【摘要】：目的肺癌是目前最常見的惡性腫瘤之一,其死亡率已位居各種惡性腫瘤之首。同步放化療是局部晚期非小細胞肺癌的標準治療,但由于放化療的相關(guān)毒性及耐藥的產(chǎn)生等多種因素,肺癌的治療現(xiàn)狀仍不容樂觀。因此尋找全新結(jié)構(gòu)和作用機理的高效低毒的抗腫瘤藥物是當今肺癌治療的重要任務(wù)之一。本小組既往研究證明細梗香草總皂苷對非小細胞肺癌具有抗癌活性且無明顯的毒副作用,因此,本課題擬對細梗香草總皂苷在非小細胞肺癌中的放療增敏潛能進行探索。方法第一部分：MTS法分析細梗香草總皂苷對非小細胞肺癌(H460、PC-9、H1299)細胞增殖的影響,集落形成實驗、流式細胞儀和Hochest33528分別檢測細梗香草總皂苷聯(lián)合放療后細胞集落形成率和凋亡的情況,Western blot檢測細梗香草總皂苷對凋亡相關(guān)蛋白的影響。第二部分：我們首先通過細梗香草總皂苷作用于骨髓瘤基因報告細胞后,采用流式細胞術(shù)篩選出可能涉及的DNA損傷修復(fù)機制,然后Western blot檢測DNA雙鏈損傷修復(fù)過程中可能涉及的蛋白分子,同時利用小干擾RNA下調(diào)DNA-PKcs的表達后,觀察細梗香草總皂苷的放療增敏作用是否受到影響。另外,還通過免疫組化法檢測細梗香草總皂苷對放療后的肺癌細胞中組蛋白H2AX的持續(xù)時間的影響,再次驗證細梗香草總皂苷通過抑制DNA損傷修復(fù)過程中的非同源末端連接發(fā)揮放療增敏作用。第三部分本小組采用transwell小室檢測細梗香草總皂苷對肺癌細胞轉(zhuǎn)移侵襲能力的影響。結(jié)果第一部分：MTS的結(jié)果顯示細梗香草總皂苷對H460、PC-9、H1299細胞株均有增殖抑制作用,且呈劑量和時間依賴性,同時它還能增強放療引起的細胞增殖抑制和放療誘導的細胞凋亡潛能。細梗香草總皂苷(1μg/mL)預(yù)處理48h后肺癌細胞株H460、 PC-9、H1299的放射增敏比分別為：1.67±0.22、2.07±0.34、1.43±0.21,在H460細胞中細梗香草濃度4.84±0.13μg/mL和放療劑量在4.99±0.05 Gy以下時,聯(lián)合指數(shù)小于1.0,PC-9細胞中細梗香草總皂苷濃度大于0.128±0.012μg/m和放療劑量大于0.168±0.03Gy時,聯(lián)合指數(shù)也小于1.0,通過放射增敏比大于1.0和聯(lián)合指數(shù)小于1.0可以得出細梗香草總皂苷對放療具有協(xié)同作用。與流式細胞術(shù)結(jié)果想對應(yīng),Western blot的結(jié)果表明：細梗香草總皂苷能夠上調(diào)肺癌細胞株凋亡相關(guān)的蛋白表達。第二部分：我們首先通過流式細胞術(shù)篩選骨髓瘤基因報告細胞,得知細梗香草總皂苷抑制DNA損傷修復(fù)過程中的非同源末端連接,Western blot檢測結(jié)果提示細梗香草總皂苷抑制非同源末端連接過程中相關(guān)的蛋白的表達水平,緊接著利用小干擾RNA下調(diào)DNA-PKcs的表達后,細梗香草總皂苷的放療增敏作用大大減弱,因此可以得出細梗香草總皂苷通過抑制非同源末端鏈接的修復(fù)能力發(fā)揮放療增敏活性。另外,免疫組化結(jié)果表明細梗香草總皂苷預(yù)處理后,DNA雙鏈斷點附近的組蛋白H2AX的持續(xù)時間延長,即DNA雙鏈斷裂修復(fù)能力下降,再次證實了細梗香草總皂苷的作用機理。最后本研究還發(fā)現(xiàn)細梗香草總皂苷抑制肺癌細胞侵襲轉(zhuǎn)移能力。結(jié)論在非小細胞肺癌中細梗香草總皂苷通過抑制DNA損傷修復(fù)過程中的非同源末端連接而發(fā)揮放療增敏作用,且抑制肺癌細胞的侵襲轉(zhuǎn)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.
【學位授予單位】：浙江中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2

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