發(fā)布時間：2018-05-24 12:07

  本文選題：乳腺腫瘤 + 三陰性��； 參考：《濟(jì)南大學(xué)》2017年碩士論文

【摘要】：目的與背景:乳腺癌處于女性惡性腫瘤的首要位置。三陰性乳腺癌(triple negative breast cancer,TNBC)屬于乳腺癌luminal分型中的一種特殊的分子亞型,其特點(diǎn)為雌激素受體、孕激素受體及表皮生長因子受體2均陰性表達(dá)。TNBC侵襲性高,具有首發(fā)年齡小、病情進(jìn)一步發(fā)展時間快、影像學(xué)表現(xiàn)各不相同、非常容易復(fù)發(fā)轉(zhuǎn)移等特點(diǎn)。據(jù)相關(guān)的研究表明在中國女性中,TNBC的發(fā)病率大約占到了乳腺癌發(fā)病率的17%。由于相關(guān)受體均陰性表達(dá),TNBC沒有內(nèi)分泌和HER-2靶向治療的機(jī)會,目前化療是TNBC主要的治療方法。TNBC惡性程度很高、病人預(yù)后很差,這些特點(diǎn)與表皮生長因子受體(Epidermal growth factor receptor,EGFR)在TNBC患者中高度表達(dá)密切相關(guān)。但是最近發(fā)現(xiàn)EGFR靶向治療的相關(guān)研究并沒有取得理想的療效,療效不理想的原因主要和EGFR分子靶向治療過程中耐藥性的產(chǎn)生相關(guān)。如何增強(qiáng)TNBC靶向治療敏感性成為目前研究的新熱點(diǎn)。有報(bào)道表明在EGFR異常表達(dá)的腫瘤中,可以檢測到自噬表達(dá)水平發(fā)生了明顯改變。自噬是真核生物所特有生理病理現(xiàn)象,它可被外界多種刺激(如放療、化療、靶向治療)激活。在腫瘤形成過程中,自噬的激活能夠?yàn)榱鲶w提供必需的營養(yǎng)及能量供給,使腫瘤組織免于治療損傷而得以繼續(xù)存活,這也是腫瘤發(fā)生耐藥的重要機(jī)制。阻斷腫瘤細(xì)胞在靶向治療過程中增加的自噬水平,可以顯著提高腫瘤治療的敏感性。目前相關(guān)研究表明,在異常表達(dá)EGFR的非小細(xì)胞肺癌細(xì)胞中,抑制自噬的表達(dá)水平,可以明顯增強(qiáng)非小細(xì)胞肺癌細(xì)胞對吉非替尼治療的敏感性。然而,在TNBC細(xì)胞中,自噬與EGFR靶向治療的研究目前缺乏相關(guān)研究報(bào)道。方法:本研究通過吉非替尼單獨(dú)用藥或者聯(lián)合兩種作用機(jī)制不同的自噬抑制劑3甲基腺嘌呤(3-methyladenine,3MA)和巴弗洛霉素A1(Bafilomycin A1,BAF)處理TNBC細(xì)胞系MDA-MB-468和MDA-MB-231細(xì)胞,采用MTT實(shí)驗(yàn)方法、克隆形成實(shí)驗(yàn)方法檢測腫瘤細(xì)胞的增殖抑制率與克隆形成率,通過應(yīng)用流式細(xì)胞儀檢測腫瘤細(xì)胞的周期分布、Western blot免疫印跡法檢測與DNA損傷相關(guān)的標(biāo)志性蛋白和線粒體凋亡相關(guān)標(biāo)志性蛋白的表達(dá),進(jìn)一步驗(yàn)證在TNBC細(xì)胞系MDA-MB-468和MDA-MB-231中,自噬抑制劑對EGFR抑制劑吉非替尼治療敏感性的具體機(jī)制。通過ROS染色測定實(shí)驗(yàn)明確自噬抑制劑與吉非替尼聯(lián)合用藥時導(dǎo)致的凋亡與線粒體凋亡的關(guān)系。建立BALB/c裸鼠移植瘤模型,通過裸鼠體內(nèi)實(shí)驗(yàn)驗(yàn)證自噬抑制劑對吉非替尼治療敏感性的影響,免疫組化方法檢測移植瘤中Cleaved-CASP 3的表達(dá)。結(jié)果:吉非替尼+3MA組和吉非替尼+BAF組MDA-MB-468細(xì)胞的IC50分別為(4.1±0.2)μmol/L和(3.8±0.3)μmol/L,均明顯低于吉非替尼組[(7.0±0.2)μmol/L,均P0.05]。吉非替尼+3MA組和吉非替尼+BAF組MDA-MB-231細(xì)胞的IC50分別為(9.7±0.1)μmol/L和(7.7±0.2)μmol/L,均明顯低于吉非替尼組[(14.7±0.1)μmol/L,均P0.05]�？寺⌒纬蓪�(shí)驗(yàn)顯示,吉非替尼與自噬抑制劑聯(lián)合用藥組MDA-MB-468細(xì)胞克隆形成率分別為(29.85±5.54)%和(33.58±6.31)%,顯著低于單用吉非替尼組(63.46±8.32)%(Z=-2.309,P0.017)。吉非替尼與自噬抑制劑聯(lián)合用藥組MDA-MB-231細(xì)胞克隆形成率分別為(22.63±6.27)%和(31.54±5.81)%,顯著低于單用吉非替尼組(49.35±6.56)%(Z=-2.309,P0.017)。吉非替尼聯(lián)兩種作用機(jī)制不同的自噬抑制劑3MA和BAF,誘導(dǎo)了TNBC細(xì)胞系MDA-MB-468和MDA-MB-231細(xì)胞DNA發(fā)生損傷,誘導(dǎo)了TNBC細(xì)胞發(fā)生G0/G1期細(xì)胞周期阻滯。吉非替尼聯(lián)合3MA和BAF處理TNBC細(xì)胞,導(dǎo)致細(xì)胞氧自由基ROS的表達(dá)水平明顯增加。Western Blot免疫印跡實(shí)驗(yàn)發(fā)現(xiàn)線粒體凋亡相關(guān)蛋白細(xì)胞色素C、BAX、cleaved CASP 3表達(dá)水平明顯增加。裸鼠移植瘤模型實(shí)驗(yàn)發(fā)現(xiàn)自噬抑制劑聯(lián)合吉非替尼處理后,MDA-MB-468細(xì)胞腫瘤的生長受到明顯的抑制。結(jié)論:本研究表明自噬抑制劑可以增強(qiáng)TNBCMDA-MB-468和MDA-MB-231細(xì)胞對EGFR靶向治療的敏感性。并且這種作用與線粒體凋亡相關(guān)途徑的激活有關(guān)。
