【摘要】：放療，即利用電離輻射如X射線、γ射線或電子束等殺傷腫瘤細(xì)胞，是目前治療惡性腫瘤的主要手段之一。但是由于腫瘤細(xì)胞的生物學(xué)特性及局部微環(huán)境等的影響，導(dǎo)致某些腫瘤細(xì)胞對射線的敏感性較低，射線不能有效地殺傷腫瘤細(xì)胞，因而放療的效果較差。為了改善放療效果，臨床上將某些藥物與放療聯(lián)合使用，稱之為放射增敏劑（radiosensitizingagents），其與放療聯(lián)用，通過選擇性地殺傷乏氧腫瘤細(xì)胞、抑制放射損傷修復(fù)、調(diào)節(jié)細(xì)胞周期、誘導(dǎo)腫瘤細(xì)胞凋亡等，來增強腫瘤細(xì)胞的放射敏感性，降低照射劑量，減少對正常細(xì)胞的損傷。 卵巢癌是婦科常見的惡性腫瘤之一，其患者死亡率居各類婦科腫瘤的首位。卵巢癌依病期不同，可采用手術(shù)、放療、化療或以上方式綜合治療。但在放療中，由于各類卵巢癌細(xì)胞對射線的敏感性不同，且癌細(xì)胞致死所需放射量和正常組織的耐受量差異很小，故有放療效果欠佳或?qū)φ＝M織的損傷較重等弊端，因此，有必要對放射增敏劑在卵巢癌放療中應(yīng)用的可能性進行探討。 本實驗選用染料木黃酮（Genistein，Gen）、槲皮素（Quercetin，Que）和白楊素（Chrysin，Chr）三種代表性的黃酮類化合物作為增敏藥物，以對放療不敏感的人卵巢漿液性乳頭狀囊腺癌細(xì)胞株SKOV3作為卵巢癌的細(xì)胞模型，采用X射線照射，從提高腫瘤細(xì)胞放射敏感性的方法入手，并結(jié)合黃酮類化合物自身的藥理學(xué)特性，研究黃酮類化合物的放射增敏作用及其機制，并探討了黃酮類化合物作為放射增敏劑的可能性，以期為黃酮類化合物在放射增敏方面的進一步研究提供實驗依據(jù)。具體內(nèi)容如下：（一）黃酮類化合物對SKOV3細(xì)胞的毒性 采用MTT法篩選出三種黃酮類化合物對SKOV3細(xì)胞的無毒濃度范圍。結(jié)果表明，超出一定濃度范圍的染料木黃酮、槲皮素和白楊素作用于SKOV3細(xì)胞24，48，72h后，與同時間點內(nèi)的正常對照組相比，SKOV3細(xì)胞存活率均顯著性下降（P0.05），且呈現(xiàn)一定的濃度和時間依賴性（P0.05）。染料木黃酮、槲皮素和白楊素作用于SKOV3細(xì)胞72h的無毒濃度應(yīng)分別小于31.12，15.30，20.77μmol·L-1，說明染料木黃酮的細(xì)胞毒性相對較低。 （二）黃酮類化合物對SKOV3細(xì)胞的放射增敏作用 無毒濃度范圍內(nèi)的黃酮類化合物，即2.5～30μmol·L-1Gen、1.25～15μmol·L-1Que和1.25～20μmol·L-1Chr，分別于照射前2h預(yù)作用于SKOV3細(xì)胞，X射線（2，4，6，8，10Gy）單次照射后72h，采用MTT法檢測放射增敏作用。三種黃酮類化合物對SKOV3細(xì)胞的放射增敏比（SER）分別為1.37～2.06，1.20～1.80和1.23～1.83，三種黃酮類化合物的各藥物濃度組的SER值均有顯著性差異（P0.01），放射生物學(xué)參數(shù)D0、Dq和N值均較單純放射組顯著性減�。≒0.05），且隨藥物濃度的增大，SER值增大（P0.01），D0、Dq和N值減�。≒0.05）。結(jié)果表明，一定濃度范圍內(nèi)的染料木黃酮、槲皮素和白楊素均對SKOV3細(xì)胞有放射增敏作用，使SKOV3細(xì)胞的放射損傷修復(fù)能力降低，放射敏感性增強，且染料木黃酮的放射增敏作用相對較強。 （三）染料木黃酮對SKOV3細(xì)胞放射增敏機制的研究 5,10,20μmol·L-1Gen分別于照射前2h預(yù)作用于SKOV3細(xì)胞，X射線(2,4,6Gy)單次照射后24，48h，采用試劑盒檢測細(xì)胞內(nèi)ROS、MDA含量及T-AOC水平，采用PI單染法結(jié)合流式細(xì)胞儀檢測細(xì)胞周期分布，采用Hoechst33342單染法結(jié)合熒光顯微鏡檢測細(xì)胞凋亡，采用RT-PCR及Western Blot檢測Bcl-2、Bax、Survivin、NF-κB mRNA及蛋白表達。 照射后24，48h，單純加藥組、單純放射組以及加藥聯(lián)合放射組的SKOV3細(xì)胞內(nèi)ROS、MDA含量及細(xì)胞凋亡率均顯著性高于同時間點內(nèi)的正常對照組（P0.05），加藥聯(lián)合放射組的ROS、MDA含量及細(xì)胞凋亡率比相同濃度單純加藥組或相同劑量單純放射組的顯著增加（P0.05），且隨藥物濃度和放射劑量的增大，ROS、MDA含量及細(xì)胞凋亡率相應(yīng)增加（P0.05）；與同時間點內(nèi)的正常對照組相比，單純放射組和加藥聯(lián)合放射組的T-AOC水平、G1期細(xì)胞比例顯著性降低（P0.05），，G2/M期細(xì)胞比例上升（P0.05），且與放射劑量存在一定的相關(guān)性（P0.05），但無藥物濃度依賴性（P0.05）。 照射后48h，與正常對照組相比，6Gy單純放射和20μmol·L-1Gen+6Gy聯(lián)合作用均使SKOV3細(xì)胞的Bcl-2、Bax mRNA和NF-κB mRNA及蛋白表達顯著性上調(diào)，Bcl-2蛋白表達顯著性下調(diào)（P0.01），且聯(lián)合作用組的Bax mRNA、NF-κB mRNA及蛋白表達水平顯著性高于單純放射組（P0.05），Bcl-2蛋白表達水平顯著性低于單純放射組（P0.01）；20μmol·L-1Gen和20μmol·L-1Gen+6Gy聯(lián)合作用均使Bax蛋白表達高于正常對照組（P0.05），且聯(lián)合作用組的Bax蛋白表達水平顯著性高于20μmol·L-1Gen組和單純放射組（P0.01）；將各組之間的Bcl-2/Bax mRNA及蛋白表達比值進行比較，20μmol·L-1Gen、6Gy單純放射和20μmol·L-1Gen+6Gy組的Bcl-2/Bax比值均較正常對照組減�。≒0.05），且聯(lián)合作用組顯著性小于20μmol·L-1Gen組以及6Gy單純放射組（P0.01）；20μmol·L-1Gen和6Gy單純放射均使Survivin蛋白表達顯著性低于正常對照組（P0.01），但20μmol·L-1Gen+6Gy聯(lián)合作用組卻無顯著性變化（P0.05）。 結(jié)果表明，染料木黃酮對SKOV3細(xì)胞的放射增敏機制可能是通過增加ROS、MDA含量來加劇SKOV3細(xì)胞的放射損傷，最終通過下調(diào)Bcl-2蛋白、上調(diào)Bax mRNA及蛋白表達水平和降低Bcl-2/Bax比值來誘導(dǎo)SKOV3細(xì)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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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