聚蛋白多糖酶和MMP在OA和RA关节中的表达及其意义
发布时间：2018-07-21 15:49
【摘要】： 实验背景和目的:关节软骨破坏的重要病理表现是软骨细胞外基质(ECM)的减少,其中,聚蛋白多糖丢失是关节炎的主要病理表现之一。聚蛋白多糖可以被基质金属蛋白酶(MMP)和聚蛋白多糖酶(Aggrecanase)分别在核心蛋白球间区(IGD)的Asn~(341)-Phe~(342)位点和Glu~(373)-Ala~(374)位点降解,并分别产生FFGxx和ARGxx的新肽链。迄今为止被广泛认可的聚蛋白多糖酶有ADAMTS-4和ADAMTS-5,在MMP家族中MMP-2有广泛的作用底物,包括聚蛋白多糖和Ⅱ型胶原,MMP-3则是MMP家族中降解聚蛋白多糖最强者。尽管聚蛋白多糖酶和MMP都能裂解聚蛋白多糖的核心蛋白,而在疾病状态下哪种酶起主要作用以及聚蛋白多糖酶的上调因素仍然是近年来争论的热点问题。 有体外软骨培养体系证实最初几周降解聚蛋白多糖的酶是聚蛋白多糖酶,MMP在几周后才发挥作用,聚蛋白多糖酶在软骨降解的后期的作用尚不清楚,而且影响软骨细胞表达聚蛋白多糖酶的因素更加复杂,甚至在体外试验中的研究结果也有很大差异,比如有报道用IL-1处理培养的正常人软骨,不影响ADAMTS-4和ADAMTS-5的表达,而另有报道人软骨细胞经IL-1刺激后,增加了ADAMTS-4的表达,却不影响ADAMTS-5的表达。 骨关节炎(OA)和类风湿性关节炎(RA)都有关节软骨破坏的病理表现,但是二者的疾病性质却不相同。OA是非炎性的退行性关节炎,RA则是自身免疫性疾病,是炎性关节炎,炎症因子,如IL-1,在其中起了重要作用。对两种疾病的比较研究不仅可以比较聚蛋白多糖酶和MMP的作用,还对揭示人类疾病状态下聚蛋白多糖酶的表达调节有重要意义。本文通过对ADAMTS-4、ADAMTS-5、MMP-2和MMP-3以及它们的产物在OA和RA病变关节的软骨、滑膜和关节液中的表达比较,探讨了聚蛋白多糖酶和MMP在人类疾病状态下的不同作用以及可能的激活机制。 材料和方法:本研究所有标本均取自人类膝关节股骨髁,共有OA软骨和滑膜21例;OA关节液32例;RA软骨9例、滑膜12例、关节液28例;正常软骨3例。15例OA软骨的进行了组织块培养(5天),并在第一天应用10ug/ml的IL-1β对培养的软骨块进行了刺激。应用免疫组织化学方法观察了3例正常软骨、15例OA培养前后的软骨的ADAMTS-4的表达,比较了ADAMTS-4在不同软骨的表层、中层和深层分布;应用半定量RT-PCR方法检测了ADAMTS-4mRNA在3例正常软骨和15例OA培养前后的软骨中的表达差异。还应用免疫组织化学方法观察了ADAMTS-4和ADAMTS-5在21例OA软骨和滑膜、9例RA软骨、12例RA滑膜中的表达比例和分布区域,并进行了比较;应用酶联免疫吸附实验(ELISA)观察了ADAMTS-4、ADAMTS-5、MMP-2和MMP-3以及ARGxx和FFGxx在32例OA关节液和28例RA关节液中的表达,以ELISA吸光度(OD)值代表所测各项在关节液中的浓度,对结果进行t检验和相关性分析。 结果:在3例正常软骨的免疫组化切片中,只有极少数软骨细胞中有ADAMTS-4表达;OA软骨的ADAMTS-4表达区域主要分布在软骨表层的细胞内,中层细胞表达较少,深层软骨细胞则几乎没有表达;当OA软骨表层磨损后,ADAMTS-4阳性细胞主要分布在中层靠近关节表面的区域,靠近深层的区域很少有阳性细胞,深层的软骨细胞仍然几乎没有ADAMTS-4的表达;当软骨进一步磨损,中层也消失后,深层细胞可见增生并表达ADAMTS;当OA软骨在经过与IL-1β共同培养后,ADAMTS-4表达量增加;统计结果显示培养前后的OA软骨的表层和中层ADAMTS-4阳性细胞数均显著高于正常软骨;OA软骨培养前后相比较可见表层阳性细胞数没有差异,而培养后的OA软骨中层阳性细胞数明显增多。RT-PCR的结果显示ADAMTS-4 mRNA的表达在OA软骨中明显高于正常软骨,IL-1刺激后的软骨明显高于OA软骨。 所有OA和RA软骨中均有ADAMTS-4和ADAMTS-5的表达,但表达部位不同,ADAMTS-4、5在OA软骨中主要表达在表层,RA软骨的表层已被血管翳替代,其细胞强烈表达聚蛋白多糖酶,而且软骨中层出现阳性细胞的例数明显高于OA;OA滑膜出现ADAMTS-4和ADAMTS-5阳性细胞的比例分别为52%(11/21)和43%(9/21)与RA的50%(6/12)和58%(7/12)相比较没有显著差异。 ELISA结果显示ADAMTS-4、ADAMTS-5、MMP-2和MMP-3以及ARGxx和FFGxx在RA关节液中的浓度均高于OA关节液,结果有显著差异(p＜0.01)。RA和OA关节液中ARGxx的浓度明显高于FFGxx(p＜0.01);ADAM7S-4的浓度均高于ADAMTS-5(p＜0.01);MMP-3的浓度均高于MMP-2(p＜0.01)。OA关节液中ADAMTS-4的浓度与ARGxx成正相关(r=0.38,p＜0.05);ADAMTS-5与ARGxx以及MMP-2、MMP-3与FFGxx均无明显相关关系。