Tat-SmacN7融合肽對腫瘤細(xì)胞輻射增敏作用的研究
發(fā)布時間：2018-10-25 14:39
【摘要】：腫瘤是嚴(yán)重威脅人類健康和生命的疾病之一,目前臨床上主要治療手段為手術(shù)治療、放射治療、化療和生物治療,以及一些輔助治療,如中醫(yī)中藥治療、內(nèi)分泌治療、射頻消融和熱療等。其中,放療在腫瘤治療上發(fā)揮的作用也是越來越大。但是,由于腫瘤的發(fā)生、發(fā)展是一個多因素和多階段參與的復(fù)雜過程,造成了腫瘤對放射治療的敏感性不同；另外,接受過放療的腫瘤細(xì)胞可對其產(chǎn)生耐受,導(dǎo)致療效降低,因此如何提高腫瘤細(xì)胞對放療的敏感性,是腫瘤治療一個迫切需要解決的問題。腫瘤細(xì)胞內(nèi)凋亡信號通路調(diào)控異常,往往是腫瘤細(xì)胞對放射治療誘導(dǎo)的凋亡產(chǎn)生耐受性的原因之一。腫瘤細(xì)胞內(nèi)凋亡抑制蛋白家族(IAPs)的主要作用是通過與有活性的caspase-3、-7和-9結(jié)合,繼而阻礙它們的功能,最終抑制細(xì)胞的凋亡,使腫瘤細(xì)胞產(chǎn)生輻射耐受性。IAPs家族的主要成員有XIAP. cIAP-1和cIAP-2。 Smac/DIABLO是一種線粒體蛋白,在凋亡刺激下可以從線粒體中釋放,擾亂IAPs與caspase的相互作用,解除IAPs對腫瘤細(xì)胞的凋亡抑制作用,從而提高腫瘤細(xì)胞對輻射的敏感性。SmacN7是Smac蛋白促凋亡功能的最小活性肽段,但是SmacN7不能穿透細(xì)胞膜,而連接可以穿透細(xì)胞膜的引導(dǎo)肽Tat之后,Tat-SmacN7融合肽可以發(fā)揮較明顯的輻射增敏作用。目的：本項目探討了Tat-SmacN7在體內(nèi)外對食管癌Ec109細(xì)胞和非小細(xì)胞肺癌H460細(xì)胞兩種不同腫瘤細(xì)胞株的輻射增敏作用,闡明了Tat-SmacN7融合肽的輻射增敏作用機(jī)制。方法：(1)細(xì)胞分為對照組、Tat-SmacN7組、照射組和聯(lián)合組。通過細(xì)胞克隆形成實驗和流式細(xì)胞術(shù)檢測細(xì)胞凋亡,評價Tat-SmacN7對兩種腫瘤細(xì)胞的輻射增敏作用。(2)PCR和Western blot實驗觀察細(xì)胞內(nèi)caspase分子水平和XIAP蛋白水平的變化,ELISA方法檢測caspase活性。利用caspase抑制劑阻斷Tat-SmacN7的放射增敏作用,來探討Tat-SmacN7的輻射增敏作用機(jī)制。(3)通過體內(nèi)實驗,觀察不同處理組裸鼠的腫瘤增長情況、是否能夠耐受Tat-SmacN7融合肽的體內(nèi)應(yīng)用。結(jié)果：(1)Tat-SmacN7單獨(dú)使用對腫瘤細(xì)胞的促凋亡作用不明顯,但是可以提高細(xì)胞對輻射的敏感性,從而提高腫瘤細(xì)胞的凋亡。使用微摩爾級的Tat-SmacN7處理細(xì)胞,在輻射相對耐受(H460)和相對敏感(Ec109)細(xì)胞株都發(fā)現(xiàn)放射增敏作用,對輻射耐受的H460細(xì)胞放射增敏作用更明顯,SER分別為1.63和1.51。(2)PCR結(jié)果顯示,聯(lián)合組細(xì)胞的caspase-3、-8和.9的分子表達(dá)增高,ELASA證明caspase活性也被激活。使用caspase抑制劑(z-VAD-fmk)可阻斷Tat-SmacN7的輻射增敏作用,H460和Ec109細(xì)胞的SER分別降到1.17和1.09。(3)聯(lián)合治療組的裸鼠在Tat-SmacN7的輻射增敏作用下腫瘤生長較其它處理組最慢。結(jié)論：Tat-SmacN7是一種高效、潛在的新型放療增敏藥物。Tat-SmacN7對腫瘤細(xì)胞H460和Ec109的輻射增敏作用機(jī)制主要是通過提高caspase-3、-8和.9的分子表達(dá),并且提高caspase的活性,體內(nèi)外實驗均有明顯的輻射增敏效果。本項目將對發(fā)現(xiàn)新的輻射增敏藥物、增強(qiáng)放療效果具有重要的理論指導(dǎo)意義。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R730.55
本文編號：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R730.55
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相關(guān)期刊論文 前2條
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