强直性脊柱炎滑膜组织破骨细胞在软骨损伤中的作用及分化因素的研究
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摘要
背景
强直性脊柱炎的主要病理学特点是病变关节内早期滑膜组织异常增生,大量单、多核细胞浸润,血管翳形成,侵蚀骨、软骨表面,于相接毗邻处形成局灶性骨、软骨破坏灶,晚期脊柱周围韧带、滑膜、椎间盘骨化强直,并同时伴有全身或关节局部骨质疏松表现。骨质丢失及软骨破坏是炎性关节病及多种类风湿性疾病中的一个常见的临床特点,来自关节炎动物模型和类风湿性关节炎的研究结果己经确定,破骨细胞在炎性关节疾病发病过程中数量及活性都有所增加,在骨质破坏/丢失中起关键性作用。大量的研究证明破骨细胞及其前体来源于骨髓造血系统的单核巨噬细胞系,外周血单核巨噬细胞、肺泡巨噬细胞、脾细胞及牙槽巨噬细胞等都可以被分化成有活性的的破骨细胞。在破骨细胞前体分化为成熟的破骨细胞过程中,成骨细胞/基质细胞的作用是必需的,是破骨细胞成熟、发育及分化过程中的重要调节细胞。没有成骨细胞/基质细胞参与并与成骨细胞/基质细胞的接触,前体破骨细胞就不能分化为成熟破骨细胞,成熟破骨细胞也不能活化为功能性破骨细胞。分子水平研究破骨细胞的分化也是近年来研究的热点,护骨素/细胞核因子κB受体活化因子配基/细胞核因子κB受体活化因子系统、巨噬细胞集落刺激因子、血管内皮细胞生长因子、肿瘤坏死因子-α等都可以促进破骨细胞的分化成熟。分化成熟的破骨细胞通过分泌酸、溶酶体酶及蛋白酶来降解骨基质,其中基质金属蛋白酶-3是软骨基质降解中的一种较为重要的酶,并在强直性脊柱炎外周血及关节液中都有表达。在强直性脊柱炎滑膜部位聚集大量的单核巨噬细胞组成了滑膜增生的重要组成成份,这些细胞是否是破骨前体细胞,他们是否参与了局灶性软骨的破坏,其破坏途径是否与基质金属蛋白酶-3有关,滑膜组织中是否存在与其分化有关的因子,都不十分明确。
目的
本研究采用强直性脊柱炎患者骶髂关节及髋关节部位的滑膜组织,通过检测及培养滑膜组织中破骨细胞、基质金属蛋白酶-3及其与软骨破坏之间的关系,探讨破骨细胞对软骨的破坏作用及破坏途径;通过检测强直性脊柱炎滑膜组织中成骨细胞及破骨细胞分化因子肿瘤坏死因子-α、血管内皮细胞生长因子的表达,探讨强直性脊柱炎滑膜组织破骨细胞的分化因素及分化途径。
方法
采用强直性脊柱炎患者骶髂关节或髋关节部位的滑膜组织及软骨,制备石蜡切片和冰冻切片,经HE染色及甲苯胺蓝染色观察软骨组织病理学改变并进行Mankin评分,酶组织化学TRAP染色观察TRAP+细胞的阳性染色面积及分布,采用Spearman相关分析分析其与Mankin评分间的相关性;原位杂交技术检测滑膜组织中基质金属蛋白酶-3mRNA的表达,采用图像分析系统比较与正常对照组间图像灰度值的差异;酶组织化学联合原位杂交技术检测TRAP+细胞上基质金属蛋白酶的表达,分析TRAP+细胞与基质金属蛋白酶-3的关系;分离纯化培养强直性脊柱炎滑膜单核巨噬细胞,并与正常关节软骨共培养,HE染色及电镜检测软骨的破坏程度,进一步探讨TRAP+细胞对软骨破坏的破坏作用及破坏途径。
原位杂交技术检测强直性脊柱炎滑膜组织中肿瘤坏死因子-αmRNA、血管内皮生长因子mRNA的表达,采用图像分析系统比较与正常对照组图像灰度值之间的差异,与TRAP+染色面积及基质金属蛋白酶-3mRNA进行Spearman相关性分析;分离纯化培养滑膜成纤维细胞,取第3代滑膜成纤维细胞进行成骨鉴定:钙钴法检测碱性磷酸酶、免疫组织化学法检测I型胶原,茜素红染色检测钙结节及放射免疫法检测骨钙素的含量。
结果
1.强直性脊柱炎滑膜组织酶组织化学-TRAP染色可见滑膜组织衬里层及滑膜软骨交界部部分细胞被染成红色,对照组阴性,两组阳性细胞染色面积比具有统计学差异(P<0.01);HE染色及甲苯胺蓝染色强直性脊柱炎软骨破坏程度,并行组织病理学Mankin评分,评分与TRAP阳性染面积比具有显著相关性(r=0.712,P=0.001);
2.原位杂交技术检测强直性脊柱炎滑膜组织中基质金属蛋白酶-3mRNA的表达可见滑膜衬里层及滑膜下层细胞有棕黄色颗位沉着,对照组阴性表达,两组图象灰度值比较有统计学差异(P<0.01),强直性脊柱炎组基质金属蛋白酶-3mRNA阳性细胞灰度值与TRAP阳性染色面积比及Mankin评分具有相关性(r=0.491,P=0.024;r=0.728,P=0.001)原位杂交技术联合酶组织化学检测可见在细胞内的棕黄色颗粒变成棕红色;
3.成功分离强直性脊柱炎滑膜组织中单核巨噬细胞, TRAP染色可见细胞大而多核,细胞浆内有红色颗粒;滑膜单核巨噬细胞与正常关节软骨共培养,HE染色及电镜检测可见软骨表面不定型物质脱落,不平整,胶原暴露、断裂;
4.原位杂交技术检测肿瘤坏死因子-αmRNA、血管内皮生长因子mRNA的表达,可见强直性脊柱炎滑膜组织衬里层及衬里下层细胞有棕黄色颗位沉着,对照组阴性表达,两组图象灰度值比较有统计学差异(P<0.01),肿瘤坏死因子-αmRNA阳性细胞灰度值与TRAP阳性染色面积比及Mankin评分具有相关性(r=0.634,P=0.002;r=0.851,P=0.001)、血管内皮细胞生长因子mRNA阳性细胞灰度值与TRAP阳性染色面积比及Mankin评分具有显著相关性(r=0.598,P=0.004;r=0.851,P=0.001)。
5.第3代滑膜成纤维细胞成骨鉴定:钙钴法检测碱性磷酸酶表达,可见成纤维细胞内有黑色颗粒,对照组阴性表达;免疫组织化学染色检测I型胶原,可见细胞内有黄色颗粒,对照组阴性表达;放免法检测细胞内骨钙素含量,与对照组比较有统计学差异(P<0.01);滑膜成纤维细胞培养第18天,茜素红染色可见钙结节形成。