RA关节液中所测各酶与其产物之间均无相关关系。 结论: (1)ADAMTS-4在严重磨损的软骨中大量表达,说明它不仅在OA早期起着重要作用,在中晚期OA的软骨降解中仍然有重要作用;抑制ADAMTS-4对治疗中晚期OA仍然有意义; (2)聚蛋白多糖酶总是集中表达在软骨的靠近关节面的区域,强烈支持OA发病机制的机械应力学说,并且说明机械应力并非简单地引起的磨损,同时可能是摩擦力激活了聚蛋白多糖酶,两者的共同作用导致了软骨的逐渐变薄; (3)关节液中ARGxx多于FFGxx提示对OA和RA关节软骨聚蛋白多糖的丢失,聚蛋白多糖酶比MMP起了更大的作用; (4)OA病变关节中软骨细胞是聚蛋白多糖酶的主要来源,在RA病变关节中聚蛋白多糖酶主要来源于软骨细胞和血管翳;OA和RA的滑膜均少量表达聚蛋白多糖酶; (5)IL-1β可以上调ADAMTS-4的基因表达,与机械摩擦力相比两者作用的部位是不同的,提示OA出现滑膜炎症时可以加速软骨的降解; (6)RA关节液中聚蛋白多糖酶和MMP以及其代谢产物均高于OA关节液,提示在人类关节炎疾病过程中炎症因子不仅参与上调MMP,同时上调了聚蛋白多糖酶的表达。
[Abstract]:Experimental background and purpose: the important pathological manifestation of articular cartilage destruction is the reduction of the cartilage extracellular matrix (ECM), in which the loss of polyproteoglycan is one of the main pathological manifestations of arthritis. Polyproteoglycan can be Asn~ (341) of matrix metalloproteinases (MMP) and polyprotease (Aggrecanase) in the core protein interzone (IGD), respectively. -Phe~ (342) site and Glu~ (373) -Ala~ (374) site degrade and produce new peptide chains of FFGxx and ARGxx respectively. So far the widely recognized polyprotease has ADAMTS-4 and ADAMTS-5. MMP-2 has a wide range of substrates in MMP family, including polyproteoglycan and type II collagen, MMP-3 is the strongest proteoglycan degradation in MMP family. Although polyprotease and MMP can both cleave the core protein of polyproteoglycan, which enzyme plays a major role in the disease state and the up-regulated factor of polyprotease is still a hot issue in recent years.
In vitro cartilage culture system confirms that the enzyme that degrades polyproteoglycan in the first few weeks is polyprotease, MMP plays a role in a few Zhou Houcai. The role of polyprotease in the late stage of cartilage degradation is not clear, and the factors that affect the expression of polyprotease in cartilage cells are more complex, even in the study in vitro. There are also great differences. For example, it is reported that the normal human cartilage treated with IL-1 does not affect the expression of ADAMTS-4 and ADAMTS-5, but the expression of ADAMTS-4 is increased after IL-1 stimulation in human chondrocytes, but it does not affect the expression of ADAMTS-5.
