黄酮类化合物对人卵巢癌细胞株SKOV3的放射增敏作用及其机制研究

发布时间：2019-07-08 16:42

【摘要】：放疗，即利用电离辐射如X射线、γ射线或电子束等杀伤肿瘤细胞，是目前治疗恶性肿瘤的主要手段之一。但是由于肿瘤细胞的生物学特性及局部微环境等的影响，导致某些肿瘤细胞对射线的敏感性较低，射线不能有效地杀伤肿瘤细胞，因而放疗的效果较差。为了改善放疗效果，临床上将某些药物与放疗联合使用，称之为放射增敏剂（radiosensitizingagents），其与放疗联用，通过选择性地杀伤乏氧肿瘤细胞、抑制放射损伤修复、调节细胞周期、诱导肿瘤细胞凋亡等，来增强肿瘤细胞的放射敏感性，降低照射剂量，减少对正常细胞的损伤。卵巢癌是妇科常见的恶性肿瘤之一，其患者死亡率居各类妇科肿瘤的首位。卵巢癌依病期不同，可采用手术、放疗、化疗或以上方式综合治疗。但在放疗中，由于各类卵巢癌细胞对射线的敏感性不同，且癌细胞致死所需放射量和正常组织的耐受量差异很小，故有放疗效果欠佳或对正常组织的损伤较重等弊端，因此，有必要对放射增敏剂在卵巢癌放疗中应用的可能性进行探讨。本实验选用染料木黄酮（Genistein，Gen）、槲皮素（Quercetin，Que）和白杨素（Chrysin，Chr）三种代表性的黄酮类化合物作为增敏药物，以对放疗不敏感的人卵巢浆液性乳头状囊腺癌细胞株SKOV3作为卵巢癌的细胞模型，采用X射线照射，从提高肿瘤细胞放射敏感性的方法入手，并结合黄酮类化合物自身的药理学特性，研究黄酮类化合物的放射增敏作用及其机制，并探讨了黄酮类化合物作为放射增敏剂的可能性，以期为黄酮类化合物在放射增敏方面的进一步研究提供实验依据。具体内容如下：（一）黄酮类化合物对SKOV3细胞的毒性采用MTT法筛选出三种黄酮类化合物对SKOV3细胞的无毒浓度范围。结果表明，超出一定浓度范围的染料木黄酮、槲皮素和白杨素作用于SKOV3细胞24，48，72h后，与同时间点内的正常对照组相比，SKOV3细胞存活率均显著性下降（P0.05），且呈现一定的浓度和时间依赖性（P0.05）。染料木黄酮、槲皮素和白杨素作用于SKOV3细胞72h的无毒浓度应分别小于31.12，15.30，20.77μmol·L-1，说明染料木黄酮的细胞毒性相对较低。（二）黄酮类化合物对SKOV3细胞的放射增敏作用无毒浓度范围内的黄酮类化合物，即2.5～30μmol·L-1Gen、1.25～15μmol·L-1Que和1.25～20μmol·L-1Chr，分别于照射前2h预作用于SKOV3细胞，X射线（2，4，6，8，10Gy）单次照射后72h，采用MTT法检测放射增敏作用。三种黄酮类化合物对SKOV3细胞的放射增敏比（SER）分别为1.37～2.06，1.20～1.80和1.23～1.83，三种黄酮类化合物的各药物浓度组的SER值均有显著性差异（P0.01），放射生物学参数D0、Dq和N值均较单纯放射组显著性减小（P0.05），且随药物浓度的增大，SER值增大（P0.01），D0、Dq和N值减小（P0.05）。结果表明，一定浓度范围内的染料木黄酮、槲皮素和白杨素均对SKOV3细胞有放射增敏作用，使SKOV3细胞的放射损伤修复能力降低，放射敏感性增强，且染料木黄酮的放射增敏作用相对较强。（三）染料木黄酮对SKOV3细胞放射增敏机制的研究 5,10,20μmol·L-1Gen分别于照射前2h预作用于SKOV3细胞，X射线(2,4,6Gy)单次照射后24，48h，采用试剂盒检测细胞内ROS、MDA含量及T-AOC水平，采用PI单染法结合流式细胞仪检测细胞周期分布，采用Hoechst33342单染法结合荧光显微镜检测细胞凋亡，采用RT-PCR及Western Blot检测Bcl-2、Bax、Survivin、NF-κB mRNA及蛋白表达。照射后24，48h，单纯加药组、单纯放射组以及加药联合放射组的SKOV3细胞内ROS、MDA含量及细胞凋亡率均显著性高于同时间点内的正常对照组（P0.05），加药联合放射组的ROS、MDA含量及细胞凋亡率比相同浓度单纯加药组或相同剂量单纯放射组的显著增加（P0.05），且随药物浓度和放射剂量的增大，ROS、MDA含量及细胞凋亡率相应增加（P0.05）；与同时间点内的正常对照组相比，单纯放射组和加药联合放射组的T-AOC水平、G1期细胞比例显著性降低（P0.05），，G2/M期细胞比例上升（P0.05），且与放射剂量存在一定的相关性（P0.05），但无药物浓度依赖性（P0.05）。照射后48h，与正常对照组相比，6Gy单纯放射和20μmol·L-1Gen+6Gy联合作用均使SKOV3细胞的Bcl-2、Bax mRNA和NF-κB mRNA及蛋白表达显著性上调，Bcl-2蛋白表达显著性下调（P0.01），且联合作用组的Bax mRNA、NF-κB mRNA及蛋白表达水平显著性高于单纯放射组（P0.05），Bcl-2蛋白表达水平显著性低于单纯放射组（P0.01）；20μmol·L-1Gen和20μmol·L-1Gen+6Gy联合作用均使Bax蛋白表达高于正常对照组（P0.05），且联合作用组的Bax蛋白表达水平显著性高于20μmol·L-1Gen组和单纯放射组（P0.01）；将各组之间的Bcl-2/Bax mRNA及蛋白表达比值进行比较，20μmol·L-1Gen、6Gy单纯放射和20μmol·L-1Gen+6Gy组的Bcl-2/Bax比值均较正常对照组减小（P0.05），且联合作用组显著性小于20μmol·L-1Gen组以及6Gy单纯放射组（P0.01）；20μmol·L-1Gen和6Gy单纯放射均使Survivin蛋白表达显著性低于正常对照组（P0.01），但20μmol·L-1Gen+6Gy联合作用组却无显著性变化（P0.05）。结果表明，染料木黄酮对SKOV3细胞的放射增敏机制可能是通过增加ROS、MDA含量来加剧SKOV3细胞的放射损伤，最终通过下调Bcl-2蛋白、上调Bax mRNA及蛋白表达水平和降低Bcl-2/Bax比值来诱导SKOV3细胞凋亡。
[Abstract]:Radiotherapy, that is, the use of ionizing radiation, such as X-ray, X-ray or electron beam, is one of the main methods for treating malignant tumor. But due to the biological characteristics of the tumor cells and the local microenvironment, the sensitivity of certain tumor cells to the radiation is low, the radiation cannot effectively kill the tumor cells, and the effect of the radiotherapy is poor. in ord to improve that effect of the radiation