结论
1.强直性脊柱炎滑膜组织存在TRAP+细胞,对关节软骨具有破坏作用,是强直性脊柱炎关节软骨破坏的重要因素,其表达基质金属蛋白酶-3是软骨破坏的重要途径之一。
2.强直性脊柱炎滑膜组织中存在破骨细胞分化的环境:成纤维细胞表达成骨细胞特性、肿瘤坏死因子和血管内皮生长因子的表达,是构成强直性脊柱炎破骨细胞分化的重要环境因素。
3.强直性脊柱炎滑膜成纤维细胞分化为成骨细胞也是强直性脊柱炎病理性成骨的重要因素。
The chief pathological manifestations of ankylosing spondylitis (AS) includes paraplasm of synovial tissue in affected joint, massive monocyte and polykaryocytes infiltration, formation of ocular pannus with erosion on bone and cartilage surface, focal osteoclasia on adjacent spots; ossification rigidity of ligaments, synovial tissue and intervertebral disks and systemic or focal osteoporosis could be observed in later stage patients. Osteoporosis and cartilage osteoclasia are commonly observed in inflammatory arthropathy and multiple rheumatoid diseases, research on animal model of arthritis and rheumatoid arthritis have confirmed that osteoclast (OC) had a increased number and activity during the pathogenesis, which played a critical role in osteoporosis and osteoclasia. Numerous researches confirmed that OC and its precursor derives from mononuclear macrophages in hematopoietic system, active OC can originate from multiple sources including mononuclear macrophages in peripheral blood, pulmonary alveolar macrophages, spleen cell and alveolar macrophages etc. The effect of osteoblast/stroma cells are indispensable to the process of precursor differentiate into mature OC, which regulated the maturation, development and differentiation of OC. Without the involvement and contact with OB/SC, neither precursors could not differentiate into matured OC, nor matured would activate into functional OC. Approach to the differentiation of OC from molecular perspective became a hot-spot, osteprotegerin (OPG)/ receptor activator of nuclear factorκB ligand, (RANKL)/ receptor activator of nuclear factorκB (RANK), colony stimulating factor of macrophages(M-CSF), vascular endothelial cell growth factor (VEGF), tumor necrosis factor-α(TNF-α)have been found to related with the promotion of OC differentiation and maturation. Bone matrix is degraded by acid, lysosomal enzyme and protease secretion from matured OC, among which matrix metalloproteinase-3 (MMP-3) is critical to the degradation of cartilage matrix, and its expression have been found on peripheral blood and synovial fluid of AS patients. The massive mononuclear macrophages on AS synovial tissue constituted the hyperplasia of synovial tissue, whether theses cells can be identified as osteoclast precursor, do they involved the degradation of focal cartilage destruction, if the mechanism is related with MMP-3, or any differentiation related factors existed in synovial tissue still requires further investigation.
