汉黄芩素抑制高糖诱导的人乳腺癌细胞生长的机制研究

发布时间：2018-05-13 17:22

本文选题：MCF-7 + 高糖　；参考：《武汉大学》2016年博士论文

【摘要】：乳腺癌是全球妇女最常见的恶性肿瘤,2015年美国癌症协会(ACS)的年度统计数据报告显示,妇女乳腺癌的新增病例仍居首位,而乳腺癌的死亡率居女性癌症相关死亡的第二位。由于肿瘤细胞利用葡萄糖提供维持其增殖必需的营养源,因此高血糖为乳腺癌细胞的存活和生长提供了绝佳的环境。体内的高糖环境是促使肿瘤细胞增殖、分化和迁移的重要因素,高血糖或糖尿病可增加乳腺癌的发病风险和死亡风险。高血糖或糖尿病合并乳腺癌已经严重威胁人类健康,给经济社会和家庭带来了沉重的经济和精神压力。因此阐明高血糖或糖尿病与乳腺癌发生发展的关系、机制和治疗靶点,寻求一种相对安全有效的治疗方式,对于控制高血糖或糖尿病合并乳腺癌的发展具有非常重要的临床意义。近年来,传统中药在肿瘤中的应用已经成为了研究的热点。汉黄芩素是传统中草药黄芩的有效成分之一,属于黄酮类化合物,已有诸多文献报道黄酮具有多种药理作用,包括抗氧化,抗炎,抗病毒,抗肿瘤和抗过敏活性。诸多体外实验研究证实了汉黄芩素的抗肿瘤活性,包括其对骨髓性白血病细胞,原发性肝癌细胞,乳腺癌细胞,鼠肉瘤细胞,肺癌细胞,前列腺癌细胞和膀胱癌细胞的抑制作用；而对外周血单个核细胞和成纤维细胞并没有诱导细胞凋亡的作用。目前国内外文献中对汉黄芩素抑制乳腺癌的作用机制报道不一,且汉黄芩素对高糖诱导的乳腺癌细胞的抑制作用未有报道。结合已有的国内外文献中的研究报道和本研究前期实验结果,我们在体外模拟高糖环境,比较乳腺癌细胞在正常糖浓度和高糖浓度下的生长情况,观察汉黄芩素对不同环境下乳腺癌细胞的抑制作用,合理假设汉黄芩素可通过MAPKs信号通路,激活某一种或多种MAPKs亚型,从而抑制高糖诱导的人乳腺癌细胞的增殖,侵袭及转移。本研究主要包括以下两个部分：第一部分高浓度葡萄糖对人乳腺癌MCF-7细胞的影响作用目的：研究高浓度葡萄糖对人乳腺癌MCF-7细胞生存力,迁移及侵袭的影响,并探究其可能的作用机制。方法：采用MTT法检测不同浓度葡萄糖环境下MCF-7细胞的相对活力；运用Transwell法检测不同浓度葡萄糖环境下MCF-7细胞的侵袭能力；采用细胞划痕实验观察不同浓度葡萄糖环境下MCF-7细胞的迁移情况；采用TUNEL和DAPI双染法观察并分析不同浓度葡萄糖环境下MCF-7细胞的凋亡情况；运用蛋白免疫印迹(Western-blotting)技术检测不同浓度葡萄糖环境下MCF-7细胞中AKT, PKCδ,及MAPKs各亚型总蛋白和磷酸化蛋白的表达情况；应用医学专用统计学软件对上述数据进行统计学分析。结果：(1)与正常浓度(5.5mM)葡萄糖组相比,MTT法测得的高浓度(11mM, 22mM)葡萄糖组MCF-7相对细胞活力明显升高,且暴露于葡萄糖时间越长,存活的细胞数增加越明显,两者差异有统计学意义p0.05,0.01)。(2)与正常浓度(5.5mM)葡萄糖组相比,Transwell法测得的高浓度(11mM,22mM)葡萄糖组穿过基底膜侵入下室的MCF-7细胞明显增加,两者差异有统计学意义,(p0.05,0.011。(3)与正常浓度(5.5mM)葡萄糖组相比,划痕实验观察到高浓度(11mM, 22mM)葡萄糖组划痕处MCF-7细胞覆盖率增加,两者差异有统计学意义,(p0.05,0.011。(4)与正常浓度(5.5mM)葡萄糖组相比,高浓度(11mM,22mM)葡萄糖组TUNEL法显示红色荧光的MCF-7细胞数减少,DAPI法显示的蓝色荧光的细胞数增加,两者差异有统计学意义,p0.05,0.01)。(5)与正常浓度(5.5mM)葡萄糖组相比,Western-blotting检测的高浓度(11mM,22mM)葡萄糖组AKT和PKC5的磷酸化水平明显增加,两者差异有统计学意义,p0.05)。而P38MAPK的磷酸化水平降低,两者差异有统计学意义,p0.05)。结论：(1)高浓度葡萄糖可提高人乳腺癌MCF-7细胞生存活力,促进侵袭和迁移,抑制凋亡。(2)高浓度葡萄糖可通过激活AKT和PKCΠ磷酸化,抑制P38MAPK磷酸化而实现其促肿瘤生长与进展的作用。第二部分汉黄芩素对高糖诱导的MCF-7细胞生长的抑制作用及机制研究目的：研究汉黄芩素对高糖诱导的MCF-7细胞活力,侵袭和迁移的抑制及促凋亡作用,并对其作用机制进行深入探究。方法：采用MTT法检测汉黄芩素处理后不同浓度葡萄糖环境下MCF-7细胞的相对活力；运用Transwell法检测汉黄芩素处理后不同浓度葡萄糖环境下MCF-7细胞的侵袭能力；采用细胞划痕实验观察汉黄芩素处理后不同浓度葡萄糖环境下MCF-7细胞的迁移情况；采用TUNEL和DAPI双染法观察并分析汉黄芩素处理后不同浓度葡萄糖环境下MCF-7细胞的凋亡情况；运用蛋白免疫印迹(Western-blotting)技术检测汉黄芩素处理后不同浓度葡萄糖环境下MCF-7细胞中AKT, PKCδ及MAPKs各亚型总蛋白和磷酸化蛋白的表达情况；为了进一步证明P38MAPK信号通路在汉黄芩素抑制肿瘤的作用,我们加入P38 MAPK抑制剂SB203580和P38shRNA瞬时转染沉默P38基因重复上述实验,并观察结果。应用医学专用统计学软件对上述数据进行统计学分析。结果：(1)与未经处理的细胞相比,MTT法检测的经汉黄芩素处理的高浓度葡萄糖(11mM,22mM)的细胞活力明显降低,且葡萄糖浓度越高,抑制作用越明显,两者差异有显著统计学意义(p0.05,0.01)。(2)与未经处理的细胞相比,Transwell法检测到经汉黄芩素处理的高浓度葡萄糖(11mM,22mM)的培养基内侵入到小室内的细胞数明显减少,两者差异有统计学意义(p0.05)。(3)与未经处理的细胞相比,划痕实验观察到加入汉黄芩素处理的高浓度葡萄糖(11mM,22mM)的培养基内,MCF-7细胞迁移数量明显减少,接近初始状态,两者差异有统计学意义(p0.05)。(4)与未经处理的细胞相比,经汉黄芩素处理的高浓度(11mM,22mM)葡萄糖组TUNEL法显示红色荧光的MCF-7细胞数明显增加,DAPI法显示的蓝色荧光的细胞数明显减少,两者差异有显著统计学意义(p0.01)。(5)与未经处理的细胞相比,Western-blotting检测到经汉黄芩素处理的高浓度(11mM,22mM)葡萄糖组AKT和PKCδ的磷酸化水平降低,而P38磷酸化水平升高,差异有统计学意义(p0.05)。(6)加入P38 MAPK抑制剂后SB203580,高浓度葡萄糖(11mM和22mM)组P-P38与P38总蛋白比例显著下降,MCF-7细胞的相对活力增强,细胞迁移和侵袭增加,凋亡减少,差异有统计学意义(p0.05,0.01)。(7)与对照组相比,P38shRNA培养基中高浓度葡萄糖(11mM和22mM)下P-P38与P38总蛋白比例显著下降,MCF-7细胞的相对活力增强,细胞迁移和侵袭增加,凋亡减少,差异有统计学意义(p0.05,0.01)。结论：汉黄芩素可通过抑制AKT和PKCδ的磷酸化和激活P38MAPK磷酸化抑制高糖诱导的MCF-7细胞生存,迁移和侵袭,促进凋亡。
