联合应用重组人骨保护素和二膦酸盐抑制破骨细胞及治疗骨质疏松的实验研究
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摘要
骨保护素(Osteoprotegerin,OPG)是破骨细胞局部抑制因子,已成为极具潜力的治疗骨吸收类疾病药物。它作为破骨细胞分化和活性关键因子RANKL(Receptor Activator of Nuclear Factor-κB Ligand,核因子—κB受体激活因子配基)的可溶性假受体,竞争性抑制RANKL与破骨细胞及前体细胞胞膜受体RANK(Receptor Activator of Nuclear Factor-κB)结合,影响破骨细胞及前体细胞的信号传导过程,从而抑制破骨细胞活性。在治疗骨质疏松的动物实验和临床应用中,OPG均表现出卓越的抗骨吸收效能。
虽然OPG抑制破骨细胞效果显著,但其价格较昂贵(例如,酵母表达OPG目前市价约为$4.2/mg),若长期大量使用患者花费较高。而且由于人类遗传多态性的缘故,长期使用OPG有引发异常免疫反应的可能。为降低OPG生产成本、减轻患者负担并减少可能发生的副作用,本研究一方面构建新OPG-Fc表达菌株,希望减少蛋白发酵过程的降解;另一方面试图找到一种能够与OPG联合使用的药物,从而更有效的联合抑制破骨细胞。
本实验采用酵母菌SMD1168构建新的OPG-Fc表达菌株——SMD1168/OPG-Fc,并且进行了两种表达菌株(X33/OPG-Fc和SMD1168/OPG-Fc)的发酵时程和OPG-Fc产量方面的对比研究。证明X33/OPG-Fc在更短的时间内使OPG-Fc产量达到要求;而SMD1168/OPG-Fc虽发酵过程较慢,但产生的杂蛋白较少,发酵获得的OPG-Fc纯度更高。X33/OPG-Fc和SMD1168/OPG-Fc发酵获得的OPG-Fc经纯化后均可有效发挥抑制破骨细胞的活性。
在此基础上本实验进行了联合应用OPG和阿伦膦酸钠(alendronate,ALN)抑制破骨细胞的研究。二膦酸盐(bisphosphonate,BPs)类药物,如ALN可抑制破骨细胞的甲羟戊酸途径(mevalonate Pathway)中FPP合成酶(farnesyl diphosphate synthase)从而有效地抑制破骨细胞分化并引起破骨细胞凋亡。体外细胞实验显示:联合应用rhOPG-Fc和ALN可更强烈地抑制破骨细胞前体细胞分化,更有效地抑制成熟破骨细胞及原代骨巨细胞瘤多核巨细胞的骨吸收功能。这两种药物在抑制破骨细胞分化和功能方面都表现出明显地交互作用,且联合应用1/2剂量的rhOPG-Fc+ALN的抑制破骨细胞和原代骨巨细
Osteoprotegerin is an inhibitory factor of Osteoclasts. It has been the potential treatment for osteoporosis. OPG is a key factor acting as a negative regulator against osteoclasts' differentiation and activation, through competitive inhibition of RANKL's binding to its membrane receptor - Receptor Activator of Nuclear Factor-κB (RANK). In that way, OPG interferes with the signal pathway of Osteoclasts and their ancestor, and showing excellent effect of inhibiting bone resorption function not only in animal experiments but also in clinical application.Although it can inhibit Osteoclasts, OPG is expensive (For example, the market price of OPG expressed by yeast is $ 4.2/mg). It will cost a lot of monkey for the patients if they use OPG for a long period of time. On the other hand, abnormal allery immunologic reaction could be found in those patients who use the OPG for a long time. For lighting the economic load of patients and lowering the side effect, we reconstructed a new OPG-Fc yeast expression system and try to combine OPG and some other drugs to enhance the effect of inhibiting the Osteoclasts.In this study, we used the methylotrophic yeast Pichia pastoris-SMD1168 to rebuild SMD1168/OPG-Fc system. Comparing with the former system we had already built--X33/OPG-Fc, SMD1168 grow slower but express less non-target protein. And the OPG-Fc produced by both SMD1168 and X33 has inhibited Osteoclasts function.In the experiment, we accessed combined effect of rhOPG-Fc and Alendronate (ALN) on osteoclasts. ALN can inhibite farnesyl diphosphate synthase of Mevalonate Pathway of osteoclast, and induce these cells' apoptosis. The results showed that combination of rhOPG-FC and ALN had the great inhibition effect on mature osteoclasts and their precursor cells' differentiation. And half dose of OPG + ALN had greater effect on inhibiting the Osteoclast than using OPG or ALN only. In an other experiment, OPG + ALN also showed the
