X-射線輻射對(duì)乳腺癌細(xì)胞MDA-MB-231生物學(xué)特性的影響及蛋白組學(xué)研究
發(fā)布時(shí)間：2018-10-26 08:33
【摘要】：乳腺癌是危害女性健康常見惡性腫瘤之一。放射治療是乳腺癌治療的重要手段之一,然而由于個(gè)體或腫瘤細(xì)胞對(duì)射線的輻射敏感性存在差異,導(dǎo)致放療療效的差異。在放療中,利用各種輔助手段降低腫瘤細(xì)胞的輻射抗性,增加其輻射敏感性,是提高腫瘤放療療效的重要途徑。因此,研究X-射線對(duì)乳腺癌細(xì)胞生物學(xué)特性的影響及相關(guān)蛋白的表達(dá)變化,對(duì)于提高腫瘤細(xì)胞的放射敏感性并了解其相關(guān)分子調(diào)控機(jī)制具有重要的理論意義。1.X-射線輻射對(duì)乳腺癌細(xì)胞MDA-MB-231生物學(xué)特性的影響目的:比較不同劑量的X-射線輻射對(duì)乳腺癌MDA-MB-231細(xì)胞的增殖、周期和凋亡的影響,分析其生物學(xué)特性與細(xì)胞輻射敏感性關(guān)系。方法:選擇指數(shù)生長期的乳腺癌MDA-MB-231細(xì)胞,不同劑量(0、1、2、4、8、10 Gy)的X-射線輻射后24 h,48 h和72 h采用MTT法檢測MDA-MB-231的增殖能力,比較不同劑量的X-射線輻射對(duì)MDA-MB-231細(xì)胞的生長抑制作用;輻照后24 h和48 h,采用PI單染法和Annexin V/PI雙染法分別檢測細(xì)胞周期分布與凋亡,通過流式細(xì)胞儀計(jì)算并分析不同劑量的X-射線輻射對(duì)MDA-MB-231細(xì)胞周期與凋亡的影響。結(jié)果:MTT實(shí)驗(yàn)結(jié)果顯示,X-射線輻射后,在24 h、48 h和72 h時(shí)間點(diǎn)上,各劑量點(diǎn)的MDA-MB-231細(xì)胞增殖能力減弱,其抑制率隨劑量和時(shí)間的變化而變化。在48h和72h時(shí)間點(diǎn),細(xì)胞的抑制率隨劑量的增加而增加;但72 h各劑量點(diǎn)的抑制率顯著低于48 h各劑量點(diǎn)的抑制率,24 h時(shí)間點(diǎn)上各劑量點(diǎn)和對(duì)照組相比均表現(xiàn)出一定的抑制作用;在各劑量點(diǎn),細(xì)胞的抑制率都隨著時(shí)間的延長呈先上升后下降的趨勢。凋亡結(jié)果顯示,不同劑量的X-射線輻射MDA-MB-231細(xì)胞后,在24 h和48 h時(shí)間點(diǎn),細(xì)胞總凋亡率隨著劑量的升高而增加,且同一劑量點(diǎn)在照射后24 h的總凋亡率大于48 h的總凋亡率。24 h和48 h各劑量點(diǎn)的早期凋亡率無明顯差異,24 h不存在劑量依賴性,但48 h存在劑量依賴性,即隨劑量的增加細(xì)胞凋亡逐漸增多;24 h和48 h各劑量點(diǎn)的晚期凋亡率存在明顯差異,24 h和48 h均存在劑量依賴性,且同一劑量24 h的晚期凋亡率高于48 h的晚期凋亡率。細(xì)胞周期結(jié)果顯示,X-射線輻射后24 h和48 h,乳腺癌MDA-MB-231細(xì)胞的周期影響主要表現(xiàn)為G2/M期周期阻滯,24h和48h均表現(xiàn)出細(xì)胞百分比隨劑量的增加呈現(xiàn)遞增趨勢,低劑量(1,2 Gy)時(shí),G2/M期阻滯沒有顯著性差異(P0.05),高劑量(4,8,10 Gy)照射時(shí),G2/M期阻滯存在顯著性差異(P0.05)。