[Abstract]:Breast cancer is the most common malignancy of women in the world. In 2015, the annual statistics of the American Cancer Association (ACS) reported that the new cases of women's breast cancer were still the first, and the death rate of breast cancer was second of the female cancer related deaths. Hyperglycemia provides an excellent environment for the survival and growth of breast cancer cells. High glucose environment in the body is an important factor contributing to the proliferation, differentiation and migration of tumor cells. Hyperglycemia or diabetes can increase the risk and risk of death of breast cancer. Hyperglycemia or diabetes combined with breast cancer has seriously threatened human health, giving economic society to the economic society. Therefore, it is important to clarify the relationship, mechanism and target of the development of hyperglycemia or diabetes and breast cancer, and to seek a relatively safe and effective treatment for the development of hyperglycemia or diabetes with breast cancer. The application of drug in the tumor has become a hot spot of research. The baicalein is one of the effective components of the traditional Chinese herbal medicine Scutellaria baicalensis, which belongs to the flavonoids. It has been reported that flavonoids have a variety of pharmacological effects, including antioxidant, anti-inflammatory, antiviral, antitumor and antiallergic activities. A lot of experiments in vitro confirmed the Scutellaria baicalensis. Antitumor activity, including its inhibitory effects on myeloid leukemia cells, primary liver cancer cells, breast cancer cells, rat sarcomarosarcoma cells, lung cancer cells, prostate cancer cells and bladder cancer cells, while peripheral blood mononuclear cells and fibroblasts do not induce apoptosis. At present, the Chinese and foreign literature on Scutellaria baicalensis The inhibitory effect of scutellarin on breast cancer was not reported, and the inhibitory effect of scutellarin on high glucose induced breast cancer cells was not reported. Combined with the previous studies in the literature and the results of the previous study, we simulated high glucose environment in vitro, compared the growth of breast cancer cells in normal sugar concentration and high glucose concentration. In a long time, the inhibitory effect of baicalein on breast cancer cells in different environments was observed. It is assumed that baicalein can activate one or more MAPKs subtypes through MAPKs signaling pathway, thus inhibiting the proliferation, invasion and metastasis of high glucose induced human breast cancer cells. This study mainly includes the following two parts: the first part of high concentration The effect of glucose on human breast cancer MCF-7 cells Objective: To study the effect of high glucose on the survival, migration and invasion of human breast cancer MCF-7 cells and explore its possible mechanism. Methods: the relative activity of MCF-7 cells under different concentrations of glucose was detected by MTT; the Transwell method was used to detect different concentrations. The invasion ability of MCF-7 cells in the glucose environment was observed and the migration of MCF-7 cells under different concentrations of glucose was observed by the cell scratch test. The apoptosis of MCF-7 cells under different concentrations of glucose was observed and analyzed by TUNEL and DAPI double