[Abstract]:Objective and background: breast cancer is the primary position of female malignant tumor. Three triple negative breast cancer (TNBC) belongs to a special molecular subtype in the luminal classification of breast cancer, which is characterized by estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 negative expression of.TNBC, which has high invasiveness and is the first one. In Chinese women, the incidence of TNBC is about 17%. due to negative expression of related receptors, and TNBC does not have the opportunity for endocrine and HER-2 targeting therapy, and chemotherapy is present. It is the main treatment of TNBC.TNBC with high malignancy and poor prognosis. These characteristics are closely related to the high expression of Epidermal growth factor receptor (EGFR) in TNBC patients. However, the recent discovery of the related research on EGFR targeting therapy has not achieved an ideal effect. It is related to the production of drug resistance in the course of EGFR molecular targeted therapy. How to enhance the sensitivity of TNBC targeting therapy has become a new focus of research. It is reported that the level of autophagy can be detected in the abnormal expression of EGFR. Autophagy is a special physiological pathological phenomenon in eukaryotes. Stimulation (such as radiotherapy, chemotherapy, targeting therapy) activation. In the process of tumor formation, activation of autophagy can provide the necessary nutrition and energy supply for the tumor and keep the tumor tissue from treatment damage and continue to survive, which is an important mechanism of tumor resistance. Leveling can significantly increase the sensitivity of tumor therapy. Current studies have shown that inhibition of autophagy expression in non small cell lung cancer cells with abnormal expression of EGFR can significantly enhance the sensitivity of non small cell lung cancer cells to gefitinib treatment. However, there is a lack of research on autophagy and EGFR targeting therapy in TNBC cells. Methods: This study treated TNBC cell lines MDA-MB-468 and MDA-MB-231 cells by using gefitinib alone or combined with two different mechanisms of autophagic inhibitor 3-methyladenine (3MA) and buffalamycin A1 (Bafilomycin A1, BAF). The experimental method of MTT was used to clone and form an experimental method for detection of swelling. The proliferation inhibition rate and clone formation rate of tumor cells were detected by flow cytometry, and the expression of marker proteins associated with DNA damage and mitochondrial apoptosis related proteins were detected by Western blot