Objective: the research utilized synovial tissue from sacroiliac and iliac joints of AS patients, approach to the routes and effect of OC on cartilage damage through cultivate OC and analyze its relationship; and approach to the factors and routes of OC differentiation through observation the expression of differentiation factor TNF-αand VEGF and OB in synovial tissue of AS.
Method: synovial tissue and cartilage separated from sacroiliac joint of AS patients were prepared into paraffin and frozen section, cartilage pathological changes were observed through HE and toluidine blue stain, and mankin score was made; the distribution of TRAP+ cells as well as the positive areas on TRAP enzyme histochemical staining were recorded, and its relationship with Mankin score were inspected by Spearman correlation analysis; the expression of MMP-3 mRNA in synovial tissue were conducted by in situ hybridization, and the grayscale difference with normal tissue were compared by a image analysis system; expression of MMP on TRAP+ cells and the relationship between TRAP+ cells and MMP-3 were analyzed through enzyme histochemistry combined with in sity hybridization; the separated mononuclear macrophage in synovial tissue of AS were cultivated with normal joint cartilage in vitro, osteoclasia was inspected by HE staining and electron microscope, which further investigated the effect and route of cartilage damage from TRAP+ cells. Expression of mRNA of TNF-αand VEGF in synovial tissue were inspected by in situ hybridizatin, and the grayscale difference with normal tissue were compared by a image analysis system; a Spearman correlation analysis was conducted to determine its relationship with TRAP+ area and MMP-3mRNA; synovial fibroblast were separated and cultivated in vitro, the 3rd generation were collected for osteoblast identification: alkaline phosphatase by calcium-cobalt method, type I collagen by immunohistochemistry, calcium nodule by alizarin red staining and amount of osteocalcin by radio-immunity.
