本文選題：乳腺癌 + 整合素連接激酶��； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:乳腺癌是世界范圍內(nèi)女性中最常見(jiàn)的惡性腫瘤之一,其發(fā)病率位于女性所有腫瘤中的第一位。盡管乳腺癌主要的治療策略是外科手術(shù)結(jié)合放療和化療,但近年來(lái)一種全新的治療手段即分子靶向治療逐步被人們認(rèn)識(shí)并接受,特別是廣泛應(yīng)用于對(duì)上述療法無(wú)效的廣大患者。然而,由于調(diào)控乳腺癌進(jìn)程的分子機(jī)制仍然沒(méi)有完全闡明,故導(dǎo)致分子靶向治療缺乏有效的治療靶點(diǎn)。因此,探索乳腺癌細(xì)胞生長(zhǎng)的調(diào)控機(jī)制,將有利于提高乳腺癌分子靶向治療的效率和為其提供潛在的治療靶點(diǎn)。腫瘤細(xì)胞在侵襲和擴(kuò)散的過(guò)程中,依賴于腫瘤細(xì)胞與細(xì)胞外基質(zhì)之間粘附的缺失,而該過(guò)程可由整合素所介導(dǎo)。整合素連接激酶(Integrin-linked kinase,ILK)是一種重要的整合素絲氨酸-蘇氨酸蛋白激酶,其廣泛表達(dá)于人體各個(gè)組織中。先前的研究已經(jīng)表明,在多種腫瘤中均觀察到整合素連接激酶的表達(dá)上調(diào),并且整合素連接激酶還參與調(diào)節(jié)腫瘤細(xì)胞的增殖、細(xì)胞周期進(jìn)程、侵襲和遷移、細(xì)胞活性及腫瘤血管的發(fā)生等多種生理功能。在乳腺癌中,活化的整合素連接激酶可通過(guò)結(jié)合LIMD 2而促進(jìn)乳腺癌細(xì)胞的運(yùn)動(dòng)和轉(zhuǎn)移能力。然而,目前還未見(jiàn)關(guān)于整合素連接激酶參與乳腺癌細(xì)胞增殖的作用及其機(jī)制的報(bào)道。本研究利用基因轉(zhuǎn)染和RNAi來(lái)研究過(guò)表達(dá)及沉默整合素連接激酶基因?qū)θ橄侔┘?xì)胞增殖的作用及其機(jī)制,該研究將有助于人們進(jìn)一步認(rèn)識(shí)乳腺癌的發(fā)生機(jī)制,并為乳腺癌標(biāo)志物和靶點(diǎn)的確立提供重要的參考依據(jù)。方法:1、利用基因轉(zhuǎn)染的方法建立穩(wěn)定轉(zhuǎn)染ILK的MCF-7乳腺癌細(xì)胞株;WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)研究過(guò)表達(dá)ILK對(duì)MCF-7細(xì)胞增殖、活力及凋亡的影響;Western blot實(shí)驗(yàn)檢測(cè)MCF-7細(xì)胞過(guò)表達(dá)ILK對(duì)Akt、p-Akt1(Thr308)和PCNA基因蛋白表達(dá)的影響。2、利用RNAi方法建立低表達(dá)ILK的MDA-MB-231乳腺癌細(xì)胞株;WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)研究低表達(dá)ILK對(duì)MDA-MB-231細(xì)胞增殖、活力及凋亡的影響;Western blot實(shí)驗(yàn)檢測(cè)MDA-MB-231細(xì)胞低表達(dá)ILK對(duì)Akt、p-Akt1(Thr308)和PCNA基因蛋白表達(dá)的影響。3、采用Akt抑制劑LY294002來(lái)抑制過(guò)表達(dá)ILK的MCF-7細(xì)胞中p-Akt1(Thr308)的表達(dá);WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)研究在過(guò)表達(dá)ILK的MCF-7細(xì)胞中抑制PI3K/Akt通路對(duì)細(xì)胞增殖、活力及凋亡的影響;Western blot實(shí)驗(yàn)檢測(cè)封閉PI3K/Akt通路對(duì)過(guò)表達(dá)ILK的MCF-7細(xì)胞PCNA基因蛋白表達(dá)的影響。4、通過(guò)瞬時(shí)轉(zhuǎn)染HA-AKT使低表達(dá)ILK的MDA-MB-231細(xì)胞中PI3K/Akt通路被活化;WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)研究活化PI3K/Akt通路對(duì)低表達(dá)ILK的MDA-MB-231細(xì)胞增殖、活力及凋亡的影響;Western blot實(shí)驗(yàn)檢測(cè)活化PI3K/Akt通路對(duì)低表達(dá)ILK的MDA-MB-231細(xì)胞PCNA基因蛋白表達(dá)的影響。結(jié)果:1、WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)表明過(guò)表達(dá)ILK可以促進(jìn)MCF-7細(xì)胞的增殖和活力、抑制其凋亡;Western blot實(shí)驗(yàn)表明過(guò)表達(dá)ILK可上調(diào)MCF-7細(xì)胞中p-Akt1(Thr308)和PCNA基因的蛋白表達(dá),但Akt蛋白的表達(dá)量沒(méi)有受到ILK基因轉(zhuǎn)染的影響。2、WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)表明低表達(dá)ILK可以抑制MDA-MB-231細(xì)胞的增殖和活力、促進(jìn)其凋亡;Western blot實(shí)驗(yàn)表明MDA-MB-231細(xì)胞低表達(dá)ILK可下調(diào)p-Akt1(Thr308)和PCNA基因的蛋白表達(dá),但Akt蛋白的表達(dá)量沒(méi)有受到ILK基因沉默的影響。3、10μM的LY294002可顯著抑制過(guò)表達(dá)ILK的MCF-7細(xì)胞中p-Akt1(Thr308)的表達(dá),但Akt的表達(dá)不受影響,表明LY294002可有效地阻斷MCF-7細(xì)胞中PI3K/Akt通路;WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)表明封閉PI3K/Akt通路可抑制過(guò)表達(dá)ILK的MCF-7細(xì)胞的增殖和活力,促進(jìn)其凋亡。另外,Western blot實(shí)驗(yàn)表明封閉PI3K/Akt通路可下調(diào)過(guò)表達(dá)ILK的MCF-7細(xì)胞PCNA蛋白的表達(dá)。4、瞬時(shí)轉(zhuǎn)染HA-AKT可顯著上調(diào)低表達(dá)ILK的MDA-MB-231細(xì)胞中p-Akt1(Thr308)的表達(dá),但Akt的表達(dá)不受影響,表明瞬時(shí)轉(zhuǎn)染HA-AKT可有效活化MDA-MB-231細(xì)胞中PI3K/Akt通路;WST-1、BrdU摻入和LDH釋放實(shí)驗(yàn)表明活化PI3K/Akt通路可促進(jìn)低表達(dá)ILK的MDA-MB-231細(xì)胞的增殖和活力,抑制其凋亡。另外,Western blot實(shí)驗(yàn)表明活化PI3K/Akt通路可上調(diào)低表達(dá)ILK的MDA-MB-231細(xì)胞PCNA蛋白的表達(dá)。結(jié)論:本研究論證了ILK可通過(guò)活化PI3K/Akt通路而促進(jìn)乳腺癌細(xì)胞的增殖和活力,并抑制其凋亡,這表明ILK作為致癌基因在乳腺癌的發(fā)展中扮演著重要的作用。該研究成果將有助于人們對(duì)乳腺癌發(fā)生機(jī)制的進(jìn)一步認(rèn)識(shí),并可為乳腺癌的分子靶向治療提供有效的潛在靶點(diǎn)。
[Abstract]:Objective: breast cancer is one of the most common malignant tumors in women worldwide. The incidence of breast cancer is the first in all women's tumors. Although the main treatment strategy for breast cancer is surgery combined with radiotherapy and chemotherapy, a new treatment, molecular targeting therapy, is gradually recognized and accepted in recent years, especially in molecular targeting therapy. However, the molecular mechanism of the regulation of breast cancer is still not fully elucidated, which leads to the lack of effective therapeutic targets for molecular targeting therapy. Therefore, the study of the regulatory mechanism of the growth of breast cancer cells will be beneficial to the efficiency of molecular targeting therapy for breast cancer and to improve the efficiency of molecular targeting therapy for breast cancer. For potential therapeutic targets, tumor cells are dependent on the loss of adhesion between tumor cells and extracellular matrix during invasion and diffusion, and this process can be mediated by integrin. Integrin linked kinase (Integrin-linked kinase, ILK) is an important integrin serine - threonine protein kinase, which is widely expressed in human body In various tissues, previous studies have shown that the expression of integrin linked kinase is up-regulated in a variety of tumors, and integrin linked kinase is also involved in regulating the proliferation of tumor cells, cell cycle processes, invasion and migration, cell activity and the development of tumor vessels. In breast cancer, activation integration Hormone linked kinase can promote the movement and metastasis of breast cancer cells by combining LIMD 2. However, there is no report on the role and mechanism of integrin linked kinase in the proliferation of breast cancer cells. This study uses gene transfection and RNAi to study the expression and silencing of integrin linked kinase gene to breast cancer cells The effect and mechanism of proliferation will help people to further understand the mechanism of breast cancer and provide important reference for the establishment of markers and targets of breast cancer. Methods: 1, a MCF-7 breast cancer cell line stably transfected with ILK is established by gene transfection; WST-1, BrdU incorporation and LDH release experiment study the expression of I The effect of LK on the proliferation, vitality and apoptosis of MCF-7 cells. Western blot test detected the effect of MCF-7 cells over expression of ILK on the expression of Akt, p-Akt1 (Thr308) and PCNA gene protein. The effect of Western blot on the expression of Akt, p-Akt1 (Thr308) and PCNA gene protein by the low expression of ILK in MDA-MB-231 cells was detected by the Akt inhibitor LY294002. The effect of PI3K/Akt pathway on cell proliferation, activity and apoptosis; Western blot test detected the effect of closed PI3K/Akt pathway on the expression of PCNA gene protein in MCF-7 cells overexpressing ILK,.4 was activated by transient transfection of HA-AKT to the PI3K/Akt pathway in MDA-MB-231 cells with low expression of ILK. The effect of K/Akt pathway on the proliferation, activity and apoptosis of MDA-MB-231 cells with low expression of ILK. Western blot test was used to detect the effect of activated PI3K/Akt pathway on the expression of PCNA gene protein in MDA-MB-231 cells with low expression of ILK. Results: 1, WST-1, BrdU incorporation and LDH release experiments showed that overexpression could promote the proliferation and vitality of the cells and inhibit it. Apoptosis, Western blot test showed that overexpression of ILK could up regulate the protein expression of p-Akt1 (Thr308) and PCNA genes in MCF-7 cells, but the expression of Akt protein was not affected by ILK gene transfection,.2, WST-1. The results showed that low expression of ILK in MDA-MB-231 cells could down regulate the protein expression of p-Akt1 (Thr308) and PCNA gene, but the expression of Akt protein was not affected by ILK gene silencing, and LY294002 could inhibit the expression of ILK MCF-7 cells that overexpressed ILK, but the expression was not affected. It indicated that the Akt could be effectively blocked. The PI3K/Akt pathway in MCF-7 cells, WST-1, BrdU incorporation and LDH release experiments showed that closed PI3K/Akt pathway could inhibit the proliferation and vitality of MCF-7 cells overexpressing ILK and promote its apoptosis. The expression of p-Akt1 (Thr308) in MDA-MB-231 cells expressed in ILK was lowered, but the expression of Akt was not affected, which indicated that transient transfection of HA-AKT could effectively activate PI3K/Akt pathway in MDA-MB-231 cells; WST-1, BrdU incorporation and LDH release experiment showed that activation PI3K/Akt pathway could promote the proliferation and vitality of low expression cells and inhibit its apoptosis. In addition, the Western blot experiment showed that activated PI3K/Akt pathway could increase the expression of PCNA protein in MDA-MB-231 cells with low expression of ILK. Conclusion: This study demonstrated that ILK can promote the proliferation and activity of breast cancer cells by activating PI3K/Akt pathway and inhibit its apoptosis, which indicates that ILK as a oncogene plays an important role in the development of breast cancer. The results will help people to further understand the mechanism of breast cancer and provide a potential target for molecular targeting therapy for breast cancer.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 高海玉;吳爽;高世勇;季宇彬;;乳腺癌雌激素受體信號(hào)通路研究[J];哈爾濱商業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2016年06期

