【摘要】：順鉑是最常使用的化療藥,能殺死多種腫瘤細胞,但順鉑同時又對多種器官具有毒副作用,尤其是肝臟。順鉑導致的肝臟毒性機制至今尚未完全明了,但順鉑引起的細胞內ROS量的增加是其導致肝損傷的重要原因之一。本所構建的PTD-SOD融合蛋白能以活性蛋白的形式跨膜進入細胞內,清除細胞內多余的ROS,減輕氧化應激所致?lián)p傷。本課題分別建立了順鉑損傷的細胞模型和動物模型,探討了PTD-SOD融合蛋白對體外和體內順鉑損傷的防護效應。體外實驗結果表明：順鉑會在肝細胞內誘導產(chǎn)生ROS,上調TNF-α、COX-2和細胞色素C的表達量,顯著增加損傷細胞MDA含量,顯著降低SOD活力、GST活力和T-AOC能力,顯著降低細胞抗氧化體系的抗氧化能力,從而抑制損傷細胞的增殖能力,導致細胞凋亡。預給予天然SOD對順鉑所致的各項細胞損傷基本無保護作用。預給予PTD-SOD則可顯著降低損傷細胞內多余的ROS,使損傷細胞TNF-α、COX-2、細胞色素C表達量下調,而Bcl-2表達量上調,顯著降低損傷細胞MDA含量,顯著增加SOD活力、GST活力和T-AOC能力,從而顯著增加損傷細胞的增殖能力,抑制損傷細胞的凋亡。由于PTD-SOD是專一性清除超氧陰離子的酶,由本實驗結果可推測順鉑誘導細胞產(chǎn)生的ROS中包含超氧陰離子,且其可能是順鉑引起細胞損傷的重要原因。PTD-SOD通過清除損傷細胞胞內多余的超氧陰離子,改變該細胞氧化應激狀態(tài)并調節(jié)細胞凋亡信號通路,從而實現(xiàn)細胞防護效應。在體外實驗的基礎上,我們進行了初步的活體實驗考察PTD-SOD對順鉑所致的肝損傷的保護效應。實驗結果表明：順鉑會顯著增加損傷小鼠肝臟的MDA含量,顯著降低SOD活力、GST活力和GSG-Px活力,從而損傷肝臟,降低小鼠體重。給予PTD-SOD可顯著降低損傷小鼠肝臟的MDA含量,顯著提高SOD水平、GST活力、GSH-Px活力,顯著增強損傷小鼠抗氧化能力,提高小鼠的生存率,對肝臟具有很好的保護效果,促進小鼠生長。綜上所述,PTD-SOD融合蛋白無論在細胞水平還是活體水平,都表現(xiàn)出顯著的抗順鉑損傷能力。此外,PTD-SOD已證實毒副作用小,體內分布廣,而且具有優(yōu)異的放射防護能力,因此,PTD-SOD有望成為優(yōu)質的新型腫瘤放化療防護藥物。
[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- 偽, 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- 偽 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.
【學位授予單位】：福州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96

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