Results: 1.part cells in synovial lining and synovial-cartilage border became red on enzyme-histochemistry-TRAP staining, with negative results in control group, the difference between staining areas in two positive groups was of statistical significance (P<0.01); caritilage damage were inspected by HE and toluidine blue staining, and histopathology Mankin score were made, the score was significantly correlate with TRAP positive area (r=0.712,P=0.001). 2. MMP-3 mRNA expression by in situ hybridization observed brown particles in synovial lining and inferior cells, with negative results in control group, the grayscale comparison returned a statistical significant difference (P<0.01); the grayscale of MMP-3 mRNA positive cells, TRAP positive areas and Mankin score were correlated (r=0.491,P=0.024 ; r=0.728,P=0.001), in situ hybridization combined with enzyme histochemistry observed that brown particles within cells changed into copper-brown. 3. the separation of mononuclear macrophage in synovial tissue of AS was successful, microscopy on TRAP staining found cells in big size and multiple nucleus, and red particles in plasma; the co-cultivation of macrophage and normal joint cartilage discovered irregular shed on cartilage surface, collagen exposure and break-down in HE staining and electro microscope. 4. TNF-αand VEGF mRNA expression test by in situ hybridization observed brown particle in sinovial lining and inferior cells, with negative results in control group, the grayscale difference between two groups were of statistical significance (P<0.01), the grayscale of TNF-αmRNA positive cells correlated with TRAP positive area ratio and Mankin score (r=0.634,P=0.002;r=0.851,P=0.001), while significant correlation between gray scale of VEGF mRNA positive cells between TRAP positive area ratio and Mankin score were recored (r=0.598,P=0.004;r=0.851,P=0.001). 5. osteoblast identification of 3rd generation fibroblasts: alkaline phosphatase by calcium-cobalt revealed black particles in fibroblasts, negative in control group; while yellow particles were found in type I collagen test by immunohistochemistry, negative in control group; the amount of osteocalcin in experiment group is significantly different with control group by radio-immuno method (P<0.01); on day 18 of fibroblast cultivation, calcium nodule formation was observed by alizarin red staining.
Conclusion: 1. the presence of OC in synovial tissue correlated with cartilage damage in AS, the expression of MMP-3 is a key route. 2. The synovial tissue in AS formed a environment for the differentiation of OC: osteoblast-like characterization of fibroblasts, expression of TNF and VEGF are major environmental factors for OC differentiation. 3. the differentiation of fibroblasts into osteoblasts in AS also contributed to the pathological bone formation.
