【摘要】：背景:肺癌是全世界常見(jiàn)的惡性腫瘤之一,在我國(guó),男性和女性的惡性腫瘤死亡率排第1位者均為肺癌。雖然對(duì)肺癌的治療有很多新的方法,但從目前情況來(lái)看,肺癌的治療效果仍然不理想,其5年生存率低于15%。大量研究表明,腫瘤干細(xì)胞(cancer stem cells,CSC)雖然在腫瘤細(xì)胞中占比很少,但卻是腫瘤發(fā)生、發(fā)展的關(guān)鍵,同時(shí)也是形成腫瘤并促成腫瘤不斷生長(zhǎng)、侵襲、局部復(fù)發(fā)和遠(yuǎn)處轉(zhuǎn)移的根源,也是影響肺癌預(yù)后效果的根本原因。針對(duì)性根除CSCs的藥物和治療方法仍在探索中。目的:本研究旨在觀(guān)察紫花牡荊素(Casticin)能選擇性地增強(qiáng)TRAIL表達(dá)以誘導(dǎo)肺癌干細(xì)胞樣細(xì)胞凋亡,并對(duì)其作用機(jī)制進(jìn)行探討,為Casticin的臨床應(yīng)用提供更加充分的理論依據(jù)。方法:采用干細(xì)胞條件培養(yǎng)基在低粘附的6孔板中培養(yǎng)及擴(kuò)增H446干細(xì)胞;用不同濃度Casticin和TRAIL蛋白以及固定濃度比例的Casticin與TRAIL合用將H446干細(xì)胞及普通細(xì)胞培養(yǎng)分成9組,分別用Casticin或TRAIL作用于這些細(xì)胞;第1組為未分選的普通H446細(xì)胞,培養(yǎng)基中未加Casticin或TRAIL;第2組為通過(guò)干細(xì)胞條件培養(yǎng)基分選的的H446干細(xì)胞樣細(xì)胞,培養(yǎng)基在不加Casticin或TRAIL;第3組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為1μmol/m L的Casticin;第4組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為3μmol/m L的Casticin;第5組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為10μmol/m L的Casticin;第6組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為1ng/L的TRAIL;第7組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為10ng/L的TRAIL;第8組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為100ng/L的TRAIL;第9組為H446干細(xì)胞樣細(xì)胞,培養(yǎng)基終濃度為100ng/L的TRAIL和3μmo L/L的Casticin;分別培養(yǎng)24h,然后在顯微鏡比較各組的成球大小與數(shù)目,并用ELISA,FACS,Western-bolt等實(shí)驗(yàn)方法檢測(cè)其對(duì)H446干細(xì)胞樣細(xì)胞的凋亡作用。用慢病毒過(guò)表達(dá)與敲除Fox M1的方式來(lái)探討Casticin選擇性增強(qiáng)TRAIL誘導(dǎo)H446干細(xì)胞凋亡的機(jī)制。結(jié)果:利用干細(xì)胞條件培養(yǎng)基培養(yǎng)成功獲得H446干細(xì)胞樣細(xì)胞并通過(guò)蛋白質(zhì)免疫印跡法(Western-bolt)和流式細(xì)胞儀檢測(cè)得到驗(yàn)證,其干細(xì)胞樣細(xì)胞標(biāo)志物UPAR明顯高于普通H446細(xì)胞;用不同濃度Casticin和TRAIL蛋白單獨(dú)及聯(lián)合作用于H446干細(xì)胞樣細(xì)胞,結(jié)果顯示,Casticin與TRAIL具有抑制H446干細(xì)胞樣細(xì)胞成球數(shù)目與成球大小的能力,且Casticin能選擇性增強(qiáng)TRAIL的作用,兩者合用可更加明顯的抑制H446干細(xì)胞樣細(xì)胞成球能力;通過(guò)Western-bolt,ELISA,FACS等實(shí)驗(yàn)方法發(fā)現(xiàn)Casticin可選擇性的增強(qiáng)TRAIL誘導(dǎo)H446干細(xì)胞樣細(xì)胞的凋亡,抗凋亡蛋白c-Filp,P56等的表達(dá)明顯降低;并采用慢病毒的過(guò)表達(dá)與敲除的方式確定Fox M1是Casticin選擇性增強(qiáng)TRAIL誘導(dǎo)H446干細(xì)胞樣細(xì)胞凋亡的重要基因。結(jié)論:1、首次證實(shí)Casticin具有促進(jìn)H446干細(xì)胞樣細(xì)胞凋亡的作用;2、Casticin可通過(guò)選擇性增強(qiáng)TRAIL引起H446干細(xì)胞樣細(xì)胞的凋亡,其機(jī)制可能與Fox M1有關(guān)。Fox M1是Casticin引起H446干細(xì)胞凋亡的可能靶基因之一。
[Abstract]:BACKGROUND: Lung cancer is one of the most common malignant tumors in the world. Although there are many new methods for the treatment of lung cancer, the therapeutic effect of lung cancer is still not ideal, and the 5-year survival rate is less than 15%. A large number of studies have shown that the tumor stem cells (CSC), while occupying less than a few in tumor cells, are the key to the tumorigenesis and development, and at the same time are the causes of the formation of tumors and the growth, invasion, local recurrence and distant metastasis of the tumor. And also is the root cause of the effect of the prognosis of the lung cancer. The drug and treatment methods of the targeted eradication of CSCs are still in the process of exploration. Objective: The purpose of this study was to observe the ability of Casticin to selectively enhance the expression of TRAIL in order to induce the apoptosis of stem cell-like cells of lung