Osteoarthritis (OA) and rheumatoid arthritis (RA) have the pathological manifestations of articular cartilage destruction, but the two diseases are not the same as non inflammatory degenerative arthritis, and RA is autoimmune disease, inflammatory arthritis, and inflammatory factors, such as IL-1, play an important role. The comparative study of the two diseases is not only possible. To compare the effects of polyprotease and MMP, it is also important to reveal the expression of polyprotease in human disease. By comparing the expression of ADAMTS-4, ADAMTS-5, MMP-2 and MMP-3 and their products in cartilage, synovium and joint fluid of the OA and RA lesions, the polyprotease and MMP are discussed in this paper. The different roles and possible activation mechanisms of human disease.
Materials and methods: all specimens of this study were taken from the human knee joint femoral condyle, including 21 cases of OA cartilage and synovial membrane, 32 cases of OA joint fluid, 9 cases of RA cartilage, 12 cases of synovial membrane, 28 cases of articular fluid and 3 cases of OA cartilage of normal cartilage for 5 days (5 days), and the first day the 10ug/ml IL-1 beta was used to stimulate the cultured cartilaginous block. Immunohistochemistry was used to observe the expression of ADAMTS-4 in 3 normal cartilages and 15 cases of OA before and after culture. The distribution of ADAMTS-4 in the surface, middle and deep layers of different cartilage was compared. The differential expression of ADAMTS-4mRNA in 3 normal cartilages and 15 cases of OA before and after cultured OA was detected by semi quantitative RT-PCR. The expression of ADAMTS-4 and ADAMTS-5 in 21 cases of OA cartilage and synovium, 9 cases of RA cartilage and 12 cases of RA synovium were observed and compared. The expression of ADAMTS-4, ADAMTS-5, MMP-2 and MMP-3, and ARGxx and FFGxx in 32 cases of articular fluid and 28 joint fluid were observed by enzyme linked immunosorbent assay (ELISA). The absorbance (OD) value of ELISA represented the concentration of the tested substances in the joint fluid, and the results were tested by T and correlation analysis.
Results: in the immunohistochemical sections of 3 cases of normal cartilage, only a few chondrocytes were expressed in ADAMTS-4; the ADAMTS-4 expression area of OA cartilage mainly distributed in the cells of the cartilage surface, the expression of the medium layer cells was less, and the deep cartilage cells were almost not expressed. When the surface of the OA soft bone was worn, the ADAMTS-4 positive cells were mainly distributed in the cells. In the area near the surface of the joint, there are few positive cells near the deep layer, and the deep cartilage cells still have almost no expression of ADAMTS-4. When the cartilage is further worn and the middle layer is disappearing, the deep cells can be seen to proliferate and express ADAMTS; when the OA cartilage is co cultured with IL-1 beta, the expression of ADAMTS-4 is increased; the statistical results are increased. The number of ADAMTS-4 positive cells in the surface and middle layer of OA cartilage before and after culture was significantly higher than that of normal cartilage. There was no difference in the number of positive cells before and after OA cartilage culture, while the number of positive cells in the middle layer of OA cartilage increased obviously after the culture, and the result of.RT-PCR showed that the expression of ADAMTS-4 mRNA in OA cartilage was obviously higher than that of normal cartilage. Cartilage, after IL-1 stimulation, was significantly higher than that of OA cartilage.
The expression of ADAMTS-4 and ADAMTS-5 in all OA and RA cartilage, but the expression site is different, ADAMTS-4,5 is mainly expressed in the surface of OA cartilage, the surface of RA cartilage is replaced by pannus, and the cells strongly express polyprotease, and the number of positive cells in the middle cartilage layer is obviously higher than that of OA; OA synovium appears ADAMTS-4 and ADAMTS-5. The proportion of positive cells was 52% (11/21) and 43% (9/21) respectively, and there was no significant difference compared with 50% (6/12) and 58% (7/12) of RA.
ELISA results showed that the concentration of ADAMTS-4, ADAMTS-5, MMP-2 and MMP-3 as well as ARGxx and FFGxx in RA joint solution was higher than that of OA joint solution. The results showed significant difference (P < 0.01).RA and OA joint concentration was higher than that of 0.01. The concentration of ADAMTS-4 in the liquid is positively correlated with ARGxx (r=0.38, P < 0.05); ADAMTS-5 and ARGxx and MMP-2, MMP-3 and FFGxx have no significant correlation. There is no correlation between the enzymes measured in.RA joint fluid and their products.