强直性脊柱炎的主要病理学特点是病变关节内早期滑膜组织异常增生,大量单、多核细胞浸润,血管翳形成,侵蚀骨、软骨表面,于相接毗邻处形成局灶性骨、软骨破坏灶,晚期脊柱周围韧带、滑膜、椎间盘骨化强直,并同时伴有全身或关节局部骨质疏松表现。骨质丢失及软骨破坏是炎性关节病及多种类风湿性疾病中的一个常见的临床特点,来自关节炎动物模型和类风湿性关节炎的研究结果己经确定,破骨细胞在炎性关节疾病发病过程中数量及活性都有所增加,在骨质破坏/丢失中起关键性作用。大量的研究证明破骨细胞及其前体来源于骨髓造血系统的单核巨噬细胞系,外周血单核巨噬细胞、肺泡巨噬细胞、脾细胞及牙槽巨噬细胞等都可以被分化成有活性的的破骨细胞。在破骨细胞前体分化为成熟的破骨细胞过程中,成骨细胞/基质细胞的作用是必需的,是破骨细胞成熟、发育及分化过程中的重要调节细胞。没有成骨细胞/基质细胞参与并与成骨细胞/基质细胞的接触,前体破骨细胞就不能分化为成熟破骨细胞,成熟破骨细胞也不能活化为功能性破骨细胞。分子水平研究破骨细胞的分化也是近年来研究的热点,护骨素/细胞核因子κB受体活化因子配基/细胞核因子κB受体活化因子系统、巨噬细胞集落刺激因子、血管内皮细胞生长因子、肿瘤坏死因子-α等都可以促进破骨细胞的分化成熟。分化成熟的破骨细胞通过分泌酸、溶酶体酶及蛋白酶来降解骨基质,其中基质金属蛋白酶-3是软骨基质降解中的一种较为重要的酶,并在强直性脊柱炎外周血及关节液中都有表达。在强直性脊柱炎滑膜部位聚集大量的单核巨噬细胞组成了滑膜增生的重要组成成份,这些细胞是否是破骨前体细胞,他们是否参与了局灶性软骨的破坏,其破坏途径是否与基质金属蛋白酶-3有关,滑膜组织中是否存在与其分化有关的因子,都不十分明确。
目的
本研究采用强直性脊柱炎患者骶髂关节及髋关节部位的滑膜组织,通过检测及培养滑膜组织中破骨细胞、基质金属蛋白酶-3及其与软骨破坏之间的关系,探讨破骨细胞对软骨的破坏作用及破坏途径;通过检测强直性脊柱炎滑膜组织中成骨细胞及破骨细胞分化因子肿瘤坏死因子-α、血管内皮细胞生长因子的表达,探讨强直性脊柱炎滑膜组织破骨细胞的分化因素及分化途径。
方法
采用强直性脊柱炎患者骶髂关节或髋关节部位的滑膜组织及软骨,制备石蜡切片和冰冻切片,经HE染色及甲苯胺蓝染色观察软骨组织病理学改变并进行Mankin评分,酶组织化学TRAP染色观察TRAP+细胞的阳性染色面积及分布,采用Spearman相关分析分析其与Mankin评分间的相关性;原位杂交技术检测滑膜组织中基质金属蛋白酶-3mRNA的表达,采用图像分析系统比较与正常对照组间图像灰度值的差异;酶组织化学联合原位杂交技术检测TRAP+细胞上基质金属蛋白酶的表达,分析TRAP+细胞与基质金属蛋白酶-3的关系;分离纯化培养强直性脊柱炎滑膜单核巨噬细胞,并与正常关节软骨共培养,HE染色及电镜检测软骨的破坏程度,进一步探讨TRAP+细胞对软骨破坏的破坏作用及破坏途径。
原位杂交技术检测强直性脊柱炎滑膜组织中肿瘤坏死因子-αmRNA、血管内皮生长因子mRNA的表达,采用图像分析系统比较与正常对照组图像灰度值之间的差异,与TRAP+染色面积及基质金属蛋白酶-3mRNA进行Spearman相关性分析;分离纯化培养滑膜成纤维细胞,取第3代滑膜成纤维细胞进行成骨鉴定:钙钴法检测碱性磷酸酶、免疫组织化学法检测I型胶原,茜素红染色检测钙结节及放射免疫法检测骨钙素的含量。
结果
1.强直性脊柱炎滑膜组织酶组织化学-TRAP染色可见滑膜组织衬里层及滑膜软骨交界部部分细胞被染成红色,对照组阴性,两组阳性细胞染色面积比具有统计学差异(P<0.01);HE染色及甲苯胺蓝染色强直性脊柱炎软骨破坏程度,并行组织病理学Mankin评分,评分与TRAP阳性染面积比具有显著相关性(r=0.712,P=0.001);
2.原位杂交技术检测强直性脊柱炎滑膜组织中基质金属蛋白酶-3mRNA的表达可见滑膜衬里层及滑膜下层细胞有棕黄色颗位沉着,对照组阴性表达,两组图象灰度值比较有统计学差异(P<0.01),强直性脊柱炎组基质金属蛋白酶-3mRNA阳性细胞灰度值与TRAP阳性染色面积比及Mankin评分具有相关性(r=0.491,P=0.024;r=0.728,P=0.001)原位杂交技术联合酶组织化学检测可见在细胞内的棕黄色颗粒变成棕红色;
3.成功分离强直性脊柱炎滑膜组织中单核巨噬细胞, TRAP染色可见细胞大而多核,细胞浆内有红色颗粒;滑膜单核巨噬细胞与正常关节软骨共培养,HE染色及电镜检测可见软骨表面不定型物质脱落,不平整,胶原暴露、断裂;
4.原位杂交技术检测肿瘤坏死因子-αmRNA、血管内皮生长因子mRNA的表达,可见强直性脊柱炎滑膜组织衬里层及衬里下层细胞有棕黄色颗位沉着,对照组阴性表达,两组图象灰度值比较有统计学差异(P<0.01),肿瘤坏死因子-αmRNA阳性细胞灰度值与TRAP阳性染色面积比及Mankin评分具有相关性(r=0.634,P=0.002;r=0.851,P=0.001)、血管内皮细胞生长因子mRNA阳性细胞灰度值与TRAP阳性染色面积比及Mankin评分具有显著相关性(r=0.598,P=0.004;r=0.851,P=0.001)。
5.第3代滑膜成纤维细胞成骨鉴定:钙钴法检测碱性磷酸酶表达,可见成纤维细胞内有黑色颗粒,对照组阴性表达;免疫组织化学染色检测I型胶原,可见细胞内有黄色颗粒,对照组阴性表达;放免法检测细胞内骨钙素含量,与对照组比较有统计学差异(P<0.01);滑膜成纤维细胞培养第18天,茜素红染色可见钙结节形成。
结论
1.强直性脊柱炎滑膜组织存在TRAP+细胞,对关节软骨具有破坏作用,是强直性脊柱炎关节软骨破坏的重要因素,其表达基质金属蛋白酶-3是软骨破坏的重要途径之一。
2.强直性脊柱炎滑膜组织中存在破骨细胞分化的环境:成纤维细胞表达成骨细胞特性、肿瘤坏死因子和血管内皮生长因子的表达,是构成强直性脊柱炎破骨细胞分化的重要环境因素。
3.强直性脊柱炎滑膜成纤维细胞分化为成骨细胞也是强直性脊柱炎病理性成骨的重要因素。
