人骨保护素（hOPG）基因的生物活性及防治人工关节无菌性松动的实验研究
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摘要
研究背景:近年来研究发现OPG/RANKL/RANK系统在破骨细胞分化增殖中起着重要的作用,这是骨骼生理研究领域的重大进展之一。骨保护素(osteoprotegerin, OPG)是1997年发现的分泌型糖蛋白,能抑制破骨细胞分化、抑制成熟破骨细胞的活性并诱导其凋亡。成骨细胞、骨髓基质细胞及活化的T淋巴细胞均表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子如TNF-α、TGF-β、1,25(OH)2D3、雌激素、骨形态发生蛋白-2以及PTH、PGE2、糖皮质激素等均通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。
此外,随着现代人工关节技术与相关研究的飞速发展,人工关节置换术已成为治疗各种关节疾病终末期病变最普遍而有效的方法。但假体周围骨质溶解导致人工关节无菌性松动这一问题尚未得到很好的解决,成为这项技术进一步发展的瓶颈和研究热点。近十年的研究认为:人工关节无菌性松动主要与假体长期磨损或离解产生的颗粒所诱导的生物学反应有关。这生物学反应过程包括:(1)磨损颗粒的产生,(2)假体—骨界面间异物反应膜的形成,(3)破骨细胞性骨溶解,导致假体的骨性支持结构力学性能下降。尤其是破骨细胞性骨溶解在关节假体松动中的作用被认为意义重大。
研究目的:通过深入研究OPG基因的表达、生物活性以及假体周围破骨细胞性骨溶解的机制,采用基因治疗方法、应用种子细胞工程及ex vivo技术,调控OPG基因在关节松动的假体周围达到较持续的OPG蛋白表达,达到抑制假体周围破骨细胞性骨溶解的目的。可望为防治人工关节无菌性松动提供新的思路和治疗手段。
本研究工作分为以下三部分:
1. Ad-hOPG的构建、制备、及表达鉴定;
2.大鼠骨髓基质细胞(rMSCs)的生物学特性及Ad-hOPG在rMSCs中的表达;
3.转染Ad-hOPG的rMSCs对大鼠人工关节无菌性松动的防治作用。
实验方法:
Research background: These years, some researches concerned found that the osteoprotegerin/ receptor activitorof NF-kappa B ligand/ receptor activitorof nuclear factor-kappaB system(OPG/RANKL/RANK system) plays a crucial important role in osteoclast of cytodifferentiation and generation. This is a great improvement in the area of bones physiology. Osteoprotegerin(OPG) is a secretde glycoprotein which was found in 1977 which could restrain not only the differentiation of osteoclast, but the activity of mature osteoclast, what’s more, induce it’s apoptosis. Osteoblast、mesenchymal stem cell and activated T lymphocyte all could express RANKL. When RANKL was combined with RANK, which is in the surface of the osteoclast’s original cell or mature osteoblast, it could accelerate the differentiation of osteoclast and the activity of bone’s absorbtion. OPG which excreted by Osteoblast and mesenchymal stem cell can competitively bind RANK with RANKL,so OPG can block the bindation between RANK and RANKL. . Many hormones or factors in the body regulate the metabolism of bone by influence the rate of OPG/RANKL in the bone marrow’s microenvironment, such as estrogen、TNF-α、TGF-β、1,25(OH)2D3、bone morphogenetic protein-2、PTH、PGE2、glucocorticoid and so on.
Furthermore, as the flying development of modern artificial joint technique and some researches concerned, artificial joint replacement has become the most popular and effective method to treat every kinds of joint diseases’pathological changes in the last period. However, the problem that the osteolysis around the artificial joint lead to the asepsis looseness of joint have not been solved appropriately, which become the bottleneck for the further development of this technique and also a hotspot for research. The research in this decade confirm that the asepsis looseness of artificial joint due to the biology reaction which is induced by the particle, generated from long period of artificial body’s
此外,随着现代人工关节技术与相关研究的飞速发展,人工关节置换术已成为治疗各种关节疾病终末期病变最普遍而有效的方法。但假体周围骨质溶解导致人工关节无菌性松动这一问题尚未得到很好的解决,成为这项技术进一步发展的瓶颈和研究热点。近十年的研究认为:人工关节无菌性松动主要与假体长期磨损或离解产生的颗粒所诱导的生物学反应有关。这生物学反应过程包括:(1)磨损颗粒的产生,(2)假体—骨界面间异物反应膜的形成,(3)破骨细胞性骨溶解,导致假体的骨性支持结构力学性能下降。尤其是破骨细胞性骨溶解在关节假体松动中的作用被认为意义重大。
研究目的:通过深入研究OPG基因的表达、生物活性以及假体周围破骨细胞性骨溶解的机制,采用基因治疗方法、应用种子细胞工程及ex vivo技术,调控OPG基因在关节松动的假体周围达到较持续的OPG蛋白表达,达到抑制假体周围破骨细胞性骨溶解的目的。可望为防治人工关节无菌性松动提供新的思路和治疗手段。
本研究工作分为以下三部分:
1. Ad-hOPG的构建、制备、及表达鉴定;
2.大鼠骨髓基质细胞(rMSCs)的生物学特性及Ad-hOPG在rMSCs中的表达;
3.转染Ad-hOPG的rMSCs对大鼠人工关节无菌性松动的防治作用。
实验方法:
Research background: These years, some researches concerned found that the osteoprotegerin/ receptor activitorof NF-kappa B ligand/ receptor activitorof nuclear factor-kappaB system(OPG/RANKL/RANK system) plays a crucial important role in osteoclast of cytodifferentiation and generation. This is a great improvement in the area of bones physiology. Osteoprotegerin(OPG) is a secretde glycoprotein which was found in 1977 which could restrain not only the differentiation of osteoclast, but the activity of mature osteoclast, what’s more, induce it’s apoptosis. Osteoblast、mesenchymal stem cell and activated T lymphocyte all could express RANKL. When RANKL was combined with RANK, which is in the surface of the osteoclast’s original cell or mature osteoblast, it could accelerate the differentiation of osteoclast and the activity of bone’s absorbtion. OPG which excreted by Osteoblast and mesenchymal stem cell can competitively bind RANK with RANKL,so OPG can block the bindation between RANK and RANKL. . Many hormones or factors in the body regulate the metabolism of bone by influence the rate of OPG/RANKL in the bone marrow’s microenvironment, such as estrogen、TNF-α、TGF-β、1,25(OH)2D3、bone morphogenetic protein-2、PTH、PGE2、glucocorticoid and so on.
Furthermore, as the flying development of modern artificial joint technique and some researches concerned, artificial joint replacement has become the most popular and effective method to treat every kinds of joint diseases’pathological changes in the last period. However, the problem that the osteolysis around the artificial joint lead to the asepsis looseness of joint have not been solved appropriately, which become the bottleneck for the further development of this technique and also a hotspot for research. The research in this decade confirm that the asepsis looseness of artificial joint due to the biology reaction which is induced by the particle, generated from long period of artificial body’s
引文
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