immunoblotting, and the autophagy inhibitor was further verified in the TNBC cell line MDA-MB-468 and MDA-MB-231. A specific mechanism for the sensitivity of EGFR inhibitor gefitinib in the treatment of gefitinib. The relationship between apoptosis and apoptosis induced by autophagy inhibitor and gefitinib was determined by ROS staining. A BALB/c nude mouse transplanted tumor model was established, and the effects of autophagy inhibition on gefitinib sensitivity were tested in nude mice. Immunohistochemical method was used to detect the expression of Cleaved-CASP 3 in the transplanted tumor. Results: the IC50 of MDA-MB-468 cells in gefitinib group +3MA and gefitinib group +BAF were respectively (4.1 + 0.2) mu mol/L and (3.8 + 0.3) mu mol/L, all significantly lower than that of gefitinib Group [(7 + 0.2) Mu mol/L, all P0.05]. gefitinib +3MA group and gefitinib group MDA-MB-231 cells 50 (9.7 + 0.1) mu mol/L and (7.7 + 0.2) mu mol/L were significantly lower than that of gefitinib Group [(14.7 + 0.1) mol/L. All P0.05]. clone formation experiments showed that the MDA-MB-468 cell clone formation rate of gefitinib combined with autophagy inhibitor group was (29.85 + 5.54)% and (33.58 + 9.7)% respectively, significantly lower than that of gefitinib group (63.46 + 8.32)% (63.46 + 8.32)%, respectively. Z=-2.309, P0.017). The rate of MDA-MB-231 cell clone formation in gefitinib combined with autophagic inhibitor group was (22.63 + 6.27)% and (31.54 + 5.81)% respectively, significantly lower than (49.35 + 6.56)% (Z=-2.309, P0.017) in the single use gefitinib group (Z=-2.309, P0.017). The different autophagy inhibitors, 3MA and BAF, were made by gefitinib, and TNBC cell line MDA-MB-468 was induced. The damage of MDA-MB-231 cell DNA induced the cell cycle arrest of TNBC cells at G0/G1 stage. Gefitinib combined with 3MA and BAF to treat TNBC cells, which resulted in a significant increase in the expression level of oxygen free radical ROS in the cell, and the.Western Blot immunoblotting experiment found that the mitochondrial apoptosis related protein cytochrome C was found. BAX, 3 expression level was obvious. Increase. Nude mice transplantation tumor model experiment found that the growth of MDA-MB-468 cell tumor was significantly inhibited after the autophagy inhibitor combined with gefitinib. Conclusion: This study suggests that autophagy inhibitors can enhance the sensitivity of TNBCMDA-MB-468 and MDA-MB-231 cells to EGFR targeting therapy. The activation is related.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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本文編號：1928989