The chief pathological manifestations of ankylosing spondylitis (AS) includes paraplasm of synovial tissue in affected joint, massive monocyte and polykaryocytes infiltration, formation of ocular pannus with erosion on bone and cartilage surface, focal osteoclasia on adjacent spots; ossification rigidity of ligaments, synovial tissue and intervertebral disks and systemic or focal osteoporosis could be observed in later stage patients. Osteoporosis and cartilage osteoclasia are commonly observed in inflammatory arthropathy and multiple rheumatoid diseases, research on animal model of arthritis and rheumatoid arthritis have confirmed that osteoclast (OC) had a increased number and activity during the pathogenesis, which played a critical role in osteoporosis and osteoclasia. Numerous researches confirmed that OC and its precursor derives from mononuclear macrophages in hematopoietic system, active OC can originate from multiple sources including mononuclear macrophages in peripheral blood, pulmonary alveolar macrophages, spleen cell and alveolar macrophages etc. The effect of osteoblast/stroma cells are indispensable to the process of precursor differentiate into mature OC, which regulated the maturation, development and differentiation of OC. Without the involvement and contact with OB/SC, neither precursors could not differentiate into matured OC, nor matured would activate into functional OC. Approach to the differentiation of OC from molecular perspective became a hot-spot, osteprotegerin (OPG)/ receptor activator of nuclear factorκB ligand, (RANKL)/ receptor activator of nuclear factorκB (RANK), colony stimulating factor of macrophages(M-CSF), vascular endothelial cell growth factor (VEGF), tumor necrosis factor-α(TNF-α)have been found to related with the promotion of OC differentiation and maturation. Bone matrix is degraded by acid, lysosomal enzyme and protease secretion from matured OC, among which matrix metalloproteinase-3 (MMP-3) is critical to the degradation of cartilage matrix, and its expression have been found on peripheral blood and synovial fluid of AS patients. The massive mononuclear macrophages on AS synovial tissue constituted the hyperplasia of synovial tissue, whether theses cells can be identified as osteoclast precursor, do they involved the degradation of focal cartilage destruction, if the mechanism is related with MMP-3, or any differentiation related factors existed in synovial tissue still requires further investigation.
Objective: the research utilized synovial tissue from sacroiliac and iliac joints of AS patients, approach to the routes and effect of OC on cartilage damage through cultivate OC and analyze its relationship; and approach to the factors and routes of OC differentiation through observation the expression of differentiation factor TNF-αand VEGF and OB in synovial tissue of AS.