2 杜沛玲;方佳英;賈瀟岳;徐鎮(zhèn)喜;林昆;;1994～2013年中國(guó)女性乳腺癌流行病學(xué)特征[J];汕頭大學(xué)醫(yī)學(xué)院學(xué)報(bào);2016年02期

3 彭昭;田翠時(shí);高會(huì)斌;王貴生;;PCNA、8-OHdG在胃癌組織中的表達(dá)及意義[J];實(shí)用醫(yī)學(xué)雜志;2016年08期

4 段羊羊;井明晰;徐君南;孫濤;;乳腺癌分子靶向治療現(xiàn)狀[J];中國(guó)腫瘤;2016年03期

5 齊世美;呂俊;孟宇;戚之琳;凌烈鋒;;七葉皂苷鈉通過(guò)抑制AKT,ERK上游信號(hào)SRC活性誘導(dǎo)人乳腺癌MCF-7細(xì)胞凋亡[J];中國(guó)中藥雜志;2015年16期

6 趙莉琳;劉陽(yáng);;PCNA對(duì)口腔鱗癌細(xì)胞凋亡和增殖的影響[J];解放軍醫(yī)學(xué)雜志;2014年10期

7 Danny R.Youlden;Susanna M.Cramb;Cheng Har Yip;Peter D.Baade;;Incidence and mortality of female breast cancer in the AsiaPacific region[J];Cancer Biology & Medicine;2014年02期

8 李澤豪;任小元;王世兵;孔祥東;;介導(dǎo)siRNA傳遞的非病毒載體及其研究進(jìn)展[J];生命科學(xué);2014年04期

9 李娜;;肺癌VEGF及PCNA的表達(dá)及其與預(yù)后的關(guān)系[J];陜西醫(yī)學(xué)雜志;2014年01期

10 費(fèi)洪榮;趙瑩;王桂玲;曲曉蘭;王鳳澤;;PI3K/mTOR雙重抑制劑PF-04691502誘導(dǎo)人胃癌SGC-7901細(xì)胞凋亡[J];中國(guó)病理生理雜志;2013年11期

相關(guān)碩士學(xué)位論文 前2條

1 龐雪利;IL-8促進(jìn)人乳腺癌細(xì)胞EMT的作用及其機(jī)制研究[D];重慶醫(yī)科大學(xué);2015年

2 王澤民;P53、PCNA、VEGF在膀胱尿路上皮癌中的表達(dá)及對(duì)預(yù)后的影響[D];承德醫(yī)學(xué)院;2012年

，

本文編號(hào)：1986589