Tat-SmacN7融合肽对肿瘤细胞辐射增敏作用的研究
发布时间：2018-10-25 14:39
【摘要】：肿瘤是严重威胁人类健康和生命的疾病之一,目前临床上主要治疗手段为手术治疗、放射治疗、化疗和生物治疗,以及一些辅助治疗,如中医中药治疗、内分泌治疗、射频消融和热疗等。其中,放疗在肿瘤治疗上发挥的作用也是越来越大。但是,由于肿瘤的发生、发展是一个多因素和多阶段参与的复杂过程,造成了肿瘤对放射治疗的敏感性不同；另外,接受过放疗的肿瘤细胞可对其产生耐受,导致疗效降低,因此如何提高肿瘤细胞对放疗的敏感性,是肿瘤治疗一个迫切需要解决的问题。肿瘤细胞内凋亡信号通路调控异常,往往是肿瘤细胞对放射治疗诱导的凋亡产生耐受性的原因之一。肿瘤细胞内凋亡抑制蛋白家族(IAPs)的主要作用是通过与有活性的caspase-3、-7和-9结合,继而阻碍它们的功能,最终抑制细胞的凋亡,使肿瘤细胞产生辐射耐受性。IAPs家族的主要成员有XIAP. cIAP-1和cIAP-2。 Smac/DIABLO是一种线粒体蛋白,在凋亡刺激下可以从线粒体中释放,扰乱IAPs与caspase的相互作用,解除IAPs对肿瘤细胞的凋亡抑制作用,从而提高肿瘤细胞对辐射的敏感性。SmacN7是Smac蛋白促凋亡功能的最小活性肽段,但是SmacN7不能穿透细胞膜,而连接可以穿透细胞膜的引导肽Tat之后,Tat-SmacN7融合肽可以发挥较明显的辐射增敏作用。目的：本项目探讨了Tat-SmacN7在体内外对食管癌Ec109细胞和非小细胞肺癌H460细胞两种不同肿瘤细胞株的辐射增敏作用,阐明了Tat-SmacN7融合肽的辐射增敏作用机制。方法：(1)细胞分为对照组、Tat-SmacN7组、照射组和联合组。通过细胞克隆形成实验和流式细胞术检测细胞凋亡,评价Tat-SmacN7对两种肿瘤细胞的辐射增敏作用。(2)PCR和Western blot实验观察细胞内caspase分子水平和XIAP蛋白水平的变化,ELISA方法检测caspase活性。利用caspase抑制剂阻断Tat-SmacN7的放射增敏作用,来探讨Tat-SmacN7的辐射增敏作用机制。(3)通过体内实验,观察不同处理组裸鼠的肿瘤增长情况、是否能够耐受Tat-SmacN7融合肽的体内应用。结果：(1)Tat-SmacN7单独使用对肿瘤细胞的促凋亡作用不明显,但是可以提高细胞对辐射的敏感性,从而提高肿瘤细胞的凋亡。使用微摩尔级的Tat-SmacN7处理细胞,在辐射相对耐受(H460)和相对敏感(Ec109)细胞株都发现放射增敏作用,对辐射耐受的H460细胞放射增敏作用更明显,SER分别为1.63和1.51。(2)PCR结果显示,联合组细胞的caspase-3、-8和.9的分子表达增高,ELASA证明caspase活性也被激活。使用caspase抑制剂(z-VAD-fmk)可阻断Tat-SmacN7的辐射增敏作用,H460和Ec109细胞的SER分别降到1.17和1.09。(3)联合治疗组的裸鼠在Tat-SmacN7的辐射增敏作用下肿瘤生长较其它处理组最慢。结论：Tat-SmacN7是一种高效、潜在的新型放疗增敏药物。Tat-SmacN7对肿瘤细胞H460和Ec109的辐射增敏作用机制主要是通过提高caspase-3、-8和.9的分子表达,并且提高caspase的活性,体内外实验均有明显的辐射增敏效果。本项目将对发现新的辐射增敏药物、增强放疗效果具有重要的理论指导意义。
[Abstract]:Tumor is one of the diseases that seriously threaten human health and life. At present, the main treatment methods are surgical therapy, radiation therapy, chemotherapy and biological therapy, as well as some adjuvant therapy, such as traditional Chinese medicine treatment, endocrine therapy, radio frequency ablation and thermal therapy. Radiotherapy plays an increasingly important role in tumor therapy. However, due to the occurrence and development of tumor, development is a complex process of multi-factor and multi-stage involvement, which causes the sensitivity of tumor to radiation therapy. In addition, tumor cells treated with radiotherapy can tolerate it, resulting in reduced curative effect. Therefore, how to improve the sensitivity of tumor cells to radiotherapy is an urgent problem in the treatment of tumor. The abnormal regulation of apoptosis signal pathway in tumor cells is one of the reasons why tumor cells are tolerant to the apoptosis induced by radiotherapy. The primary role of the apoptosis-inhibiting protein family (IAPs) in tumor cells is by binding to active caspase-3, -7 and -9, which in turn inhibit their function and ultimately inhibit the apoptosis of the cells, resulting in radiation tolerance for tumor cells. The main members of the IAPs family are XIAP. cIAP-1 and cIAP-2. Smac/ DIABLO is a kind of mitochondrial protein, which can be released from mitochondria under the stimulation of apoptosis, disrupt the interaction between IAPs and caspase, and release IAPs to inhibit the apoptosis of tumor cells, thus increasing the sensitivity of tumor cells to radiation. SmacN7 is the minimum active peptide segment of Smac protein pro-apoptotic function, but SmacN7 can not penetrate the cell membrane, and after ligation can penetrate the cell membrane's guide peptide Tat, the Tat-SmacN7 fusion peptide can play a more obvious radiation sensitizing effect. Objective: To investigate