雌激素与骨性关节炎发病机制关系的研究
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摘要
骨性关节炎(osteoarthritis,OA)是严重影响中老年人健康的一种疾病,其发病机制复杂,涉及年龄、性别、遗传、工作等多个因素。流行病学调查显示,雌激素可能在OA发病机制中发挥重要作用。而作为成熟软骨内唯一种类细胞,软骨细胞的正常功能对于维持软骨正常性状非常重要,软骨细胞凋亡在OA病理改变中具有核心作用。本文利用Wister大鼠模型,在体内水平研究雌激素水平变化与软骨细胞凋亡的关系;体外培养兔软骨细胞,在细胞水平研究雌激素浓度与软骨细胞凋亡的关系。我们设计以下实验:1.利用双侧卵巢切除术建立去势大鼠模型,术后第6w、10w研究软骨病理改变、软骨细胞凋亡率和不同凋亡相关基因蛋白表达情况,观察去势后雌激素水平降低对软骨病理改变及软骨细胞凋亡的影响,并了解凋亡相关基因Bcl-2、Bax、iNOS、Fas、Caspase-3在此过程中可能的作用;2.利用双侧卵巢切除术建立去势大鼠模型,同时Hulth法建立大鼠OA模型,术后第6w、10w研究软骨病理改变、软骨细胞凋亡率和iNOS基因蛋白表达情况,观察雌激素水平降低对OA病理发展的影响,以及NO在其中可能发挥的作用;3.取生长良好的第二代软骨细胞,不同浓度雌激素预处理2h后,应用NO供体硝普钠诱导体外软骨细胞凋亡,观察不同浓度雌激素对软骨细胞的保护作用,Western blot观察凋亡相关蛋白的表达情况。从实验中我们观察到: 1.去势能够导致大鼠膝关节软骨退化,出现OA样改变;雌激素水平降低后软骨细胞凋亡增加,相应的凋亡相关基因iNOS、Fas、Bax、Caspase-3表达增强,而Bcl-2表达减弱;软骨细胞凋亡增加可能在软骨病理改变中发挥重要作用;2、去势加剧OA动物模型软骨改变,软骨细胞凋亡增加,相关的凋亡基因iNOS表达增加,可能在其中发挥重要作用;3、0.2mM硝普钠作用于软骨细胞培养体系24h,能够引起软骨细胞的明显凋亡,而10-3~10-9mM雌激素预作用2h能够保护软骨细胞,减少软骨细胞的凋亡。Western blotting显示雌激素预作用组较无雌激素组,凋亡相关蛋白Bcl-2表达增加,Bax、Caspase-3表达降低,预示着在对软骨细胞的保护过程中发挥着重要的作用。我们通过体内、体外两个途径,验证了雌激素水平变化与软骨细胞凋亡及透明软骨组织退化的关系,证实雌激素可能在OA病理改变中发挥重要作用,推测为雌激素水平降低后,雌激素对软骨细胞的保护作用降低,异常的力学刺激等因素进而引起软骨细胞凋亡,并进而诱导OA出现、发展。
Purposes: OA is the most prevalent disease in middle-aged and older,a heavy burden to society, and the mechanisms of which is still not known. With the aging of the human society, OA increase rapidly. To the medical workers, especially health care workers, it is necessary to further study the pathogenesis of OA. Epidemiological studies have found that in post-menopause women the incidence of OA increase quickly, so whether post-menopausal or not is a watershed for women suffering from OA, suggesting that changes in estrogen levels may play an important role in the pathogenesis of OA. OA mainly manifested as pathological degradation of articular cartilage, and synovial membrane and subchondral bone change. As the only cell type in the mature cartilage tissue, increased chondrocyte apoptosis is considered the core of the pathogenesis of OA. NO and Fas pathways are two main channels of chondrocyte apoptosis. Others including Bcl-2, Bax, Caspase-3 and other apoptosis regulatory proteins also play important roles. Our research focuses on the relationship between changes in the level of estrogen and chondrocyte apoptosis and the pathological changes, we have assumed: First, if the level of estrogen decrease , chondrocyte apoptosis may increase through Fas and NO passways, leading to articular cartilage degradation, and may aggravate the pathological changes in the course of OA, a series of apoptosis regulatory proteins may play important roles. Second, estrogen bind to the estrogen receptors in chondrocyte surface, regulating the expression of cellular proteins, which may play a protective effect on cartilage cells. Our experiment is set up to verify whether the above assumptions, as well as the mechanisms.
