核酸适配体AS1411对肿瘤细胞辐射敏感性影响研究

发布时间：2018-06-22 02:38

  本文选题：AS1411 + 辐射敏感性　； 参考：《苏州大学》2014年硕士论文

【摘要】：目的： 本课题以HeLa细胞为研究对象，研究探讨AS1411对人宫颈癌细胞HeLa的辐射敏感性的影响，通过检测不同浓度AS1411预处理联合X射线照射条件下HeLa细胞的存活率，以及观察AS1411预处理联合X射线对HeLa细胞DNA损伤修复、周期分布和凋亡的影响，研究AS1411对HeLa细胞辐射敏感性影响的作用机理。 方法： 采用CCK-8法测定细胞活性，研究AS1411对HeLa细胞增殖-毒性的影响；克隆形成法观测不同浓度AS1411预处理联合X射线照射对HeLa细胞存活率的影响；采用抗γ-H2AX抗体标记法来检测DNA双链断裂，研究AS1411对X射线导致的HeLa细胞DNA双链断裂的影响；利用流式细胞术测定AS1411预处理联合X射线照射对HeLa细胞周期分布、细胞阻滞和细胞凋亡的影响。 结果： (1) CCK-8实验结果显示：不同浓度AS1411处理HeLa细胞24h、48h、72h，，HeLa细胞存活率均大于94%，表明AS1411的细胞毒性小。克隆形成实验显示：不同浓度AS1411预处理的HeLa细胞X射线照射后细胞存活率具有显著性差异(P0.05)。从吸收剂量-细胞存活曲线可以得出：低剂量区“肩区”同样明显缩小变窄，直线斜率部分增大，且各剂量点的存活率均低于对照组，这表明AS1411对HeLa细胞具有明显的辐射增敏作用。 (2) DNA损伤实验结果得到：经AS1411预处理的HeLa细胞细胞核内DNA断裂数明显增多。经AS1411处理HeLa细胞24h，细胞核内DNA断裂数明显多于对照组；照射剂量为4Gy、8Gy时，加药+照射组核内DNA断裂数明显多于单纯照射组，其浓度为1μmol/L照射剂量为4Gy、8Gy时细胞核内DNA断裂数分别是21.024±2.25和61.052±2.68,而单纯照射组核内DNA断裂数分别是9.681±2.13和20.789±2.73。 (3)细胞周期实验结果得出：经不同浓度AS1411处理HeLa细胞后，细胞周期（G0/G1期、S期、G2/M期）分布与对照组相比S期细胞增加；8GyX射线照射经不同浓度AS1411预处理HeLa细胞出现G2/M期阻滞，但AS1411预处理并不影响辐射引起的G2/M期阻滞，这表明AS1411增加HeLa细胞的辐射敏感性的机制与细胞周期无关。 (4)细胞凋亡实验结果显示：AS1411可以促进辐射诱导的HeLa细胞的凋亡。经AS1411预处理，500nmol/L组和1μmol/L组细胞早期凋亡率分别是7.71±1.51%、8.91±1.04%，对照组细胞早期凋亡率是5.86±0.81%；照射剂量为4Gy、8Gy时，加药+照射组细胞凋亡率高于单纯照射组，照射剂量为4Gy时500nmol/L组和1μmol/L组的细胞早期凋亡率分别是19.67±1.90%和21.31±1.74%，单纯照射组细胞早期凋亡率是15.44±2.41%；照射剂量为8Gy时500nmol/L组和1μmol/L组的细胞早期凋亡率分别是39.02±1.91%和41.99±0.65%，单纯照射组细胞早期凋亡率是35.90±2.92%。 结论： (1) AS1411对HeLa细胞没有显现出细胞毒性作用。克隆形成实验结果表明AS1411对HeLa细胞具有明显的辐射增敏作用。 (2)AS1411可以阻止辐射诱导的HeLa细胞DNA损伤的修复，这表明AS1411提高HeLa细胞的辐射增敏作用与其阻止细胞DNA损伤修复的作用有关。 (3)AS1411增加HeLa细胞的辐射敏感性的机制与细胞周期无关。 (4)AS1411可以增加HeLa细胞的凋亡率并可以促进辐射诱导的HeLa细胞的凋亡，AS1411增加HeLa细胞的辐射敏感性的机制与细胞凋亡有关。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R730.55;R737.33

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