虽然OPG抑制破骨细胞效果显著,但其价格较昂贵(例如,酵母表达OPG目前市价约为$4.2/mg),若长期大量使用患者花费较高。而且由于人类遗传多态性的缘故,长期使用OPG有引发异常免疫反应的可能。为降低OPG生产成本、减轻患者负担并减少可能发生的副作用,本研究一方面构建新OPG-Fc表达菌株,希望减少蛋白发酵过程的降解;另一方面试图找到一种能够与OPG联合使用的药物,从而更有效的联合抑制破骨细胞。
本实验采用酵母菌SMD1168构建新的OPG-Fc表达菌株——SMD1168/OPG-Fc,并且进行了两种表达菌株(X33/OPG-Fc和SMD1168/OPG-Fc)的发酵时程和OPG-Fc产量方面的对比研究。证明X33/OPG-Fc在更短的时间内使OPG-Fc产量达到要求;而SMD1168/OPG-Fc虽发酵过程较慢,但产生的杂蛋白较少,发酵获得的OPG-Fc纯度更高。X33/OPG-Fc和SMD1168/OPG-Fc发酵获得的OPG-Fc经纯化后均可有效发挥抑制破骨细胞的活性。
在此基础上本实验进行了联合应用OPG和阿伦膦酸钠(alendronate,ALN)抑制破骨细胞的研究。二膦酸盐(bisphosphonate,BPs)类药物,如ALN可抑制破骨细胞的甲羟戊酸途径(mevalonate Pathway)中FPP合成酶(farnesyl diphosphate synthase)从而有效地抑制破骨细胞分化并引起破骨细胞凋亡。体外细胞实验显示:联合应用rhOPG-Fc和ALN可更强烈地抑制破骨细胞前体细胞分化,更有效地抑制成熟破骨细胞及原代骨巨细胞瘤多核巨细胞的骨吸收功能。这两种药物在抑制破骨细胞分化和功能方面都表现出明显地交互作用,且联合应用1/2剂量的rhOPG-Fc+ALN的抑制破骨细胞和原代骨巨细
Osteoprotegerin is an inhibitory factor of Osteoclasts. It has been the potential treatment for osteoporosis. OPG is a key factor acting as a negative regulator against osteoclasts' differentiation and activation, through competitive inhibition of RANKL's binding to its membrane receptor - Receptor Activator of Nuclear Factor-κB (RANK). In that way, OPG interferes with the signal pathway of Osteoclasts and their ancestor, and showing excellent effect of inhibiting bone resorption function not only in animal experiments but also in clinical application.Although it can inhibit Osteoclasts, OPG is expensive (For example, the market price of OPG expressed by yeast is $ 4.2/mg). It will cost a lot of monkey for the patients if they use OPG for a long period of time. On the other hand, abnormal allery immunologic reaction could be found in those patients who use the OPG for a long time. For lighting the economic load of patients and lowering the side effect, we reconstructed a new OPG-Fc yeast expression system and try to combine OPG and some other drugs to enhance the effect of inhibiting the Osteoclasts.In this study, we used the methylotrophic yeast Pichia pastoris-SMD1168 to rebuild SMD1168/OPG-Fc system. Comparing with the former system we had already built--X33/OPG-Fc, SMD1168 grow slower but express less non-target protein. And the OPG-Fc produced by both SMD1168 and X33 has inhibited Osteoclasts function.In the experiment, we accessed combined effect of rhOPG-Fc and Alendronate (ALN) on osteoclasts. ALN can inhibite farnesyl diphosphate synthase of Mevalonate Pathway of osteoclast, and induce these cells' apoptosis. The results showed that combination of rhOPG-FC and ALN had the great inhibition effect on mature osteoclasts and their precursor cells' differentiation. And half dose of OPG + ALN had greater effect on inhibiting the Osteoclast than using OPG or ALN only. In an other experiment, OPG + ALN also showed the
引文
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