本文選題：AS1411 + 輻射敏感性�。� 參考：《蘇州大學》2014年碩士論文

【摘要】：目的： 本課題以HeLa細胞為研究對象，研究探討AS1411對人宮頸癌細胞HeLa的輻射敏感性的影響，通過檢測不同濃度AS1411預處理聯(lián)合X射線照射條件下HeLa細胞的存活率，以及觀察AS1411預處理聯(lián)合X射線對HeLa細胞DNA損傷修復、周期分布和凋亡的影響，研究AS1411對HeLa細胞輻射敏感性影響的作用機理。 方法： 采用CCK-8法測定細胞活性，研究AS1411對HeLa細胞增殖-毒性的影響；克隆形成法觀測不同濃度AS1411預處理聯(lián)合X射線照射對HeLa細胞存活率的影響；采用抗γ-H2AX抗體標記法來檢測DNA雙鏈斷裂，研究AS1411對X射線導致的HeLa細胞DNA雙鏈斷裂的影響；利用流式細胞術測定AS1411預處理聯(lián)合X射線照射對HeLa細胞周期分布、細胞阻滯和細胞凋亡的影響。 結(jié)果： (1) CCK-8實驗結(jié)果顯示：不同濃度AS1411處理HeLa細胞24h、48h、72h，，HeLa細胞存活率均大于94%，表明AS1411的細胞毒性小�？寺⌒纬蓪嶒烇@示：不同濃度AS1411預處理的HeLa細胞X射線照射后細胞存活率具有顯著性差異(P0.05)。從吸收劑量-細胞存活曲線可以得出：低劑量區(qū)“肩區(qū)”同樣明顯縮小變窄，直線斜率部分增大，且各劑量點的存活率均低于對照組，這表明AS1411對HeLa細胞具有明顯的輻射增敏作用。 (2) DNA損傷實驗結(jié)果得到：經(jīng)AS1411預處理的HeLa細胞細胞核內(nèi)DNA斷裂數(shù)明顯增多。經(jīng)AS1411處理HeLa細胞24h，細胞核內(nèi)DNA斷裂數(shù)明顯多于對照組；照射劑量為4Gy、8Gy時，加藥+照射組核內(nèi)DNA斷裂數(shù)明顯多于單純照射組，其濃度為1μmol/L照射劑量為4Gy、8Gy時細胞核內(nèi)DNA斷裂數(shù)分別是21.024±2.25和61.052±2.68,而單純照射組核內(nèi)DNA斷裂數(shù)分別是9.681±2.13和20.789±2.73。 (3)細胞周期實驗結(jié)果得出：經(jīng)不同濃度AS1411處理HeLa細胞后，細胞周期（G0/G1期、S期、G2/M期）分布與對照組相比S期細胞增加；8GyX射線照射經(jīng)不同濃度AS1411預處理HeLa細胞出現(xiàn)G2/M期阻滯，但AS1411預處理并不影響輻射引起的G2/M期阻滯，這表明AS1411增加HeLa細胞的輻射敏感性的機制與細胞周期無關。 (4)細胞凋亡實驗結(jié)果顯示：AS1411可以促進輻射誘導的HeLa細胞的凋亡。經(jīng)AS1411預處理，500nmol/L組和1μmol/L組細胞早期凋亡率分別是7.71±1.51%、8.91±1.04%，對照組細胞早期凋亡率是5.86±0.81%；照射劑量為4Gy、8Gy時，加藥+照射組細胞凋亡率高于單純照射組，照射劑量為4Gy時500nmol/L組和1μmol/L組的細胞早期凋亡率分別是19.67±1.90%和21.31±1.74%，單純照射組細胞早期凋亡率是15.44±2.41%；照射劑量為8Gy時500nmol/L組和1μmol/L組的細胞早期凋亡率分別是39.02±1.91%和41.99±0.65%，單純照射組細胞早期凋亡率是35.90±2.92%。 結(jié)論： (1) AS1411對HeLa細胞沒有顯現(xiàn)出細胞毒性作用�？寺⌒纬蓪嶒灲Y(jié)果表明AS1411對HeLa細胞具有明顯的輻射增敏作用。 (2)AS1411可以阻止輻射誘導的HeLa細胞DNA損傷的修復，這表明AS1411提高HeLa細胞的輻射增敏作用與其阻止細胞DNA損傷修復的作用有關。 (3)AS1411增加HeLa細胞的輻射敏感性的機制與細胞周期無關。 (4)AS1411可以增加HeLa細胞的凋亡率并可以促進輻射誘導的HeLa細胞的凋亡，AS1411增加HeLa細胞的輻射敏感性的機制與細胞凋亡有關。
[Abstract]:Objective:
In this study, the effect of AS1411 on the radiosensitivity of human cervical cancer cell HeLa was investigated by HeLa cells. The survival rate of HeLa cells under the conditions of AS1411 preconditioning combined with X ray irradiation, and the effects of AS1411 preconditioning combined with X ray on the repair of HeLa cell DNA damage, the distribution of periodic distribution and apoptosis were observed. Objective to study the mechanism of the effect of AS1411 on radiosensitivity of HeLa cells.