Conclusion:
(1) the expression of ADAMTS-4 in severely worn cartilage indicates that it not only plays an important role in the early stage of OA, but also plays an important role in the degradation of cartilage in the middle and late stages of OA, and the inhibition of ADAMTS-4 is still significant for the treatment of OA in the middle and late stages.
(2) polyprotease is always concentrated in the area near the articular surface of the cartilage, strongly supports the mechanical stress of the pathogenesis of OA, and shows that the mechanical stress is not simply caused by the wear and tear, and it may be that the friction force activates the polyprotease, and the co action of the two causes the gradual thinning of the cartilage.
(3) ARGxx in the synovial fluid is more than FFGxx, suggesting that the loss of polyproteoglycan in articular cartilage of OA and RA is more significant than that of MMP.
(4) the chondrocytes in the OA lesions are the main source of polyprotease. In the RA lesion, polyprotease is mainly derived from the chondrocytes and pannus, and the synovial membrane of OA and RA expresses a small amount of polyprotease.
(5) IL-1 beta can up regulate the gene expression of ADAMTS-4, which is different from the mechanical friction, suggesting that the degradation of cartilage can be accelerated when OA occurs in synovitis.
(6) both polyprotease and MMP and their metabolites in RA joint fluid are higher than those of OA joint fluid. It is suggested that in the process of human arthritis, inflammatory factors not only participate in the up regulation of MMP, but also up the expression of polyprotease.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R341
本文编号：2136008
[Abstract]:Experimental background and purpose: the important pathological manifestation of articular cartilage destruction is the reduction of the cartilage extracellular matrix (ECM), in which the loss of polyproteoglycan is one of the main pathological manifestations of arthritis. Polyproteoglycan can be Asn~ (341) of matrix metalloproteinases (MMP) and polyprotease (Aggrecanase) in the core protein interzone (IGD), respectively. -Phe~ (342) site and Glu~ (373) -Ala~ (374) site degrade and produce new peptide chains of FFGxx and ARGxx respectively. So far the widely recognized polyprotease has ADAMTS-4 and ADAMTS-5. MMP-2 has a wide range of substrates in MMP family, including polyproteoglycan and type II collagen, MMP-3 is the strongest proteoglycan degradation in MMP family. Although polyprotease and MMP can both cleave the core protein of polyproteoglycan, which enzyme plays a major role in the disease state and the up-regulated factor of polyprotease is still a hot issue in recent years.
In vitro cartilage culture system confirms that the enzyme that degrades polyproteoglycan in the first few weeks is polyprotease, MMP plays a role in a few Zhou Houcai. The role of polyprotease in the late stage of cartilage degradation is not clear, and the factors that affect the expression of polyprotease in cartilage cells are more complex, even in the study in vitro. There are also great differences. For example, it is reported that the normal human cartilage treated with IL-1 does not affect the expression of ADAMTS-4 and ADAMTS-5, but the expression of ADAMTS-4 is increased after IL-1 stimulation in human chondrocytes, but it does not affect the expression of ADAMTS-5.
Osteoarthritis (OA) and rheumatoid arthritis (RA) have the pathological manifestations of articular cartilage destruction, but the two diseases are not the same as non inflammatory degenerative arthritis, and RA is autoimmune disease, inflammatory arthritis, and inflammatory factors, such as IL-1, play an important role. The comparative study of the two diseases is not only possible. To compare the effects of polyprotease and MMP, it is also important to reveal the expression of polyprotease in human disease. By comparing the expression of ADAMTS-4, ADAMTS-5, MMP-2 and MMP-3 and their products in cartilage, synovium and joint fluid of the OA and RA lesions, the polyprotease and MMP are discussed in this paper. The different roles and possible activation mechanisms of human disease.
Materials and methods: all specimens of this study were taken from the human knee joint femoral condyle, including 21 cases of OA cartilage and synovial membrane, 32 cases of OA joint fluid, 9 cases of RA cartilage, 12 cases of synovial membrane, 28 cases of articular fluid and 3 cases of OA cartilage of normal cartilage for 5 days (5 days), and the first day the 10ug/ml IL-1 beta was used to stimulate the cultured cartilaginous block. Immunohistochemistry was used to observe the expression of ADAMTS-4 in 3 normal cartilages and 15 cases of OA before and after culture. The distribution of ADAMTS-4 in the surface, middle and deep layers of different cartilage was compared. The differential expression of ADAMTS-4mRNA in 3 normal cartilages and 15 cases of OA before and after cultured OA was detected by semi quantitative RT-PCR. The expression of ADAMTS-4 and ADAMTS-5 in 21 cases of OA cartilage and synovium, 9 cases of RA cartilage and 12 cases of RA synovium were observed and compared. The expression of ADAMTS-4, ADAMTS-5, MMP-2 and MMP-3, and ARGxx and FFGxx in 32 cases of articular fluid and 28 joint fluid were observed by enzyme linked immunosorbent assay (ELISA). The absorbance (OD) value of ELISA represented the concentration of the tested substances in the joint fluid, and the results were tested by T and correlation analysis.