staining. Protein immunization (Western-blotting) technique was used to detect the apoptosis of MCF-7 cells. The expression of AKT, PKC Delta, and MAPKs subtypes of total protein and phosphorylated protein in MCF-7 cells under the same concentration of glucose; the above data were statistically analyzed with special medical statistics software. Results: (1) the high concentration (11mM, 22mM) glucose group of the MTT method was relatively fine compared with the normal concentration (5.5mM) glucose group. The cell viability was significantly increased, and the longer the exposure to glucose, the more significant the number of surviving cells increased, the difference was statistically significant p0.05,0.01. (2) compared with the normal concentration (5.5mM) glucose group, the MCF-7 cells of the high concentration (11mM, 22mM) glucose group measured by the Transwell method were significantly increased through the basement membrane and the MCF-7 cells were intruded in the lower chamber. The difference between the two groups was significant. Statistical significance (p0.05,0.011. (3) compared with the normal concentration (5.5mM) glucose group, the scratch test observed that the MCF-7 cell coverage of the high concentration (11mM, 22mM) glucose group increased, and the difference was statistically significant. (p0.05,0.011. (4) compared with the normal concentration (5.5mM) glucose group, the TUNEL method of the high concentration (11mM, 22mM) glucose group was shown by TUNEL method. The number of MCF-7 cells in red fluorescence decreased and the number of blue fluorescent cells increased by DAPI method. The difference was statistically significant, p0.05,0.01). (5) compared with the normal concentration (5.5mM) glucose group, the phosphorylation level of AKT and PKC5 in the high concentration (11mM, 22mM) glucose group detected by Western-blotting was significantly increased, and the difference was statistically significant. P0.05). And the phosphorylation level of P38MAPK decreased, and the difference was statistically significant, P0.05). Conclusion: (1) high concentration glucose can improve the viability of human breast cancer MCF-7 cells, promote invasion and migration, inhibit apoptosis. (2) high concentration glucose can activate AKT and PKC phosphorylation to inhibit the phosphorylation of P38MAPK to achieve its tumor growth and growth promoting growth and tumor growth. The effect of the second part of scutellarin on the inhibition and mechanism of high glucose induced MCF-7 cell growth and its mechanism study: To study the inhibition and apoptosis effect of hutlutin on high glucose induced MCF-7 cell activity, invasion and migration, and to explore the mechanism of its action. Method: the treatment of scutellarin by MTT method after scutellarin treatment The relative vitality of MCF-7 cells in different concentration of glucose environment; Transwell assay was used to detect the invasion ability of MCF-7 cells under different concentration of glucose environment after Scutellaria baicalein treatment. The migration of MCF-7 cells under different concentration of glucose environment after scutellarin treatment was observed with Scutellaria baicalein, and TUNEL and DAPI