本文選題：MCF-7 + 高糖　； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：乳腺癌是全球婦女最常見的惡性腫瘤,2015年美國癌癥協(xié)會(ACS)的年度統(tǒng)計數(shù)據(jù)報告顯示,婦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癥相關(guān)死亡的第二位。由于腫瘤細(xì)胞利用葡萄糖提供維持其增殖必需的營養(yǎng)源,因此高血糖為乳腺癌細(xì)胞的存活和生長提供了絕佳的環(huán)境。體內(nèi)的高糖環(huán)境是促使腫瘤細(xì)胞增殖、分化和遷移的重要因素,高血糖或糖尿病可增加乳腺癌的發(fā)病風(fēng)險和死亡風(fēng)險。高血糖或糖尿病合并乳腺癌已經(jīng)嚴(yán)重威脅人類健康,給經(jīng)濟社會和家庭帶來了沉重的經(jīng)濟和精神壓力。因此闡明高血糖或糖尿病與乳腺癌發(fā)生發(fā)展的關(guān)系、機制和治療靶點,尋求一種相對安全有效的治療方式,對于控制高血糖或糖尿病合并乳腺癌的發(fā)展具有非常重要的臨床意義。近年來,傳統(tǒng)中藥在腫瘤中的應(yīng)用已經(jīng)成為了研究的熱點。漢黃芩素是傳統(tǒng)中草藥黃芩的有效成分之一,屬于黃酮類化合物,已有諸多文獻(xiàn)報道黃酮具有多種藥理作用,包括抗氧化,抗炎,抗病毒,抗腫瘤和抗過敏活性。諸多體外實驗研究證實了漢黃芩素的抗腫瘤活性,包括其對骨髓性白血病細(xì)胞,原發(fā)性肝癌細(xì)胞,乳腺癌細(xì)胞,鼠肉瘤細(xì)胞,肺癌細(xì)胞,前列腺癌細(xì)胞和膀胱癌細(xì)胞的抑制作用；而對外周血單個核細(xì)胞和成纖維細(xì)胞并沒有誘導(dǎo)細(xì)胞凋亡的作用。目前國內(nèi)外文獻(xiàn)中對漢黃芩素抑制乳腺癌的作用機制報道不一,且漢黃芩素對高糖誘導(dǎo)的乳腺癌細(xì)胞的抑制作用未有報道。結(jié)合已有的國內(nèi)外文獻(xiàn)中的研究報道和本研究前期實驗結(jié)果,我們在體外模擬高糖環(huán)境,比較乳腺癌細(xì)胞在正常糖濃度和高糖濃度下的生長情況,觀察漢黃芩素對不同環(huán)境下乳腺癌細(xì)胞的抑制作用,合理假設(shè)漢黃芩素可通過MAPKs信號通路,激活某一種或多種MAPKs亞型,從而抑制高糖誘導(dǎo)的人乳腺癌細(xì)胞的增殖,侵襲及轉(zhuǎn)移。本研究主要包括以下兩個部分：第一部分高濃度葡萄糖對人乳腺癌MCF-7細(xì)胞的影響作用目的：研究高濃度葡萄糖對人乳腺癌MCF-7細(xì)胞生存力,遷移及侵襲的影響,并探究其可能的作用機制。方法：采用MTT法檢測不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的相對活力；運用Transwell法檢測不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的侵襲能力；采用細(xì)胞劃痕實驗觀察不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的遷移情況；采用TUNEL和DAPI雙染法觀察并分析不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的凋亡情況；運用蛋白免疫印跡(Western-blotting)技術(shù)檢測不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞中AKT, PKCδ,及MAPKs各亞型總蛋白和磷酸化蛋白的表達(dá)情況；應(yīng)用醫(yī)學(xué)專用統(tǒng)計學(xué)軟件對上述數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析。結(jié)果：(1)與正常濃度(5.5mM)葡萄糖組相比,MTT法測得的高濃度(11mM, 22mM)葡萄糖組MCF-7相對細(xì)胞活力明顯升高,且暴露于葡萄糖時間越長,存活的細(xì)胞數(shù)增加越明顯,兩者差異有統(tǒng)計學(xué)意義p0.05,0.01)。(2)與正常濃度(5.5mM)葡萄糖組相比,Transwell法測得的高濃度(11mM,22mM)葡萄糖組穿過基底膜侵入下室的MCF-7細(xì)胞明顯增加,兩者差異有統(tǒng)計學(xué)意義,(p0.05,0.011。(3)與正常濃度(5.5mM)葡萄糖組相比,劃痕實驗觀察到高濃度(11mM, 22mM)葡萄糖組劃痕處MCF-7細(xì)胞覆蓋率增加,兩者差異有統(tǒng)計學(xué)意義,(p0.05,0.011。(4)與正常濃度(5.5mM)葡萄糖組相比,高濃度(11mM,22mM)葡萄糖組TUNEL法顯示紅色熒光的MCF-7細(xì)胞數(shù)減少,DAPI法顯示的藍(lán)色熒光的細(xì)胞數(shù)增加,兩者差異有統(tǒng)計學(xué)意義,p0.05,0.01)。