Method: synovial tissue and cartilage separated from sacroiliac joint of AS patients were prepared into paraffin and frozen section, cartilage pathological changes were observed through HE and toluidine blue stain, and mankin score was made; the distribution of TRAP+ cells as well as the positive areas on TRAP enzyme histochemical staining were recorded, and its relationship with Mankin score were inspected by Spearman correlation analysis; the expression of MMP-3 mRNA in synovial tissue were conducted by in situ hybridization, and the grayscale difference with normal tissue were compared by a image analysis system; expression of MMP on TRAP+ cells and the relationship between TRAP+ cells and MMP-3 were analyzed through enzyme histochemistry combined with in sity hybridization; the separated mononuclear macrophage in synovial tissue of AS were cultivated with normal joint cartilage in vitro, osteoclasia was inspected by HE staining and electron microscope, which further investigated the effect and route of cartilage damage from TRAP+ cells. Expression of mRNA of TNF-αand VEGF in synovial tissue were inspected by in situ hybridizatin, and the grayscale difference with normal tissue were compared by a image analysis system; a Spearman correlation analysis was conducted to determine its relationship with TRAP+ area and MMP-3mRNA; synovial fibroblast were separated and cultivated in vitro, the 3rd generation were collected for osteoblast identification: alkaline phosphatase by calcium-cobalt method, type I collagen by immunohistochemistry, calcium nodule by alizarin red staining and amount of osteocalcin by radio-immunity.
Results: 1.part cells in synovial lining and synovial-cartilage border became red on enzyme-histochemistry-TRAP staining, with negative results in control group, the difference between staining areas in two positive groups was of statistical significance (P<0.01); caritilage damage were inspected by HE and toluidine blue staining, and histopathology Mankin score were made, the score was significantly correlate with TRAP positive area (r=0.712,P=0.001). 2. MMP-3 mRNA expression by in situ hybridization observed brown particles in synovial lining and inferior cells, with negative results in control group, the grayscale comparison returned a statistical significant difference (P<0.01); the grayscale of MMP-3 mRNA positive cells, TRAP positive areas and Mankin score were correlated (r=0.491,P=0.024 ; r=0.728,P=0.001), in situ hybridization combined with enzyme histochemistry observed that brown particles within cells changed into copper-brown. 3. the separation of mononuclear macrophage in synovial tissue of AS was successful, microscopy on TRAP staining found cells in big size and multiple nucleus, and red particles in plasma; the co-cultivation of macrophage and normal joint cartilage discovered irregular shed on cartilage surface, collagen exposure and break-down in HE staining and electro microscope. 4. TNF-αand VEGF mRNA expression test by in situ hybridization observed brown particle in sinovial lining and inferior cells, with negative results in control group, the grayscale difference between two groups were of statistical significance (P<0.01), the grayscale of TNF-αmRNA positive cells correlated with TRAP positive area ratio and Mankin score (r=0.634,P=0.002;r=0.851,P=0.001), while significant correlation between gray scale of VEGF mRNA positive cells between TRAP positive area ratio and Mankin score were recored (r=0.598,P=0.004;r=0.851,P=0.001). 5. osteoblast identification of 3rd generation fibroblasts: alkaline phosphatase by calcium-cobalt revealed black particles in fibroblasts, negative in control group; while yellow particles were found in type I collagen test by immunohistochemistry, negative in control group; the amount of osteocalcin in experiment group is significantly different with control group by radio-immuno method (P<0.01); on day 18 of fibroblast cultivation, calcium nodule formation was observed by alizarin red staining.
Conclusion: 1. the presence of OC in synovial tissue correlated with cartilage damage in AS, the expression of MMP-3 is a key route. 2. The synovial tissue in AS formed a environment for the differentiation of OC: osteoblast-like characterization of fibroblasts, expression of TNF and VEGF are major environmental factors for OC differentiation. 3. the differentiation of fibroblasts into osteoblasts in AS also contributed to the pathological bone formation.
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