细梗香草总皂苷对非小细胞肺癌放疗增敏作用的研究
发布时间：2018-11-22 20:09
【摘要】：目的肺癌是目前最常见的恶性肿瘤之一,其死亡率已位居各种恶性肿瘤之首。同步放化疗是局部晚期非小细胞肺癌的标准治疗,但由于放化疗的相关毒性及耐药的产生等多种因素,肺癌的治疗现状仍不容乐观。因此寻找全新结构和作用机理的高效低毒的抗肿瘤药物是当今肺癌治疗的重要任务之一。本小组既往研究证明细梗香草总皂苷对非小细胞肺癌具有抗癌活性且无明显的毒副作用,因此,本课题拟对细梗香草总皂苷在非小细胞肺癌中的放疗增敏潜能进行探索。方法第一部分：MTS法分析细梗香草总皂苷对非小细胞肺癌(H460、PC-9、H1299)细胞增殖的影响,集落形成实验、流式细胞仪和Hochest33528分别检测细梗香草总皂苷联合放疗后细胞集落形成率和凋亡的情况,Western blot检测细梗香草总皂苷对凋亡相关蛋白的影响。第二部分：我们首先通过细梗香草总皂苷作用于骨髓瘤基因报告细胞后,采用流式细胞术筛选出可能涉及的DNA损伤修复机制,然后Western blot检测DNA双链损伤修复过程中可能涉及的蛋白分子,同时利用小干扰RNA下调DNA-PKcs的表达后,观察细梗香草总皂苷的放疗增敏作用是否受到影响。另外,还通过免疫组化法检测细梗香草总皂苷对放疗后的肺癌细胞中组蛋白H2AX的持续时间的影响,再次验证细梗香草总皂苷通过抑制DNA损伤修复过程中的非同源末端连接发挥放疗增敏作用。第三部分本小组采用transwell小室检测细梗香草总皂苷对肺癌细胞转移侵袭能力的影响。结果第一部分：MTS的结果显示细梗香草总皂苷对H460、PC-9、H1299细胞株均有增殖抑制作用,且呈剂量和时间依赖性,同时它还能增强放疗引起的细胞增殖抑制和放疗诱导的细胞凋亡潜能。细梗香草总皂苷(1μg/mL)预处理48h后肺癌细胞株H460、 PC-9、H1299的放射增敏比分别为：1.67±0.22、2.07±0.34、1.43±0.21,在H460细胞中细梗香草浓度4.84±0.13μg/mL和放疗剂量在4.99±0.05 Gy以下时,联合指数小于1.0,PC-9细胞中细梗香草总皂苷浓度大于0.128±0.012μg/m和放疗剂量大于0.168±0.03Gy时,联合指数也小于1.0,通过放射增敏比大于1.0和联合指数小于1.0可以得出细梗香草总皂苷对放疗具有协同作用。与流式细胞术结果想对应,Western blot的结果表明：细梗香草总皂苷能够上调肺癌细胞株凋亡相关的蛋白表达。第二部分：我们首先通过流式细胞术筛选骨髓瘤基因报告细胞,得知细梗香草总皂苷抑制DNA损伤修复过程中的非同源末端连接,Western blot检测结果提示细梗香草总皂苷抑制非同源末端连接过程中相关的蛋白的表达水平,紧接着利用小干扰RNA下调DNA-PKcs的表达后,细梗香草总皂苷的放疗增敏作用大大减弱,因此可以得出细梗香草总皂苷通过抑制非同源末端链接的修复能力发挥放疗增敏活性。另外,免疫组化结果表明细梗香草总皂苷预处理后,DNA双链断点附近的组蛋白H2AX的持续时间延长,即DNA双链断裂修复能力下降,再次证实了细梗香草总皂苷的作用机理。最后本研究还发现细梗香草总皂苷抑制肺癌细胞侵袭转移能力。结论在非小细胞肺癌中细梗香草总皂苷通过抑制DNA损伤修复过程中的非同源末端连接而发挥放疗增敏作用,且抑制肺癌细胞的侵袭转移能力。
[Abstract]:Lung cancer is one of the most common malignant tumors, and its mortality rate has been the first of all kinds of malignant tumors. Concurrent chemoradiotherapy is a standard treatment for locally advanced non-small cell lung cancer, but the treatment status of lung cancer is not optimistic due to the related toxicity of radiotherapy and chemotherapy and the production of drug resistance. So it is one of the most important tasks in the treatment of lung cancer. The previous study of this group has shown that the total soap of the fine stem and herb has anticancer activity and no obvious toxic and side effect on the non-small cell lung cancer, and therefore, the topic is to explore the potential of the total soap of the fine stem and herb in the non-small cell lung cancer. Methods The first part: MTS method was used to analyze the effect of total soap of fine stem and herb on the proliferation of non-small cell lung cancer (H460, PC-9, H1299), colony formation, flow cytometry and Hochest33528, respectively. The effect of total soap on apoptosis-related protein was detected by Western blot. In the second part, we first reported the possible DNA damage repair mechanism by flow cytometry, and then Western blot was used to detect the possible protein molecules in the DNA double-chain injury repair. At the same time, after down-regulation of the expression of DNA-PKcs by small interfering RNA, it was observed whether the effect of the total soap level of the fine stem and the total soap was affected. In addition, the effect of total soap of fine stem and vanilla on the duration of histone H2AX in lung cancer cells after radiotherapy was also detected by immunohistochemistry. The third part of this group uses the transwell chamber to detect the effect of the total soap level on the transfer and invasion ability of the lung cancer cells. Results The results of the first part: The results of MTS showed that the total soap of fine stem and vanilla had a proliferation inhibitory effect on H460, PC-9 and H1299 cell lines, and it was dose-dependent and time-dependent, and it can enhance the cell proliferation inhibition and the potential of apoptosis induced by radiotherapy. The radiosensitizing ratio of the lung cancer cell line H460, PC-9 and H1299 was 1.67, 0.22, 2.07, 0.34, 1.43 and 0.21, respectively. The combined index of the total soap in the PC-9 cells was more than 0. 128 and 0. 012.mu. g/ m and the radiation dose was more than 0. 168 to 0. 03Gy. The combined index was also less than 1.0, and the combined index was less than 1.0 and the combined index was less than 1.0. The results of the Western blot show that the total soap level of the fine stem and herb can increase the expression of the protein related to the apoptosis of the lung cancer cell line. in that second part, we first screen the myeloma gene reporter cell by flow cytometry to learn that the total soap of the fine stem and vanilla is not connected with the non-homologous end of the DNA damage repair process, The results of Western blot showed that the total soap level of the fine stem and vanilla was inhibited by the expression level of the related protein in the non-homologous end ligation, and the effect of the total soap of the fine stem and the total soap was greatly reduced after the expression of the DNA-PKcs was down-regulated by the small interfering RNA. Therefore, it can be concluded that the total soap of the fine-stem vanilla can play the radiotherapy-sensitization activity by inhibiting the repair capacity of the non-homologous end link. In addition, the results showed that the duration of the histone H2AX in the vicinity of the double-strand break point of the DNA was prolonged, that is, the double-strand break repair ability of the DNA was decreased, and the action mechanism of the total soap of the fine stem and vanilla was confirmed again. Finally, the study also found that the total soap of fine stem and herb can inhibit the invasion and metastasis of lung cancer cells. Conclusion The total soap in the non-small cell lung cancer can play the radiotherapeutic effect by inhibiting the non-homologous end connection in the DNA damage repair process, and inhibit the invasion and metastasis of the lung cancer cells.
