软骨及滑膜损伤的影响[J].骨与关节损伤杂志,2000；15(2)：121-122.
[16]Blaisdell FW,et al. The pathophysiology of skeletal muscle ischemia and the reperfusion sndrome [J] Cardiovasc Surg J,2002,10(6):620-630.
[17]陈瑗,周玫.自由基医学基础与病理生理[M]第一版,上海：人民卫生出版社,2002：265.
[18]倪江东,付荫宇,李贺君.骨骼肌缺血再灌注损伤及其保护的实验研究[J].湖南医学,1998,15(2)：92-93.
[19]Bhendel M,Volk T,Kox WJ. The influence of cellular hypoxia and reactive oxygen species on the development of endothelial cell edema, [J] Microcirc Clin Exp,1997,17:67-74.
[20]Beetsch JW, Park TS, Dugan LL, et al. Xanthine oxides-derived superoxide causes reoxygenation injury of ischemia cerebral endothelial cells[J] Brain Res, 1998,786:89-95.
[21]周君琳,凌亦凌,李陈利等.一氧化氮和过氧亚硝基阴离子在肢体缺血再灌注致肺损伤中的作用,200l,17(2)：158-160.
[22]孙宇一,韩竹,吕松岑等.一氧化氮在骨骼肌缺血再灌注损伤中的作用[J].哈尔滨医科大学学报,2002,36(6)：440-443.
[23]Lepore DA. Nitric oxide synthase-independent generation of nitric oxide in muscle ischemia-reperfusion injury[J].Nitric Oxide,2000,4 (6):541-565.
[24]郑庆丰,郭平凡.陈福真等.肿瘤坏死因子α介导骨骼肌缺血再灌注损伤的实验研究[J].中华普通外科杂志,2002,17(8)：486-488.
[25]王亦璁主编.膝关节外科的基础与临床[M].第一版,北京：人民卫生出版社,2000：2-23.
[26]孙新军,王成琪,周继红等.缺血再灌注损伤对再植肢体关节滑膜的早期影响[J].第三军医大学学报,2001,23(10)：1191-1193.
[27]Joseph A. Buckwalter,MD,MS Thomas A. Einhom, MD Sheldon R. Simon, MD主 编.陈启明,梁国穗,秦岭主译.骨科基础科学.[M].第一版,北京：人民卫生出版社,2001：459-464.
[28]钟慈声,孙安阳主编.一氧化氮的生物医学[M]第2版.上海：上海医科大学出版社,1997,281-5.
[29]Stangel M, Zettl UK, Mix E, et al.H2O2 and nitric oxide-mediated oxidative stress induce apoptosis in rat skeletal muscle myoblaster. [J] Exp Neurol,1996,55:36-43.
[30]陈瑗,周玫.自由基医学基础与病理生理.[M].第一版,上海：人民卫生出版社,2002,110-118.
[31]N.J. Goodstone, T.E. Hardingham. Tumour necrosis factor astimulates nitric oxide production more potently than interleukin-1βin porcine articular chondrocytes. Rheumatology 2002,883-891.
[32]Mel S Lee,Takashi Ikenoue, Michael C D Trindade, et al. Protective effects of intermittent hydrostatic pressure on osteoarthritic chondrocytes activated by bacterial endotoxin in vitro[J]. Journal of Orthopaedic Research,2003,21(1): 117-122.
[33]Clancy RM, Gomez PF, Abramson SB. Nitric oxide sustains nuclear factor kappa B activation in cytokine-stimulated chondrocytes[J].Osteoarthritis Cartilage,2004,12 (7):552-558.
[34]Mendes AF, Carvalho AP, Caramona MM, et al. Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS Ⅱ expression in articular chondrocytes. [J] Inflamm Res 2002,51(7):369-375.
[35]王传家,李宇,霍霞,等.一氧化氮对兔关节软骨细胞凋亡的影响[J].实用美容整形外科杂志,2002,13(6)：324-326.
[36]Ayse Karan, Mehmet Akif Karan, Pervin Vural, et al. Synovial fluid nitric oxide levels in patients with knee osteoarthritis[J]. Clin Rheumatol 2003,22:397-399.
[37]彭丹,孙材江,周江南.一氧化氮在实验性骨关节炎软骨细胞凋亡中的作用[J].中华风湿病学杂志,2000,4(4)：232-4.
[38]Studer RK, Levicoff E, Georgescu H, et al. Nitric oxide inhibits chondrocyte response to IGF-1:inhibition of IGF-IRbgr; tyrosine phosphorylation. [J] Am J Physiol 2000,279:C961-969.
[39]Taskiran D, Stefanovic-Racic M, Georgescu. et al. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1 [J]. Biochem Biophys Res Commun 1994,200:142-148.
[40]孙炜,王吉兴,秦立斌,等.一氧化氮合酶抑制剂在实验性兔关节软骨修复中的作用[J].中华风湿病学杂志,2002,6(4)：227-30.
[41]王吉兴,孙炜,金大地,等.一氧化氮合酶抑制剂对关节软骨修复的影响[J].中华骨科杂志,2003,23(2)：108-113.
[42]A Scharstuhl,HM van Beuningen,ELVitters,et al. Loss of transforming growth factor counteraction on interleukin 1 mediated effects in cartilage of old mice [J].Annals of the Rheumatic Diseases,2002,21(5):914.
[43]Pelletier JP,Lascar CV,Jovanovic D,et al. Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors. [J] Rheumatol 1999,26:2002-2014.
[44]张梅,李平,陈朝晖,等.马钱子碱对一氧化氮诱导软骨细胞凋亡的影响[J].中国临床康复,2003,7(26)：3554.
[45]R K Studer,K Decker,S Melhem,et al. Nitric oxide inhibition of IGF-1 stimulated proteoglycan synthesis:Role of cGMP [J].Journal of Orthopaedic Research, 2003,002040:468-475.
[46]van't Hof RJ, Hocking L,Wright PK,et al. Nitric oxide is a mediator of apoptosis in the rheumatoid joint [J]. Rheumatology (Oxford),2000,30(9):1004-1008.
[47]Bezerra MM,Bezerra MM,Greenacre S,et al. Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan-induced arthritis.[J] Br J Pharmacol,2004,141(1):172-182.
[1]师建国,林雨冬,闫庆国等.乳腺癌组织芯片中凋亡相关基因BAG-l的表达及其意义[J].中国普通外科杂志,2004,13(5)：369-371.
[2]徐志秀,李新功,董艳光.组织芯片的Feulgen反应和免疫组化染色[J].临床与实验病理学杂志,2003,19(2)：214-215.
[3]张彦宁,周小鸽,张长淮等.应用组织芯片技术检测抗体特异性和敏感性[J].诊断病理学杂志,2003,l0(5)：313.
[4]张建中,韩瑞刚,郑艳华等.多肿瘤组织芯片在评价肿瘤细胞增殖与凋亡状态研究中的作用[J].肿瘤防治杂志,2004,11(1)：25-28.
[5]向正华,吕军,蒋平等. 组织芯片技术及其应用[J].解剖学杂志,2002,25(4)403-407.
[6]Schrml P, Kononen J, Bubendorf L, tal.Tissue microarrays for sene amplificationsurveys in many different tumor types [J].Clin Canceer Res,1999,5(8): 1966-1975.
[7]韩瑞刚,张建中,黄英武等.肺癌石蜡包埋组织及新鲜标本组织芯片制备方法学探讨.[J].临床与实验病理学杂志,2002,18(4)：404-407.