【摘要】：背景及研究目的:DNP(2,4二硝基苯酚)是一種能抑制氧化磷酸化的解偶聯(lián)劑,能通過(guò)抑制ADP的磷酸化過(guò)程而抑制細(xì)胞的能量代謝,而只能以熱能的方式散發(fā)出去,曾經(jīng)作為減肥藥物而廣泛使用。腫瘤細(xì)胞是一種高耗能的細(xì)胞,同時(shí)腫瘤細(xì)胞受到射線照射以后,其自身的生長(zhǎng)增殖以及對(duì)輻射損傷的修復(fù)都需要大量的能量。因此可以設(shè)想使用DNP,通過(guò)抑制細(xì)胞能量的生成,抑制受損傷腫瘤細(xì)胞的修復(fù)增殖,從而達(dá)到輻射增敏的效果。本文擬通過(guò)研究DNP對(duì)Hela和KB細(xì)胞的效應(yīng),評(píng)價(jià)該藥物的輻射增敏能力,探討其產(chǎn)生增敏效應(yīng)的可能相關(guān)機(jī)制,為研發(fā)新的輻射增敏藥物提供實(shí)驗(yàn)數(shù)據(jù)。方法:MTT實(shí)驗(yàn)和細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)不同濃度的DNP對(duì)Hela以及KB的生長(zhǎng)和增殖能力的影響。然后選擇200μmol/L DNP濃度繼續(xù)實(shí)驗(yàn),通過(guò)細(xì)胞克隆形成實(shí)驗(yàn),證實(shí)DNP是否能夠誘導(dǎo)輻射增敏效應(yīng);通過(guò)流式細(xì)胞儀,檢測(cè)對(duì)Hela、KB細(xì)胞的周期分布影響和線粒體膜電位的改變;通過(guò)使用PDMPO溶酶體黃/藍(lán)色熒光探針,激光共聚焦顯微鏡觀察細(xì)胞溶酶體內(nèi)p H變化;通過(guò)透射電子顯微鏡觀察藥物作用后Hela和KB細(xì)胞的亞細(xì)胞結(jié)構(gòu)的變化,尤其是自噬小體和自噬溶酶體堆積的形成。結(jié)果:DNP對(duì)Hela和KB細(xì)胞生長(zhǎng)增殖具有明顯的抑制作用,KB細(xì)胞更為敏感;DNP具有明顯的輻射增敏作用,對(duì)兩種細(xì)胞的增敏比SERD0分別為1.45和1.18。Hela和KB細(xì)胞在單純藥物作用24h后,或者聯(lián)合4 Gy X線照射后,細(xì)胞G2/M期的細(xì)胞比例明顯上升,產(chǎn)生了G2/M期的細(xì)胞阻滯。線粒體膜電位檢測(cè)也發(fā)現(xiàn),無(wú)論是單獨(dú)藥物作用,單純4Gy照射,或者4Gy X線照射后聯(lián)合藥物作用24h,均能使低線粒體膜電位的細(xì)胞比例升高,藥物聯(lián)合射線后效果更加顯著。與對(duì)照組相比,DNP作用后的Hela、KB細(xì)胞內(nèi)均發(fā)現(xiàn)自噬小體和溶酶體聚集,KB細(xì)胞更加明顯增多;KB細(xì)胞溶酶體內(nèi)PDMPO溶酶體黃/藍(lán)色熒光探針的藍(lán)色熒光強(qiáng)度稍高于對(duì)照KB細(xì)胞組,說(shuō)明藥物作用后,溶酶體的p H值也受到了影響,而Hela細(xì)胞未能明顯觀察到熒光的顯著變化。結(jié)論:DNP能夠誘導(dǎo)產(chǎn)生輻射增敏效應(yīng),其作用機(jī)制可能包括:DNP誘導(dǎo)細(xì)胞G2/M期周期阻滯,導(dǎo)致細(xì)胞線粒體膜電位顯著下降而抑制線粒體功能,細(xì)胞溶酶體p H向偏中性變化,增加細(xì)胞自噬效應(yīng),促進(jìn)細(xì)胞啟動(dòng)死亡過(guò)程等。DNP可能是一種有前途的輻射增敏劑。
[Abstract]:Background and objective: DNP is a kind of uncoupling agent that can inhibit oxidative phosphorylation. It can inhibit the energy metabolism of cells by inhibiting the phosphorylation of ADP, but it can only be emitted in the form of heat energy. It was widely used as a weight loss drug. Tumor cell is a kind of high energy consuming cell. At the same time, the growth and proliferation of tumor cells and the repair of radiation damage require a lot of energy. It can be envisaged that DNPs can inhibit the repair and proliferation of damaged tumor cells by inhibiting the generation of cell energy, thus achieving the effect of radiosensitization. In this paper, the effects of DNP on Hela and KB cells were studied, and the radiosensitization ability of DNP was evaluated, and the possible mechanism of its sensitization effect was discussed, which could provide experimental data for the development of new radiosensitizing drugs. Methods the effects of different concentrations of DNP on the growth and proliferation of Hela and KB were detected by cell clone formation and cell MTT assay. Then the concentration of 200 渭 mol / L DNP was selected to continue the experiment, the cell clone formation test was used to confirm whether DNP could induce radiosensitization, and the effect of DNP on the cell cycle distribution and mitochondrial membrane potential was detected by flow cytometry. By using PDMPO lysosomal yellow / blue fluorescence probe, laser confocal microscopy was used to observe the changes of pH in lysosomes, and transmission electron microscopy was used to observe the changes of subcellular structure of Hela and KB cells. In particular, the formation of autophagy bodies and autophagy lysosomes. Results the growth and proliferation of Hela and KB cells were significantly inhibited by: 1: DNP. The sensitizing ratio of DNP to the two cells was 1.45 and 1.18. Hela and KB cells were respectively 1.45 and 1.18. Hela and KB cells were exposed to drugs alone for 24 hours. Or combined with 4 Gy X ray irradiation, the proportion of cells in G 2 / M phase increased significantly, resulting in cell arrest in G 2 / M phase. It was also found that the ratio of cells with low mitochondrial membrane potential (MMP) could be increased by the combination of 4 Gy alone or 4 Gy X ray irradiation for 24 h, and the effect was more significant after the combination of drug and radiation. Compared with the control group, there were more PDMPO lysosomal yellow / blue fluorescence probes in the lysosomal PDMPO fluorescence probe of the KB cells than those in the control group, and the fluorescence intensity of PDMPO lysosomal yellow / blue fluorescent probe was slightly higher than that of the control KB cell group, and the number of lysosome and lysosomal aggregating KB cells in the control group was higher than that in the control group. The results showed that the pH value of lysosome was also affected after the drug treatment, but the fluorescence of Hela cells was not observed significantly. ConclusionDNP can induce radiosensitization, and its mechanism may include G 2 / M cycle arrest induced by w DNP, resulting in a significant decrease in mitochondrial membrane potential and inhibition of mitochondrial function, and the neutral change of lysosome pH in cells. DNP may be a promising radiosensitizer to increase autophagy and promote cell death.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R73-36

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本文編號(hào)：2123160