X-射线辐射对乳腺癌细胞MDA-MB-231生物学特性的影响及蛋白组学研究
发布时间：2018-10-26 08:33
【摘要】：乳腺癌是危害女性健康常见恶性肿瘤之一。放射治疗是乳腺癌治疗的重要手段之一,然而由于个体或肿瘤细胞对射线的辐射敏感性存在差异,导致放疗疗效的差异。在放疗中,利用各种辅助手段降低肿瘤细胞的辐射抗性,增加其辐射敏感性,是提高肿瘤放疗疗效的重要途径。因此,研究X-射线对乳腺癌细胞生物学特性的影响及相关蛋白的表达变化,对于提高肿瘤细胞的放射敏感性并了解其相关分子调控机制具有重要的理论意义。1.X-射线辐射对乳腺癌细胞MDA-MB-231生物学特性的影响目的:比较不同剂量的X-射线辐射对乳腺癌MDA-MB-231细胞的增殖、周期和凋亡的影响,分析其生物学特性与细胞辐射敏感性关系。方法:选择指数生长期的乳腺癌MDA-MB-231细胞,不同剂量(0、1、2、4、8、10 Gy)的X-射线辐射后24 h,48 h和72 h采用MTT法检测MDA-MB-231的增殖能力,比较不同剂量的X-射线辐射对MDA-MB-231细胞的生长抑制作用;辐照后24 h和48 h,采用PI单染法和Annexin V/PI双染法分别检测细胞周期分布与凋亡,通过流式细胞仪计算并分析不同剂量的X-射线辐射对MDA-MB-231细胞周期与凋亡的影响。结果:MTT实验结果显示,X-射线辐射后,在24 h、48 h和72 h时间点上,各剂量点的MDA-MB-231细胞增殖能力减弱,其抑制率随剂量和时间的变化而变化。在48h和72h时间点,细胞的抑制率随剂量的增加而增加;但72 h各剂量点的抑制率显著低于48 h各剂量点的抑制率,24 h时间点上各剂量点和对照组相比均表现出一定的抑制作用;在各剂量点,细胞的抑制率都随着时间的延长呈先上升后下降的趋势。凋亡结果显示,不同剂量的X-射线辐射MDA-MB-231细胞后,在24 h和48 h时间点,细胞总凋亡率随着剂量的升高而增加,且同一剂量点在照射后24 h的总凋亡率大于48 h的总凋亡率。24 h和48 h各剂量点的早期凋亡率无明显差异,24 h不存在剂量依赖性,但48 h存在剂量依赖性,即随剂量的增加细胞凋亡逐渐增多;24 h和48 h各剂量点的晚期凋亡率存在明显差异,24 h和48 h均存在剂量依赖性,且同一剂量24 h的晚期凋亡率高于48 h的晚期凋亡率。细胞周期结果显示,X-射线辐射后24 h和48 h,乳腺癌MDA-MB-231细胞的周期影响主要表现为G2/M期周期阻滞,24h和48h均表现出细胞百分比随剂量的增加呈现递增趋势,低剂量(1,2 Gy)时,G2/M期阻滞没有显著性差异(P0.05),高剂量(4,8,10 Gy)照射时,G2/M期阻滞存在显著性差异(P0.05)。结论:X-射线辐射能有效的抑制乳腺癌MDA-MB-231细胞的增殖,使细胞周期阻滞在G2/M期,并诱导细胞凋亡。2.X-射线辐射后乳腺癌细胞MDA-MB-231差异蛋白表达质谱分析目的:分析X-射线辐射对MDA-MB-231细胞蛋白表达谱的影响,通过比较蛋白表达图谱,筛选、鉴定差异表达蛋白质,进而得到X-射线辐射对MDA-MB-231细胞作用的相关的分子标志物,为以后明确肿瘤放射敏感性的相关机制和相应辐射增敏产品的开发提供帮助。方法:选择对数生长期的MDA-MB-231细胞,4 Gy X-射线辐射(实验组)和不经X-射线辐射(对照组)后48h,对实验组和对照组的细胞总蛋白进行双向电泳,2D凝胶通过Versadoc4000 images system捕获图像。分别在凝胶上选取几个差异明显的蛋白点进行挖点、酶解、萃取、上样,随后使用API 4800串联飞行时间质谱仪MALDI-TOF/TOF(Applied Biosystems)进行分析。结果:通过分析比较,本实验一共筛选出32个蛋白差异点。与对照组相比,实验组细胞中共有17个蛋白点表现为上调,15个蛋白点表现为下调。分别在凝胶上选取32个差异明显的蛋白点进行质谱鉴定,蛋白质谱结果显示8个差异蛋白的具体信息,它们分别是:HSC70,GRP78,IMPDH2,EIF4H,GAPDH,VIM和微管相关蛋白(TUBA1B,TUBA8)。其中HSC70,GRP78,IMPDH2蛋白表达上调,EIF4H,GAPDH,VIM和微管相关蛋白(TUBA1B,TUBA8)表达下调。结论:X-射线辐射影响MDA-MB-231细胞蛋白表达谱。差异表达蛋白质谱鉴定结果显示,X-射线辐射诱导MDA-MB-231细胞热休克蛋白70家族成员HSC70,次黄嘌呤核苷酸脱氢酶(IMPDH2),葡萄糖调节蛋白78(GRP78),真核翻译起始因子(EIF4H),甘油醛-3-磷酸脱氢酶(GAPDH),波形蛋白(VIM)和微管相关蛋白(TUBA1B,TUBA8)表达变化。说明这些蛋白作为MDA-MB-231细胞对X-射线辐射的效应物,可能参与细胞的辐射敏感性的调节,同时,这些蛋白可能成为乳腺癌临床放射治疗的生物靶点。
[Abstract]:Breast cancer is one of the most common malignant tumors in women. Radiotherapy is one of the most important methods in the treatment of breast cancer. However, there is a difference in radiation sensitivity of the individual or tumor cells to radiation, which leads to the difference in the therapeutic effect of radiotherapy. In the radiotherapy, the radiation resistance of tumor cells is reduced by various auxiliary means, and the radiation sensitivity of the tumor cells is increased, which is an important way to improve the curative effect of tumor radiotherapy. Therefore, the effects of X-ray on the biological characteristics of breast cancer cells and the expression of related proteins were studied. The effect of X-ray radiation on the biological characteristics of breast cancer cells MDA-MB-231 was studied: the proliferation of MDA-MB-231 cells in breast cancer was compared with X-ray radiation at different doses. The effects of cycle and apoptosis were analyzed, and the relationship between biological characteristics and cell radiation sensitivity was analyzed. Methods: The proliferation ability of MDA-MB-231 cells, different doses (0, 1, 2, 4, 8, 10 Gy) was detected by MTT assay at 24 h, 48 h and 72 h after irradiation of breast cancer MDA-MB-231 cells in exponential growth phase. The growth of MDA-MB-231 cells was inhibited by different doses of X-ray radiation, 24 h and 48 h after irradiation. Cell cycle distribution and apoptosis were