therapy, some drugs are used in combination with radiation therapy, which is called a radiation-sensitization agent, which is used in combination with the radiotherapy, and the radiation damage repair is inhibited, the cell cycle is regulated, the apoptosis of the tumor cells is induced, So that the radiation sensitivity of the tumor cells can be enhanced, the irradiation dose is reduced, and the damage to the normal cells is reduced. Ovarian cancer is one of the most common malignant tumors of the gynaecology, and the patient's death rate is the first of all kinds of gynecological tumors. The patients with ovarian cancer may be treated by operation, radiotherapy, chemotherapy or the above method depending on the stage of the disease. In the radiation therapy, the sensitivity of various ovarian cancer cells to the radiation is different, the radiation quantity required by the cancer cell death and the tolerance amount of the normal tissue are small, so that the defects of poor radiation therapy effect or serious damage to the normal tissues and the like are overcome, It is necessary to explore the possibility of the radiosensitizer in the treatment of ovarian cancer. In this paper, three representative flavonoids of genistein (Genistein, Gen), Quercetin, Que and Chryl (Chrysin, Chr) were used as sensitizing drugs to treat human ovarian serous papillary cystadenocarcinoma cell line SKOV3 not sensitive to radiotherapy as the fine of ovarian cancer. In order to improve the radiosensitivity of the tumor cells, the radiosensitizing effect and the mechanism of the flavonoid compounds were studied by X-ray irradiation and the pharmacological properties of the flavonoids. Possibility, with a view to providing a solid basis for further research on the radiosensitization of flavonoids The specific content is as follows: (1) the flavonoid compound is fine for SKOV3 The cytotoxicity of three flavonoids to SKOV3 cells was screened by MTT method. The results showed that, after 24,48 and 72 h of SKOV3 cells, the survival rate of SKOV3 cells decreased significantly after 24,48 and 72 h of SKOV3 cells (P <0.05). 0.05) The non-toxic concentration of genistein, quercetin and chrysin on SKOV3 cells for 72 h should be less than 31.12, 15.30, 20.77. mu.mol 路 L-1, respectively. relatively low in sex. (ii) the flavonoids are fine for SKOV3 The radiosensitizing effect of the cells was in the range of 2.5 to 30. m u.mol 路 L-1Gen, 1.25 to 15.mu. mol 路 L-1Que and 1.25 to 20. m u.mol 路 L-1Chr, which were pre-acted on SKOV3 cells, X-ray (2,4,6,8,10 Gy) before the irradiation. ) MTT after single exposure for 72 h The radiosensitizing effect of three flavonoids on SKOV3 cells was 1.37-2.06, 1.20-1.80 and 1.23-1.83, respectively. The value of SER increased with the increase of drug concentration (P0.01), D0, Dq and N. The results showed that the radiosensitizing effect of the genistein, quercetin and chrysin on SKOV3 cells in a certain concentration range, the radiosensitivity of SKOV3 