【学位授予单位】：浙江中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2350433
[Abstract]:Lung cancer is one of the most common malignant tumors, and its mortality rate has been the first of all kinds of malignant tumors. Concurrent chemoradiotherapy is a standard treatment for locally advanced non-small cell lung cancer, but the treatment status of lung cancer is not optimistic due to the related toxicity of radiotherapy and chemotherapy and the production of drug resistance. So it is one of the most important tasks in the treatment of lung cancer. The previous study of this group has shown that the total soap of the fine stem and herb has anticancer activity and no obvious toxic and side effect on the non-small cell lung cancer, and therefore, the topic is to explore the potential of the total soap of the fine stem and herb in the non-small cell lung cancer. Methods The first part: MTS method was used to analyze the effect of total soap of fine stem and herb on the proliferation of non-small cell lung cancer (H460, PC-9, H1299), colony formation, flow cytometry and Hochest33528, respectively. The effect of total soap on apoptosis-related protein was detected by Western blot. In the second part, we first reported the possible DNA damage repair mechanism by flow cytometry, and then Western blot was used to detect the possible protein molecules in the DNA double-chain injury repair. At the same time, after down-regulation of the expression of DNA-PKcs by small interfering RNA, it was observed whether the effect of the total soap level of the fine stem and the total soap was affected. In addition, the effect of total soap of fine stem and vanilla on the duration of histone H2AX in lung cancer cells after radiotherapy was also detected by immunohistochemistry. The third part of this group uses the transwell chamber to detect the effect of the total soap level on the transfer and invasion ability of the lung cancer cells. Results The results of the first part: The results of MTS showed that the total soap of fine stem and vanilla had a proliferation inhibitory effect on H460, PC-9 and H1299 cell lines, and it was dose-dependent and time-dependent, and it can enhance the cell proliferation inhibition and the potential of apoptosis induced by radiotherapy. The radiosensitizing ratio of the lung cancer cell line H460, PC-9 and H1299 was 1.67, 0.22, 2.07, 0.34, 1.43 and 0.21, respectively. The combined index of the total soap in the PC-9 cells was more than 0. 128 and 0. 012.mu. g/ m and the radiation dose was more than 0. 168 to 0. 03Gy. The combined index was also less than 1.0, and the combined index was less than 1.0 and the combined index was less than 1.0. The results of the Western blot show that the total soap level of the fine stem and herb can increase the expression of the protein related to the apoptosis of the lung cancer cell line. in that second part, we first screen the myeloma gene reporter cell by flow cytometry to learn that the total soap of the fine stem and vanilla is not connected with the non-homologous end of the DNA damage repair process, The results of Western blot showed that the total soap level of the fine stem and vanilla was inhibited by the expression level of the related protein in the non-homologous end ligation, and the effect of the total soap of the fine stem and the total soap was greatly reduced after the expression of the DNA-PKcs was down-regulated by the small interfering RNA. Therefore, it can be concluded that the total soap of the fine-stem vanilla can play the radiotherapy-sensitization activity by inhibiting the repair capacity of the non-homologous end link. In addition, the results showed that the duration of the histone H2AX in the vicinity of the double-strand break point of the DNA was prolonged, that is, the double-strand break repair ability of the DNA was decreased, and the action mechanism of the total soap of the fine stem and vanilla was confirmed again. Finally, the study also found that the total soap of fine stem and herb can inhibit the invasion and metastasis of lung cancer cells. Conclusion The total soap in the non-small cell lung cancer can play the radiotherapeutic effect by inhibiting the non-homologous end connection in the DNA damage repair process, and inhibit the invasion and metastasis of the lung cancer cells.
【学位授予单位】：浙江中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
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