detected by PI single-stain method and Annexin V/ PI double staining method. The effects of X-ray radiation on the cell cycle and apoptosis of MDA-MB-231 were calculated and analyzed by flow cytometry. Results: After X-ray irradiation, MDA-MB-231 cell proliferation ability was decreased at 24 h, 48 h and 72 h after X-ray irradiation, and the inhibition rate was changed with dose and time. At 48h and 72h, the inhibition rate of cells increased with the increase of dosage; however, the inhibition rate of each dose point at 72 hours was significantly lower than that of each dose point at 48h, and the inhibition rate of each dose point in 24h time point showed a certain inhibitory effect compared with the control group; at each dose point, The inhibition rate of cells decreased with time. Apoptosis results showed that the total apoptosis rate increased with the increase of dose at 24 h and 48 h after irradiation with different doses of X-ray radiation MDA-MB-231 cells. At the same dose point, the total apoptosis rate of 24 h after irradiation was more than 48 h. There was no significant difference in the early apoptotic rate of 24 h and 48 h, but there was no dose-dependent dose in 24 h. There was a significant difference in the late apoptotic rate between 24 h and 48 h, and there was dose-dependent ratio between 24 h and 48 h, and the late apoptotic rate of the same dose of 24 h was higher than that in late 48 h. Cell cycle results showed that the cycle effect of MDA-MB-231 cells in breast cancer was mainly expressed as G2/ M period block after irradiation for 24 h and 48 h after X-ray irradiation, and both 24h and 48h showed that the percentage of cells increased with the increase of dose, and at low dose (1, 2 Gy), There was no significant difference between G2/ M phase block and G2/ M block (P0.05). Conclusion: X-ray radiant energy can effectively inhibit the proliferation of MDA-MB-231 cells in breast cancer, arrest the cell cycle arrest in G2/ M phase and induce apoptosis. The expression mass spectrum analysis of breast cancer cell MDA-MB-231 after X-ray radiation is analyzed. The effect of X-ray radiation on the expression profile of MDA-MB-231 cells is analyzed. By comparing the protein expression patterns, screening and identifying differentially expressed proteins, the molecular markers related to the action of the X-ray radiation on MDA-MB-231 cells are obtained, and help is provided for the relevant mechanisms for determining the radiosensitivity of the tumor and the development of corresponding radiation sensitizing products. Methods: MDA-MB-231 cells, 4 Gy X-ray radiation (experimental group) and X-ray radiation (control group) were selected for 48h after the logarithmic growth phase, and the total proteins in the experimental group and the control group were subjected to two-dimensional electrophoresis, and the 2D gel was used to capture images through the Veradoc4000images system. Several distinct protein spots were selected on the gel for digging, enzymatic hydrolysis, extraction, and the like, followed by analysis using an API 4,800 series flight time mass spectrometer Mach-TOF/ TOF (Applied Biosystems). Results: A total of 32 protein difference points were screened through analysis and comparison. Compared with the control group, 17 protein spots in the experimental group showed up-regulation and 15 protein spots were down-regulated. Thirty-two distinct protein spots on the gel were identified by mass spectrometry. The results showed that the specific information of 8 differential proteins were