the radiation sensitizing effect of Tat-SmacN7 on human esophageal carcinoma EC109 cells and non-small cell lung cancer H460 cells in vitro and to elucidate the radiation sensitizing effect mechanism of Tat-SmacN7 fusion peptide. Methods: (1) The cells were divided into control group, Tat-SmacN7 group, irradiation group and joint group. The effect of Tat-SmacN7 on the radiation sensitizing effect of Tat-SmacN7 on two tumor cells was evaluated by cell cloning and flow cytometry. (2) The levels of caspase molecules and the levels of XIAP protein were observed by PCR and Western blot, and caspase activity was detected by ELISA. The radiosensitizing effect of Tat-SmacN7 was blocked by caspase inhibitor to explore the mechanism of radiation sensitizing action of Tat-SmacN7. (3) In vivo experiments, the tumor growth of nude mice in different treatment groups was observed, and whether the in vivo application of Tat-SmacN7 fusion peptide can be tolerated. Results: (1) Tat-SmacN7 had no obvious pro-apoptotic effect on tumor cells, but it could increase the sensitivity of cells to radiation, thus increasing the apoptosis of tumor cells. A micromolar Tat-SmacN7 was used to treat the cells. Radiosensitization was found in the radiation-tolerant (H460) and relatively sensitive (Ec109) cell lines. The radiosensitization of H460 cells with radiation tolerance was more evident, and SER was 1.63 and 1.51, respectively. (2) The results showed that the expression of caspase-3, -8, and. 9 in the combined cells increased and the caspase activity was also activated by ELASA. The use of caspase inhibitors (z-VAD-fmk) blocked the radiation sensitizing effect of Tat-SmacN7, and the SER of H460 and Ec109 cells decreased to 1. 17 and 1. 09, respectively. (3) The tumor growth was the slowest in the nude mice treated with Tat-SmacN7. Conclusion: Tat-SmacN7 is a highly effective and potential radiosensitizer. The mechanism of Tat-SmacN7 on the radiation sensitizing action of tumor cells H460 and E109 was mainly by increasing the molecular expression of caspase-3, -8 and. 9, and increasing caspase activity. This project will be of great theoretical significance for the discovery of new radiation sensitizing drugs and enhancing the effect of radiotherapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R730.55
本文编号：2293990
[Abstract]:Tumor is one of the diseases that seriously threaten human health and life. At present, the main treatment methods are surgical therapy, radiation therapy, chemotherapy and biological therapy, as well as some adjuvant therapy, such as traditional Chinese medicine treatment, endocrine therapy, radio frequency ablation and thermal therapy. Radiotherapy plays an increasingly important role in tumor therapy. However, due to the occurrence and development of tumor, development is a complex process of multi-factor and multi-stage involvement, which causes the sensitivity of tumor to radiation therapy. In addition, tumor cells treated with radiotherapy can tolerate it, resulting in reduced curative effect. Therefore, how to improve the sensitivity of tumor cells to radiotherapy is an urgent problem in the treatment of tumor. The abnormal regulation of apoptosis signal pathway in tumor cells is one of the reasons why tumor cells are tolerant to the apoptosis induced by radiotherapy. The primary role of the apoptosis-inhibiting protein family (IAPs) in tumor cells is by binding to active caspase-3, -7 and -9, which in turn inhibit their function and ultimately inhibit the apoptosis of the cells, resulting in radiation tolerance for tumor cells. The main members of the IAPs family are XIAP. cIAP-1 and cIAP-2. Smac/ DIABLO is a kind of mitochondrial protein, which can be released from mitochondria under the stimulation of apoptosis, disrupt the interaction between IAPs and caspase, and release IAPs to inhibit the apoptosis of tumor cells, thus increasing the sensitivity of tumor cells to radiation. SmacN7 is the minimum active peptide segment of Smac protein pro-apoptotic function, but SmacN7 can not penetrate the cell membrane, and after ligation can penetrate the cell membrane's guide peptide Tat, the Tat-SmacN7 fusion peptide can play a more obvious radiation sensitizing effect. Objective: To investigate the radiation sensitizing effect of Tat-SmacN7 on human esophageal carcinoma EC109 cells and non-small cell lung cancer H460 cells in vitro and to elucidate the radiation sensitizing effect mechanism of Tat-SmacN7 fusion peptide. Methods: (1) The cells were divided into control group, Tat-SmacN7 group, irradiation group and joint group. The effect of Tat-SmacN7 on the radiation sensitizing effect of Tat-SmacN7 on two tumor cells was evaluated by cell cloning and flow cytometry. (2) The levels of caspase molecules and the levels of XIAP protein were observed by PCR and Western blot, and caspase activity was detected by ELISA. The radiosensitizing effect of Tat-SmacN7 was blocked by caspase inhibitor to explore the mechanism of radiation sensitizing action of Tat-SmacN7. (3) In vivo experiments, the tumor growth of nude mice in different treatment groups was observed, and whether the in vivo application of Tat-SmacN7 fusion peptide can be tolerated. Results: (1) Tat-SmacN7 had no obvious pro-apoptotic effect on tumor cells, but it could increase the sensitivity of cells to radiation, thus increasing the apoptosis of tumor cells. A micromolar Tat-SmacN7 was used to treat the cells. Radiosensitization was found in the radiation-tolerant (H460) and relatively sensitive (Ec109) cell lines. The radiosensitization of H460 cells with radiation tolerance was more evident, and SER was 1.63 and 1.51, respectively. (2) The results showed that the expression of caspase-3, -8, and. 9 in the combined cells increased and the caspase activity was also activated by ELASA. The use of caspase inhibitors (z-VAD-fmk) blocked the radiation sensitizing effect of Tat-SmacN7, and the SER of H460 and Ec109 cells decreased to 1. 17 and 1. 09, respectively. (3) The tumor growth was the slowest in the nude mice treated with Tat-SmacN7. Conclusion: Tat-SmacN7 is a highly effective and potential radiosensitizer. The mechanism of Tat-SmacN7 on the radiation sensitizing action of tumor cells H460 and E109 was mainly by increasing the molecular expression of caspase-3, -8 and. 9, and increasing caspase activity. This project will be of great theoretical significance for the discovery of new radiation sensitizing drugs and enhancing the effect of radiotherapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R730.55
【参考文献】
相关期刊论文 前2条
1 王娴静;孙慧;;Livin和Smac在肿瘤发生中的相互关系及研究进展[J];肿瘤基础与临床;2007年03期
2 梅泽如,项其昌,吴维继,蒋向民,冯存伟,穆怀德;食管癌术后预防性放疗前瞻性研究[J];中华放射肿瘤学杂志;1997年03期
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