Method:
The effects of AS1411 on proliferation and toxicity of HeLa cells were measured by CCK-8 method, and the effect of AS1411 preconditioning with X ray irradiation on the survival rate of HeLa cells was observed by cloned formation method, and DNA double strand breaks were detected by anti -H2AX antibody labeling method, and HeLa cell DNA double strand breaks caused by AS1411 on X rays were studied. Effects of AS1411 pretreatment and X ray irradiation on cell cycle distribution, cell block and apoptosis of HeLa cells were measured by flow cytometry.
Result:
(1) CCK-8 experimental results showed that the survival rates of 24h, 48h, 72h, HeLa cells were more than 94% in HeLa cells treated with different concentrations of AS1411, indicating that the cytotoxicity of AS1411 was small. The clone formation experiment showed that the cell survival rate of HeLa cells with different concentrations of AS1411 pretreated X ray of HeLa cells had significant difference (P0.05). The line can be concluded that the "shoulder area" in the low dose area is also narrowed and narrowed obviously, the line slope part increases, and the survival rate of each dose point is lower than that of the control group, which indicates that AS1411 has an obvious radiosensitization effect on HeLa cells.
(2) the results of DNA damage experiment showed that the number of DNA breakages in the nucleus of HeLa cells pretreated by AS1411 increased obviously. The number of DNA breaking in the nucleus of HeLa cells treated by AS1411 was obviously more than that of the control group; the dose of irradiation was 4Gy and 8Gy, the number of DNA fracture in the addition and irradiation group was obviously more than that in the simple irradiation group, and the concentration was 1 mu irradiation dose. For 4Gy and 8Gy, the number of DNA breakages in the nucleus was 21.024 + 2.25 and 61.052 + 2.68 respectively, while the DNA breakage in the pure irradiation group was 9.681 + 2.13 and 20.789 + 2.73. respectively.
(3) the results of cell cycle experiments showed that after HeLa cells treated with different concentrations of AS1411, the distribution of cell cycle (G0/G1, S, G2/M) was increased in S phase compared with that of the control group; 8GyX rays irradiated HeLa cells with AS1411 pretreated with different concentrations in the G2/M phase block, but AS1411 pretreatment did not affect the G2/M phase block induced by radiation, which showed that The mechanism by which AS1411 increases the radiosensitivity of HeLa cells is independent of cell cycle.
(4) the apoptosis experiment showed that AS1411 could promote the apoptosis of radiation induced HeLa cells. After AS1411 pretreatment, the early apoptosis rate of 500nmol/L and 1 mol/L groups was 7.71 + 1.51% and 8.91 + 1.04% respectively. The early apoptosis rate of the control group was 5.86 + 0.81%, the dose of irradiation agent was 4Gy, 8Gy, and the apoptosis rate of the addition + irradiation group was higher than that of the irradiation group. The early apoptosis rate of 500nmol/L group and 1 u mol/L group was 19.67 + 1.90% and 21.31 + 1.74% respectively. The early apoptosis rate of cells in simple irradiation group was 15.44 + 2.41%, and the early apoptosis rate of 500nmol/L group and 1 u mol/L group was 39.02 + 1.91% and 41.99 + 0.65%, respectively, when the irradiation dose was 8Gy, respectively. The early apoptosis rate of the group was 35.90 + 2.92%.
Conclusion:
(1) AS1411 showed no cytotoxic effect on HeLa cells. The results of clone formation showed that AS1411 had a significant radiosensitization effect on HeLa cells.
(2) AS1411 can prevent the repair of DNA damage in HeLa cells induced by radiation, which indicates that AS1411 increases the radiation sensitization of HeLa cells and its role in preventing cell DNA damage and repair.
(3) the mechanism by which AS1411 increases the radiosensitivity of HeLa cells is independent of cell cycle.
(4) AS1411 can increase the apoptosis rate of HeLa cells and promote the apoptosis of HeLa cells induced by radiation. The mechanism of AS1411 to increase the radiosensitivity of HeLa cells is related to the apoptosis.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R730.55;R737.33

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