double staining were used. The apoptosis of MCF-7 cells in different concentration of glucose environment after baicalein treatment was observed and analyzed. The expression of AKT, PKC Delta and MAPKs subtypes of total protein and phosphorylated protein in MCF-7 cells of different concentrations of MCF-7 cells treated with baicalein treated with baicalein (Western-blotting) were detected by protein immunoblotting. One step proved the effect of P38MAPK signaling pathway in scutellarin inhibiting the tumor. We added the P38 MAPK inhibitor SB203580 and P38shRNA transient transfection of the silent P38 gene to repeat the above experiments and observed the results. The statistical analysis of the above data was carried out with the special medical statistics software. (1) the MTT method was compared with the untreated cells. The cell viability of high concentration glucose (11mM, 22mM) treated by baicalein was significantly reduced, and the higher the glucose concentration, the more significant the inhibitory effect was, the difference was significant (p0.05,0.01). (2) compared with the untreated cells, the Transwell method detected the high concentration of glucose (11mM, 22mM) treated by Scutellaria baicalein. The number of cells in the medium intruded into the small chamber was significantly reduced, and the difference was statistically significant (P0.05). (3) compared with the untreated cells, the number of MCF-7 cells in the culture base of the high concentration glucose (11mM, 22mM), which was treated with Scutellaria baicalein, was significantly reduced, close to the initial state, and the difference was statistically significant. Significance (P0.05). (4) compared with untreated cells, the high concentration (11mM, 22mM) glucose group treated with baicalein TUNEL showed that the number of MCF-7 cells in red fluorescence increased significantly, and the number of blue fluorescent cells displayed by DAPI decreased significantly (P0.01). (5) Western-, compared with untreated cells, (P0.01). Blotting detected the phosphorylation level of AKT and PKC Delta in the high concentration (11mM, 22mM) glucose group treated with baicalein, and the level of P38 phosphorylation increased, and the difference was statistically significant (P0.05). (6) after P38 MAPK inhibitor was added to SB203580, the proportion of high concentration glucose (11mM and 22mM) and total protein decreased significantly. The cell migration and invasion increased and the apoptosis decreased, the difference was statistically significant (p0.05,0.01). (7) compared with the control group, the proportion of P-P38 and P38 total protein in the P38shRNA medium was significantly decreased, the relative viability of MCF-7 cells increased, the cell migration and invasion increased, the apoptosis decreased, and the difference was statistically significant. Study significance (p0.05,0.01). Conclusion: baicalein can inhibit the survival, migration and invasion of high glucose induced MCF-7 cells by inhibiting the phosphorylation of AKT and PKC Delta and activating P38MAPK phosphorylation and promoting apoptosis.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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