cells was reduced, the radiosensitivity was enhanced, and the release of the genistein was enhanced. The effect of radiosensitization is relatively strong. (3) the effect of genistein on SKOV 5,10,20 & mu; mol 路 L-1Gen of 3-cell radiosensitization mechanism were pre-acted on SKOV3 cells and X-ray (2,4,6 Gy) for 24 and 48 h before irradiation, and the ROS in the cells was detected by using the kit. The content of MDA and T-AOC were measured by flow cytometry. The cell cycle distribution was detected by means of PI single-staining and flow cytometry. The cell apoptosis was detected by using the Hoechst33342 single-dye method in combination with the fluorescence microscope. The expression of Bcl-2, Bax, Survivin and N was detected by RT-PCR and Western Blot. The levels of ROS, MDA and apoptosis in the SKOV3 cells of the combined radiation group were significantly higher than those in the same time point (P0. (05) The content of ROS, MDA and the apoptosis rate of the combined radiation group were significantly higher than that of the same concentration alone or in the same dose group (P0.05), and the content of ROS, MDA and the rate of apoptosis were increased with the increase of the drug concentration and the radiation dose (P0.05). Compared with the normal control group in the same time point, the T-AOC level of the combined radiation group and the proportion of the G1 phase cells decreased significantly (P0.05), and the proportion of the G2/ M cells increased (P0.05), and there was a certain correlation with the radiation dose (P0.05). The expression of Bcl-2, Bax mRNA and NF-EMAB mRNA and protein in SKOV3 cells was up-regulated and the expression of Bcl-2 protein was significantly reduced (P <0.05). 1) The expression of Bax mRNA, NF-EMAB mRNA and protein in the combined action group was significantly higher than that of the pure radiation group (P0.05). The expression level of Bcl-2 protein was significantly lower than that of the pure radiation group (P0.01). The combination of 20 & mu; mol 路 L-1Gen and 20 & mu; mol 路 L-1Gen + 6Gy all made Bax egg The expression of Bax in the combination group was significantly higher than that in the normal control group (P0.05). The expression of Bax protein in the combined group was significantly higher than that of the control group (P 0.01). The expression of Bcl-2/ Bax mRNA and protein in the group was compared, and the Bcl-2/ Bax ratio of 20. m u.mol 路 L-1Gen,6 Gy alone and 20. m u.mol 路 L-1Gen + 6 Gy group was compared. The expression of Survivin was significantly lower than that of control group (P 0.01), but the expression of Survivin was significantly lower than that in control group (P0.01), but 20. mu.mol 路 L-1Gen + 6Gy combined. The results showed that the radiosensitization mechanism of the genistein on SKOV3 cells could increase the radiation damage of SKOV3 cells by increasing the content of ROS and MDA.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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