結(jié)論:X-射線輻射能有效的抑制乳腺癌MDA-MB-231細(xì)胞的增殖,使細(xì)胞周期阻滯在G2/M期,并誘導(dǎo)細(xì)胞凋亡。2.X-射線輻射后乳腺癌細(xì)胞MDA-MB-231差異蛋白表達(dá)質(zhì)譜分析目的:分析X-射線輻射對(duì)MDA-MB-231細(xì)胞蛋白表達(dá)譜的影響,通過比較蛋白表達(dá)圖譜,篩選、鑒定差異表達(dá)蛋白質(zhì),進(jìn)而得到X-射線輻射對(duì)MDA-MB-231細(xì)胞作用的相關(guān)的分子標(biāo)志物,為以后明確腫瘤放射敏感性的相關(guān)機(jī)制和相應(yīng)輻射增敏產(chǎn)品的開發(fā)提供幫助。方法:選擇對(duì)數(shù)生長期的MDA-MB-231細(xì)胞,4 Gy X-射線輻射(實(shí)驗(yàn)組)和不經(jīng)X-射線輻射(對(duì)照組)后48h,對(duì)實(shí)驗(yàn)組和對(duì)照組的細(xì)胞總蛋白進(jìn)行雙向電泳,2D凝膠通過Versadoc4000 images system捕獲圖像。分別在凝膠上選取幾個(gè)差異明顯的蛋白點(diǎn)進(jìn)行挖點(diǎn)、酶解、萃取、上樣,隨后使用API 4800串聯(lián)飛行時(shí)間質(zhì)譜儀MALDI-TOF/TOF(Applied Biosystems)進(jìn)行分析。結(jié)果:通過分析比較,本實(shí)驗(yàn)一共篩選出32個(gè)蛋白差異點(diǎn)。與對(duì)照組相比,實(shí)驗(yàn)組細(xì)胞中共有17個(gè)蛋白點(diǎn)表現(xiàn)為上調(diào),15個(gè)蛋白點(diǎn)表現(xiàn)為下調(diào)。分別在凝膠上選取32個(gè)差異明顯的蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定,蛋白質(zhì)譜結(jié)果顯示8個(gè)差異蛋白的具體信息,它們分別是:HSC70,GRP78,IMPDH2,EIF4H,GAPDH,VIM和微管相關(guān)蛋白(TUBA1B,TUBA8)。其中HSC70,GRP78,IMPDH2蛋白表達(dá)上調(diào),EIF4H,GAPDH,VIM和微管相關(guān)蛋白(TUBA1B,TUBA8)表達(dá)下調(diào)。結(jié)論:X-射線輻射影響MDA-MB-231細(xì)胞蛋白表達(dá)譜。差異表達(dá)蛋白質(zhì)譜鑒定結(jié)果顯示,X-射線輻射誘導(dǎo)MDA-MB-231細(xì)胞熱休克蛋白70家族成員HSC70,次黃嘌呤核苷酸脫氫酶(IMPDH2),葡萄糖調(diào)節(jié)蛋白78(GRP78),真核翻譯起始因子(EIF4H),甘油醛-3-磷酸脫氫酶(GAPDH),波形蛋白(VIM)和微管相關(guān)蛋白(TUBA1B,TUBA8)表達(dá)變化。說明這些蛋白作為MDA-MB-231細(xì)胞對(duì)X-射線輻射的效應(yīng)物,可能參與細(xì)胞的輻射敏感性的調(diào)節(jié),同時(shí),這些蛋白可能成為乳腺癌臨床放射治療的生物靶點(diǎn)。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9
本文編號(hào)：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9
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