cancer. Methods: H446 stem cells were cultured and amplified in a 6-well plate with low adhesion by stem cell conditioned medium. H446 stem cells and normal cell culture were divided into 9 groups with Castcin and TRAIL protein at different concentrations and a fixed concentration ratio of Castcin and TRAIL, and the cells were treated with Castcin or TRAIL, respectively. The first group was the unsorted normal H446 cells, and no Castcin or TRAIL was found in the culture medium; the second group was H446 stem cell-like cells sorted by the stem cell conditioned medium, and the medium was not added with Castcin or TRAIL; the third group was H446 stem cell-like cells, and the final concentration of the culture medium was 1. m The fourth group was the H446 stem cell-like cell, the final concentration of the medium was 3. m u.mol/ m L of Casticin, the fifth group was the H446 stem cell-like cell, the final concentration of the medium was 10. m u.mol/ m L, and the sixth group was the H446 stem cell-like cell, the final concentration of the medium was 1 ng/ L of TRAIL, and the seventh group was H446 stem cell-like cells, The final concentration of the medium was 10 ng/ L of TRAIL, the 8th group was H446 stem cell-like cells, the final concentration of the medium was 100 ng/ L of TRAIL, the 9th group was H446 stem cell-like cells, the final concentration of the medium was 100 ng/ L of TRAIL and 3. m The apoptosis of H446 cell-like cells was detected by ELISA, FACS, Western-blot and other experimental methods. The mechanism of the selective enhancement of TRAIL-induced apoptosis of H446 stem cells was discussed with lentiviral overexpression and the way of knock-out Fox M1. Results: H446 stem cell-like cells were successfully obtained by the culture of stem cell conditioned medium, and the cell-like cell marker UPAR was significantly higher than that of normal H446 cells by Western-blot and flow cytometry. Castcin and TRAIL were used alone and in combination on H446 stem cell-like cells. The results showed that Castcin and TRAIL had the ability to inhibit the number of cells of H446 cell-like cells and the size of the ball, and Castcin can selectively enhance the effect of TRAIL. By Western-bolt, ELISA, FACS and other experimental methods, Castcin can selectively enhance the apoptosis of H446 stem cell-like cells, and the expression of anti-apoptotic protein c-Filp, P56 and so on is obviously reduced. And the overexpression and knockout of the lentivirus is used to determine that the Fox M1 is an important gene for the selective enhancement of TRAIL to induce the apoptosis of the H446 stem cell-like cell. Conclusion:1. The first time that Castcin has the effect of promoting the apoptosis of H446 stem cell-like cells, and 2, Castcin can induce the apoptosis of H446 stem cell-like cells by selective enhancement of TRAIL, and the mechanism may be related to Fox M1. Fox M1 is one of the possible target genes for the apoptosis of H446 stem cells.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R734.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張濤;李東;漆仲春;彭晶晶;陳滔;譚勇;周進(jìn)軍;許濤;付曾強(qiáng);劉煜;李華;;肝癌Huh7干細(xì)胞樣細(xì)胞的富集培養(yǎng)及鑒定[J];中國(guó)腫瘤生物治療雜志;2013年06期