HSC70, GRP78, IMPDH2, EIF4H, GAPDH, VIM and microtubule-associated proteins (TUBA1B, TUBA8). wherein HSC70, GRP78, IMPDH2 protein expression upregulated, EIF4H, GAPDH, VIM and tubulin-related proteins (TUBA1B, TUBA8) were downregulated. Conclusion: X-ray radiation affects the expression of MDA-MB-231 cell protein. The results of differential expression of protein spectrum showed that the X-ray radiation induced MDA-MB-231 cell heat shock protein 70 family member HSC70, submandibular gland dehydrogenase (IMPDH2), glucose regulation protein 78 (GRP78), true nuclear translation initiation factor (EIF4H), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Changes in expression of p27 protein (VIM) and tubulin-associated protein (TUBA1B, TUBA8). The effects of these proteins as MDA-MB-231 cells on X-ray radiation may be involved in the regulation of radiation sensitivity of cells, and these proteins may be targets for clinical radiotherapy for breast cancer.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9
本文编号：2295158
[Abstract]:Breast cancer is one of the most common malignant tumors in women. Radiotherapy is one of the most important methods in the treatment of breast cancer. However, there is a difference in radiation sensitivity of the individual or tumor cells to radiation, which leads to the difference in the therapeutic effect of radiotherapy. In the radiotherapy, the radiation resistance of tumor cells is reduced by various auxiliary means, and the radiation sensitivity of the tumor cells is increased, which is an important way to improve the curative effect of tumor radiotherapy. Therefore, the effects of X-ray on the biological characteristics of breast cancer cells and the expression of related proteins were studied. The effect of X-ray radiation on the biological characteristics of breast cancer cells MDA-MB-231 was studied: the proliferation of MDA-MB-231 cells in breast cancer was compared with X-ray radiation at different doses. The effects of cycle and apoptosis were analyzed, and the relationship between biological characteristics and cell radiation sensitivity was analyzed. Methods: The proliferation ability of MDA-MB-231 cells, different doses (0, 1, 2, 4, 8, 10 Gy) was detected by MTT assay at 24 h, 48 h and 72 h after irradiation of breast cancer MDA-MB-231 cells in exponential growth phase. The growth of MDA-MB-231 cells was inhibited by different doses of X-ray radiation, 24 h and 48 h after irradiation. Cell cycle distribution and apoptosis were detected by PI single-stain method and Annexin V/ PI double staining method. The effects of X-ray radiation on the cell cycle and apoptosis of MDA-MB-231 were calculated and analyzed by flow cytometry. Results: After X-ray irradiation, MDA-MB-231 cell proliferation ability was decreased at 24 h, 48 h and 72 h after X-ray irradiation, and the inhibition rate was changed with dose and time. At 48h and 72h, the inhibition rate of cells increased with the increase of dosage; however, the inhibition rate of each dose point at 72 hours was significantly lower than that of each dose point at 48h, and the inhibition rate of each dose point in 24h time point showed a certain inhibitory effect compared with the control group; at each dose point, The inhibition rate of cells decreased with time. Apoptosis results showed that the total apoptosis rate increased with the increase of dose at 24 h and 48 h after irradiation with different doses of X-ray radiation MDA-MB-231 cells. At the same dose point, the total apoptosis rate of 24 h after irradiation was more than 