Methods: We designed the following tests: First, bilateral ovariectomy in female rat as ovariectomized model, after 6 weeks, 10weeks, the the knees from different rats, 5μm sections after decalcification, TUNEL staining for apoptosis rates, HE staining and toluidine blue staining for Mankin score to observe the pathological changes in articular cartilage, the cartilage slices were observed by immunohistochemical expression for apoptosis-related proteins, to understand the links between the level of estrogen and chondrocyte apoptosis and cartilage pathological changes, as well as apoptosis-related proteins; Second, Hulth method to establishe OA model in female rats, simultaneously bilateral ovariectomied to establisht the OVX models.After 6w, 10w we take the rat knees, and make TUNEL staining for the rate of chondrocyte apoptosis, HE staining and toluidine blue staining for Mankin score ,immunohistochemical staining for the expression of iNOS ,to verify if the level of estrogen decrease after OVX will reduce chondrocyte apoptosis and the original impact of the pathological changes of OA, and reveale if NO change in articular cartilage may be the impact of OA-like changes; Third, take the rabbit knee joint chondrocytes in primary culture cells, using the sodium nitroprusside(SNP), a nitric oxide donor to the cell culture system to establish the in vitro model of chondrocyte apoptosis .To observe if precultured with 10-3 ~ 10-9mM estrogen for 2 hours will change chondrocyte apoptosis rates and to reveal its possible mechanisms.
Results: We found that: I: In ovariectomized rats ,articular cartilage fibrosis and cystic changes,can be seen with HE staining , toluidine blue staining of articular cartilage can even see the loss of dye staining shallow. Mankin scores were significantly higher than sham-operated group, and estrogen replacement therapy can reduce the Mankin score, improve the changes of articular cartilage. TUNEL detection see OVX increase the rate of chondrocyte apoptosis, which may play an important role in the OA pathological changes. Immuno- histochemical staining showed that OVX groups iNOS, Fas, Bax, Caspase-3- positive cells increase and Bcl-2-positive cells decrease, that means NO and Fas apoptosis pathways may be involved in OA. Apoptosis regulatory proteins Bcl-2, Bax, Caspase-3 may play important roles too; II: OVX can significantly aggravate OA changes of the articular cartilage in female rat models, the Mankin score was significantly higher than non-OVX rats, iNOS staining show that OVX increase iNOS protein expression. TUNEL staining also verify chondrocyte apoptosis increased, perhaps due to lower estrogen levels, which exacerbates the changes of articular cartilage; NO level increase in the cartilage tissue may be one reason. Third, 2mmol / L SNP can induce in vitro apoptosis of rabbit chondrocytes, and 10-3 ~ 10-9mM estrogen can inhibit this effect. That estrogen may bind to ERs ,regulate apoptosis-related proteins such as Bcl-2, Bax, Caspase-3 expression and reduce NO-induced chondrocyte apoptosis.
Conclusions: Bilateral ovariectomize in female rats can successfully establish OA model.We have found that reduce the level of estrogen will induce chondrocyte apoptosis, and cartilage degradation in animals, resulting in OA-like changes, and further studies confirmed chondrocytes apoptosis through NO and Fas ways , a variety of apoptosis and apoptosis-related proteins such Bcl-2、Bax、Caspase-3 play a regulatory role. We have found that reducing the level of estrogen will increase the original OA animals change, and further studied the possibility of occurrence of the process mechanisms. Chondrocytes cultured in the system by precultured with different concentrations of estrogen, we found estrogen can prevent the SNP-induced chondrocyte apoptosis, which may play an protective effect on chondrocytes in OA pathogenesis.
From our experiments, we speculated that estrogen levels after ovariectomy reduced may lower the protective effect from attacks outside or may be induced by increased apoptosis of articular cartilage, chondrocyte apoptosis is exacerbated by increased intra-articular changes in physiology, the results may lead to the degradation of articular cartilage and thus give rise to OA lesions. The application of exogenous estrogen to chondrocytes may reduce apoptosis and cartilage degeneration, inhibit the development of OA pathology. Therefore, estrogen application in post-menpause women should be a routine treatment for the prevention and treatment of OA.
Of course, in our tests, we also found that application of exogenous estrogen, can only reduce apoptosis in cartilage and ease the development of OA pathology and can not preven OA completely, so we think that there should be other mechanisms play important roles, it is necessary to continue more researches.
Purposes: OA is the most prevalent disease in middle-aged and older,a heavy burden to society, and the mechanisms of which is still not known. With the aging of the human society, OA increase rapidly. To the medical workers, especially health care workers, it is necessary to further study the pathogenesis of OA. Epidemiological studies have found that in post-menopause women the incidence of OA increase quickly, so whether post-menopausal or not is a watershed for women suffering from OA, suggesting that changes in estrogen levels may play an important role in the pathogenesis of OA. OA mainly manifested as pathological degradation of articular cartilage, and synovial membrane and subchondral bone change. As the only cell type in the mature cartilage tissue, increased chondrocyte apoptosis is considered the core of the pathogenesis of OA. NO and Fas pathways are two main channels of chondrocyte apoptosis. Others including Bcl-2, Bax, Caspase-3 and other apoptosis regulatory proteins also play important roles. Our research focuses on the relationship between changes in the level of estrogen and chondrocyte apoptosis and the pathological changes, we have assumed: First, if the level of estrogen decrease , chondrocyte apoptosis may increase through Fas and NO passways, leading to articular cartilage degradation, and may aggravate the pathological changes in the course of OA, a series of apoptosis regulatory proteins may play important roles. Second, estrogen bind to the estrogen receptors in chondrocyte surface, regulating the expression of cellular proteins, which may play a protective effect on cartilage cells. Our experiment is set up to verify whether the above assumptions, as well as the mechanisms.
