PTD-SOD融合蛋白对顺铂所致细胞损伤的防护效应

发布时间：2019-06-05 23:07

【摘要】：顺铂是最常使用的化疗药,能杀死多种肿瘤细胞,但顺铂同时又对多种器官具有毒副作用,尤其是肝脏。顺铂导致的肝脏毒性机制至今尚未完全明了,但顺铂引起的细胞内ROS量的增加是其导致肝损伤的重要原因之一。本所构建的PTD-SOD融合蛋白能以活性蛋白的形式跨膜进入细胞内,清除细胞内多余的ROS,减轻氧化应激所致损伤。本课题分别建立了顺铂损伤的细胞模型和动物模型,探讨了PTD-SOD融合蛋白对体外和体内顺铂损伤的防护效应。体外实验结果表明：顺铂会在肝细胞内诱导产生ROS,上调TNF-α、COX-2和细胞色素C的表达量,显著增加损伤细胞MDA含量,显著降低SOD活力、GST活力和T-AOC能力,显著降低细胞抗氧化体系的抗氧化能力,从而抑制损伤细胞的增殖能力,导致细胞凋亡。预给予天然SOD对顺铂所致的各项细胞损伤基本无保护作用。预给予PTD-SOD则可显著降低损伤细胞内多余的ROS,使损伤细胞TNF-α、COX-2、细胞色素C表达量下调,而Bcl-2表达量上调,显著降低损伤细胞MDA含量,显著增加SOD活力、GST活力和T-AOC能力,从而显著增加损伤细胞的增殖能力,抑制损伤细胞的凋亡。由于PTD-SOD是专一性清除超氧阴离子的酶,由本实验结果可推测顺铂诱导细胞产生的ROS中包含超氧阴离子,且其可能是顺铂引起细胞损伤的重要原因。PTD-SOD通过清除损伤细胞胞内多余的超氧阴离子,改变该细胞氧化应激状态并调节细胞凋亡信号通路,从而实现细胞防护效应。在体外实验的基础上,我们进行了初步的活体实验考察PTD-SOD对顺铂所致的肝损伤的保护效应。实验结果表明：顺铂会显著增加损伤小鼠肝脏的MDA含量,显著降低SOD活力、GST活力和GSG-Px活力,从而损伤肝脏,降低小鼠体重。给予PTD-SOD可显著降低损伤小鼠肝脏的MDA含量,显著提高SOD水平、GST活力、GSH-Px活力,显著增强损伤小鼠抗氧化能力,提高小鼠的生存率,对肝脏具有很好的保护效果,促进小鼠生长。综上所述,PTD-SOD融合蛋白无论在细胞水平还是活体水平,都表现出显著的抗顺铂损伤能力。此外,PTD-SOD已证实毒副作用小,体内分布广,而且具有优异的放射防护能力,因此,PTD-SOD有望成为优质的新型肿瘤放化疗防护药物。
[Abstract]:DDP is the most commonly used chemotherapeutic drug, which can kill a variety of tumor cells, but it also has toxic and side effects on a variety of organs, especially the liver. The mechanism of hepatotoxicity induced by DDP has not been fully understood, but the increase of intracellular ROS induced by DDP is one of the important causes of liver injury. The constructed PTD-SOD fusion protein can enter the cell through the membrane in the form of active protein, and remove the excess ROS, in the cell to alleviate the damage caused by oxidative stress. In this study, the cell model and animal model of platinum injury were established, and the protective effects of PTD-SOD fusion protein on platinum injury in vitro and in vivo were investigated. The results of in vitro experiments showed that platinum could induce ROS, to up-regulate the expression of TNF- 伪, COX-2 and cytochrome C in hepatocytes, significantly increase the content of MDA in injured cells, and significantly decrease the activity of SOD, the activity of GST and the ability of T-AOC. It can significantly reduce the antioxidant capacity of the cell antioxidant system, which can inhibit the proliferation of injured cells and lead to apoptosis. Preadministration of natural SOD had no protective effect on cell injury induced by DDP. Pre-administration of PTD-SOD could significantly decrease the excess ROS, in injured cells and down-regulate the expression of TNF- 伪 and COX-2, while the expression of Bcl-2 was up-regulated, the content of MDA in injured cells was significantly decreased, and the activity of SOD was significantly increased. GST activity and T-AOC ability significantly increased the proliferation of injured cells and inhibited the apoptosis of injured cells. Because PTD-SOD is a specific enzyme for scavenging superoxide anion, it can be inferred from the results of this experiment that the ROS produced by cells induced by DDP contains superoxide anion. It may be an important cause of cell injury induced by DDP. PTD-SOD can change the oxidative stress state of the injured cells and regulate the apoptosis signal pathway by removing the excess oxygen anions in the injured cells, so as to achieve the cellular protective effect. On the basis of in vitro experiments, we conducted a preliminary in vivo experiment to investigate the protective effect of PTD-SOD on liver injury induced by DDP. The results showed that DDP could significantly increase the content of MDA and decrease the activity of SOD, GST and GSG-Px in the liver of injured mice, thus damaging the liver and reducing the body weight of mice. Administration of PTD-SOD significantly decreased the content of MDA in the liver of injured mice, significantly increased the level of SOD, GST activity, GSH-Px activity, significantly enhanced the antioxidant capacity of injured mice, improved the survival rate of mice, and had a good protective effect on the liver. Promote the growth of mice. In conclusion, PTD-SOD fusion protein showed significant resistance to platinum damage both at cell level and in vivo. In addition, PTD-SOD has been proved to be less toxic and side effects, widely distributed in vivo, and has excellent radiation protection ability. Therefore, PTD-SOD is expected to become a new type of tumor radiotherapy and chemotherapy protective drug.
【学位授予单位】：福州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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