Results: in the immunohistochemical sections of 3 cases of normal cartilage, only a few chondrocytes were expressed in ADAMTS-4; the ADAMTS-4 expression area of OA cartilage mainly distributed in the cells of the cartilage surface, the expression of the medium layer cells was less, and the deep cartilage cells were almost not expressed. When the surface of the OA soft bone was worn, the ADAMTS-4 positive cells were mainly distributed in the cells. In the area near the surface of the joint, there are few positive cells near the deep layer, and the deep cartilage cells still have almost no expression of ADAMTS-4. When the cartilage is further worn and the middle layer is disappearing, the deep cells can be seen to proliferate and express ADAMTS; when the OA cartilage is co cultured with IL-1 beta, the expression of ADAMTS-4 is increased; the statistical results are increased. The number of ADAMTS-4 positive cells in the surface and middle layer of OA cartilage before and after culture was significantly higher than that of normal cartilage. There was no difference in the number of positive cells before and after OA cartilage culture, while the number of positive cells in the middle layer of OA cartilage increased obviously after the culture, and the result of.RT-PCR showed that the expression of ADAMTS-4 mRNA in OA cartilage was obviously higher than that of normal cartilage. Cartilage, after IL-1 stimulation, was significantly higher than that of OA cartilage.
The expression of ADAMTS-4 and ADAMTS-5 in all OA and RA cartilage, but the expression site is different, ADAMTS-4,5 is mainly expressed in the surface of OA cartilage, the surface of RA cartilage is replaced by pannus, and the cells strongly express polyprotease, and the number of positive cells in the middle cartilage layer is obviously higher than that of OA; OA synovium appears ADAMTS-4 and ADAMTS-5. The proportion of positive cells was 52% (11/21) and 43% (9/21) respectively, and there was no significant difference compared with 50% (6/12) and 58% (7/12) of RA.
ELISA results showed that the concentration of ADAMTS-4, ADAMTS-5, MMP-2 and MMP-3 as well as ARGxx and FFGxx in RA joint solution was higher than that of OA joint solution. The results showed significant difference (P < 0.01).RA and OA joint concentration was higher than that of 0.01. The concentration of ADAMTS-4 in the liquid is positively correlated with ARGxx (r=0.38, P < 0.05); ADAMTS-5 and ARGxx and MMP-2, MMP-3 and FFGxx have no significant correlation. There is no correlation between the enzymes measured in.RA joint fluid and their products.
Conclusion:
(1) the expression of ADAMTS-4 in severely worn cartilage indicates that it not only plays an important role in the early stage of OA, but also plays an important role in the degradation of cartilage in the middle and late stages of OA, and the inhibition of ADAMTS-4 is still significant for the treatment of OA in the middle and late stages.
(2) polyprotease is always concentrated in the area near the articular surface of the cartilage, strongly supports the mechanical stress of the pathogenesis of OA, and shows that the mechanical stress is not simply caused by the wear and tear, and it may be that the friction force activates the polyprotease, and the co action of the two causes the gradual thinning of the cartilage.
(3) ARGxx in the synovial fluid is more than FFGxx, suggesting that the loss of polyproteoglycan in articular cartilage of OA and RA is more significant than that of MMP.
(4) the chondrocytes in the OA lesions are the main source of polyprotease. In the RA lesion, polyprotease is mainly derived from the chondrocytes and pannus, and the synovial membrane of OA and RA expresses a small amount of polyprotease.
(5) IL-1 beta can up regulate the gene expression of ADAMTS-4, which is different from the mechanical friction, suggesting that the degradation of cartilage can be accelerated when OA occurs in synovitis.
(6) both polyprotease and MMP and their metabolites in RA joint fluid are higher than those of OA joint fluid. It is suggested that in the process of human arthritis, inflammatory factors not only participate in the up regulation of MMP, but also up the expression of polyprotease.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R341
【参考文献】
相关期刊论文 前2条
1 管剑龙,施桂英;基质金属蛋白酶与骨关节炎[J];中华风湿病学杂志;2000年01期
2 娄思权;骨关节炎的病理与发病因素[J];中华骨科杂志;1996年01期
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