Methods: We designed the following tests: First, bilateral ovariectomy in female rat as ovariectomized model, after 6 weeks, 10weeks, the the knees from different rats, 5μm sections after decalcification, TUNEL staining for apoptosis rates, HE staining and toluidine blue staining for Mankin score to observe the pathological changes in articular cartilage, the cartilage slices were observed by immunohistochemical expression for apoptosis-related proteins, to understand the links between the level of estrogen and chondrocyte apoptosis and cartilage pathological changes, as well as apoptosis-related proteins; Second, Hulth method to establishe OA model in female rats, simultaneously bilateral ovariectomied to establisht the OVX models.After 6w, 10w we take the rat knees, and make TUNEL staining for the rate of chondrocyte apoptosis, HE staining and toluidine blue staining for Mankin score ,immunohistochemical staining for the expression of iNOS ,to verify if the level of estrogen decrease after OVX will reduce chondrocyte apoptosis and the original impact of the pathological changes of OA, and reveale if NO change in articular cartilage may be the impact of OA-like changes; Third, take the rabbit knee joint chondrocytes in primary culture cells, using the sodium nitroprusside(SNP), a nitric oxide donor to the cell culture system to establish the in vitro model of chondrocyte apoptosis .To observe if precultured with 10-3 ~ 10-9mM estrogen for 2 hours will change chondrocyte apoptosis rates and to reveal its possible mechanisms.
Results: We found that: I: In ovariectomized rats ,articular cartilage fibrosis and cystic changes,can be seen with HE staining , toluidine blue staining of articular cartilage can even see the loss of dye staining shallow. Mankin scores were significantly higher than sham-operated group, and estrogen replacement therapy can reduce the Mankin score, improve the changes of articular cartilage. TUNEL detection see OVX increase the rate of chondrocyte apoptosis, which may play an important role in the OA pathological changes. Immuno- histochemical staining showed that OVX groups iNOS, Fas, Bax, Caspase-3- positive cells increase and Bcl-2-positive cells decrease, that means NO and Fas apoptosis pathways may be involved in OA. Apoptosis regulatory proteins Bcl-2, Bax, Caspase-3 may play important roles too; II: OVX can significantly aggravate OA changes of the articular cartilage in female rat models, the Mankin score was significantly higher than non-OVX rats, iNOS staining show that OVX increase iNOS protein expression. TUNEL staining also verify chondrocyte apoptosis increased, perhaps due to lower estrogen levels, which exacerbates the changes of articular cartilage; NO level increase in the cartilage tissue may be one reason. Third, 2mmol / L SNP can induce in vitro apoptosis of rabbit chondrocytes, and 10-3 ~ 10-9mM estrogen can inhibit this effect. That estrogen may bind to ERs ,regulate apoptosis-related proteins such as Bcl-2, Bax, Caspase-3 expression and reduce NO-induced chondrocyte apoptosis.
Conclusions: Bilateral ovariectomize in female rats can successfully establish OA model.We have found that reduce the level of estrogen will induce chondrocyte apoptosis, and cartilage degradation in animals, resulting in OA-like changes, and further studies confirmed chondrocytes apoptosis through NO and Fas ways , a variety of apoptosis and apoptosis-related proteins such Bcl-2、Bax、Caspase-3 play a regulatory role. We have found that reducing the level of estrogen will increase the original OA animals change, and further studied the possibility of occurrence of the process mechanisms. Chondrocytes cultured in the system by precultured with different concentrations of estrogen, we found estrogen can prevent the SNP-induced chondrocyte apoptosis, which may play an protective effect on chondrocytes in OA pathogenesis.
From our experiments, we speculated that estrogen levels after ovariectomy reduced may lower the protective effect from attacks outside or may be induced by increased apoptosis of articular cartilage, chondrocyte apoptosis is exacerbated by increased intra-articular changes in physiology, the results may lead to the degradation of articular cartilage and thus give rise to OA lesions. The application of exogenous estrogen to chondrocytes may reduce apoptosis and cartilage degeneration, inhibit the development of OA pathology. Therefore, estrogen application in post-menpause women should be a routine treatment for the prevention and treatment of OA.
Of course, in our tests, we also found that application of exogenous estrogen, can only reduce apoptosis in cartilage and ease the development of OA pathology and can not preven OA completely, so we think that there should be other mechanisms play important roles, it is necessary to continue more researches.
引文
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