(5)與正常濃度(5.5mM)葡萄糖組相比,Western-blotting檢測的高濃度(11mM,22mM)葡萄糖組AKT和PKC5的磷酸化水平明顯增加,兩者差異有統(tǒng)計學(xué)意義,p0.05)。而P38MAPK的磷酸化水平降低,兩者差異有統(tǒng)計學(xué)意義,p0.05)。結(jié)論：(1)高濃度葡萄糖可提高人乳腺癌MCF-7細(xì)胞生存活力,促進(jìn)侵襲和遷移,抑制凋亡。(2)高濃度葡萄糖可通過激活A(yù)KT和PKCΠ磷酸化,抑制P38MAPK磷酸化而實現(xiàn)其促腫瘤生長與進(jìn)展的作用。第二部分漢黃芩素對高糖誘導(dǎo)的MCF-7細(xì)胞生長的抑制作用及機制研究目的：研究漢黃芩素對高糖誘導(dǎo)的MCF-7細(xì)胞活力,侵襲和遷移的抑制及促凋亡作用,并對其作用機制進(jìn)行深入探究。方法：采用MTT法檢測漢黃芩素處理后不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的相對活力；運用Transwell法檢測漢黃芩素處理后不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的侵襲能力；采用細(xì)胞劃痕實驗觀察漢黃芩素處理后不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的遷移情況；采用TUNEL和DAPI雙染法觀察并分析漢黃芩素處理后不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞的凋亡情況；運用蛋白免疫印跡(Western-blotting)技術(shù)檢測漢黃芩素處理后不同濃度葡萄糖環(huán)境下MCF-7細(xì)胞中AKT, PKCδ及MAPKs各亞型總蛋白和磷酸化蛋白的表達(dá)情況；為了進(jìn)一步證明P38MAPK信號通路在漢黃芩素抑制腫瘤的作用,我們加入P38 MAPK抑制劑SB203580和P38shRNA瞬時轉(zhuǎn)染沉默P38基因重復(fù)上述實驗,并觀察結(jié)果。應(yīng)用醫(yī)學(xué)專用統(tǒng)計學(xué)軟件對上述數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析。結(jié)果：(1)與未經(jīng)處理的細(xì)胞相比,MTT法檢測的經(jīng)漢黃芩素處理的高濃度葡萄糖(11mM,22mM)的細(xì)胞活力明顯降低,且葡萄糖濃度越高,抑制作用越明顯,兩者差異有顯著統(tǒng)計學(xué)意義(p0.05,0.01)。(2)與未經(jīng)處理的細(xì)胞相比,Transwell法檢測到經(jīng)漢黃芩素處理的高濃度葡萄糖(11mM,22mM)的培養(yǎng)基內(nèi)侵入到小室內(nèi)的細(xì)胞數(shù)明顯減少,兩者差異有統(tǒng)計學(xué)意義(p0.05)。(3)與未經(jīng)處理的細(xì)胞相比,劃痕實驗觀察到加入漢黃芩素處理的高濃度葡萄糖(11mM,22mM)的培養(yǎng)基內(nèi),MCF-7細(xì)胞遷移數(shù)量明顯減少,接近初始狀態(tài),兩者差異有統(tǒng)計學(xué)意義(p0.05)。(4)與未經(jīng)處理的細(xì)胞相比,經(jīng)漢黃芩素處理的高濃度(11mM,22mM)葡萄糖組TUNEL法顯示紅色熒光的MCF-7細(xì)胞數(shù)明顯增加,DAPI法顯示的藍(lán)色熒光的細(xì)胞數(shù)明顯減少,兩者差異有顯著統(tǒng)計學(xué)意義(p0.01)。(5)與未經(jīng)處理的細(xì)胞相比,Western-blotting檢測到經(jīng)漢黃芩素處理的高濃度(11mM,22mM)葡萄糖組AKT和PKCδ的磷酸化水平降低,而P38磷酸化水平升高,差異有統(tǒng)計學(xué)意義(p0.05)。(6)加入P38 MAPK抑制劑后SB203580,高濃度葡萄糖(11mM和22mM)組P-P38與P38總蛋白比例顯著下降,MCF-7細(xì)胞的相對活力增強,細(xì)胞遷移和侵襲增加,凋亡減少,差異有統(tǒng)計學(xué)意義(p0.05,0.01)。(7)與對照組相比,P38shRNA培養(yǎng)基中高濃度葡萄糖(11mM和22mM)下P-P38與P38總蛋白比例顯著下降,MCF-7細(xì)胞的相對活力增強,細(xì)胞遷移和侵襲增加,凋亡減少,差異有統(tǒng)計學(xué)意義(p0.05,0.01)。結(jié)論：漢黃芩素可通過抑制AKT和PKCδ的磷酸化和激活P38MAPK磷酸化抑制高糖誘導(dǎo)的MCF-7細(xì)胞生存,遷移和侵襲,促進(jìn)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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.9

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