48 h. There was no significant difference in the early apoptotic rate of 24 h and 48 h, but there was no dose-dependent dose in 24 h. There was a significant difference in the late apoptotic rate between 24 h and 48 h, and there was dose-dependent ratio between 24 h and 48 h, and the late apoptotic rate of the same dose of 24 h was higher than that in late 48 h. Cell cycle results showed that the cycle effect of MDA-MB-231 cells in breast cancer was mainly expressed as G2/ M period block after irradiation for 24 h and 48 h after X-ray irradiation, and both 24h and 48h showed that the percentage of cells increased with the increase of dose, and at low dose (1, 2 Gy), There was no significant difference between G2/ M phase block and G2/ M block (P0.05). Conclusion: X-ray radiant energy can effectively inhibit the proliferation of MDA-MB-231 cells in breast cancer, arrest the cell cycle arrest in G2/ M phase and induce apoptosis. The expression mass spectrum analysis of breast cancer cell MDA-MB-231 after X-ray radiation is analyzed. The effect of X-ray radiation on the expression profile of MDA-MB-231 cells is analyzed. By comparing the protein expression patterns, screening and identifying differentially expressed proteins, the molecular markers related to the action of the X-ray radiation on MDA-MB-231 cells are obtained, and help is provided for the relevant mechanisms for determining the radiosensitivity of the tumor and the development of corresponding radiation sensitizing products. Methods: MDA-MB-231 cells, 4 Gy X-ray radiation (experimental group) and X-ray radiation (control group) were selected for 48h after the logarithmic growth phase, and the total proteins in the experimental group and the control group were subjected to two-dimensional electrophoresis, and the 2D gel was used to capture images through the Veradoc4000images system. Several distinct protein spots were selected on the gel for digging, enzymatic hydrolysis, extraction, and the like, followed by analysis using an API 4,800 series flight time mass spectrometer Mach-TOF/ TOF (Applied Biosystems). Results: A total of 32 protein difference points were screened through analysis and comparison. Compared with the control group, 17 protein spots in the experimental group showed up-regulation and 15 protein spots were down-regulated. Thirty-two distinct protein spots on the gel were identified by mass spectrometry. The results showed that the specific information of 8 differential proteins were HSC70, GRP78, IMPDH2, EIF4H, GAPDH, VIM and microtubule-associated proteins (TUBA1B, TUBA8). wherein HSC70, GRP78, IMPDH2 protein expression upregulated, EIF4H, GAPDH, VIM and tubulin-related proteins (TUBA1B, TUBA8) were downregulated. Conclusion: X-ray radiation affects the expression of MDA-MB-231 cell protein. The results of differential expression of protein spectrum showed that the X-ray radiation induced MDA-MB-231 cell heat shock protein 70 family member HSC70, submandibular gland dehydrogenase (IMPDH2), glucose regulation protein 78 (GRP78), true nuclear translation initiation factor (EIF4H), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Changes in expression of p27 protein (VIM) and tubulin-associated protein (TUBA1B, TUBA8). The effects of these proteins as MDA-MB-231 cells on X-ray radiation may be involved in the regulation of radiation sensitivity of cells, and these proteins may be targets for clinical radiotherapy for breast cancer.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9
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