2 易善永;南克俊;陳靜;姜麗麗;;吉西他濱聯(lián)合YH-16對(duì)肝癌干細(xì)胞樣細(xì)胞的影響[J];中國(guó)腫瘤生物治療雜志;2010年05期

3 趙麗琴;郭偉劍;張曉偉;屈曉飛;王小鳳;倪蘇婕;黃明主;;CBX7在胃癌干細(xì)胞樣細(xì)胞中的表達(dá)及其功能研究[J];中國(guó)癌癥雜志;2013年02期

4 周蓓;肖立紅;肖蕎;任凱群;曹建國(guó);;8-溴-7-甲氧基白楊素對(duì)Huh-7細(xì)胞系肝癌干細(xì)胞樣細(xì)胞自我更新的影響[J];湖南師范大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2013年01期

5 王欣榮;單保恩;艾軍;劉麗華;劉月彩;張超;張海譜;劉登湘;;小鼠乳腺癌4T1細(xì)胞中腫瘤干細(xì)胞樣細(xì)胞的富集和鑒定[J];中國(guó)腫瘤生物治療雜志;2010年04期

6 呂新全;索振河;馬長(zhǎng)路;趙阿紅;宋一民;李惠翔;;干細(xì)胞樣細(xì)胞在乳腺良惡性病變中的數(shù)量與分布[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2008年04期

7 張洪也;程勇;胡祥;呂原;武乃金;;LoVo細(xì)胞系中結(jié)腸癌干細(xì)胞樣細(xì)胞的分離、培養(yǎng)及鑒定[J];中國(guó)腫瘤生物治療雜志;2010年02期

8 朱小青;;膠質(zhì)瘤干細(xì)胞樣細(xì)胞的自我更新和致癌性與PI3K/mTOR及MEK/ERK信號(hào)通路的相互作用有關(guān)[J];中國(guó)病理生理雜志;2010年12期

9 張小麗;高建;賈茜;鄧濤;;肝癌干細(xì)胞樣細(xì)胞的分離及其耐藥性受PI3K/Akt通路調(diào)節(jié)[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2013年02期

10 仇波;林毅;王勇;陶鈞;王運(yùn)杰;;Bcl-2在人腦膠質(zhì)瘤干細(xì)胞樣細(xì)胞中的表達(dá)及病理相關(guān)性[J];中華神經(jīng)外科疾病研究雜志;2013年04期

相關(guān)會(huì)議論文 前4條

1 米彥軍;符立梧;;Axitinib對(duì)腫瘤干細(xì)胞樣細(xì)胞的作用及其機(jī)制研究[A];2011醫(yī)學(xué)科學(xué)前沿論壇第十二屆全國(guó)腫瘤藥理與化療學(xué)術(shù)會(huì)議論文集[C];2011年

2 焦?jié)?張松法;葉楓;呂衛(wèi)國(guó);程曉東;王新宇;謝幸;;CyclinD1參與卵巢癌干細(xì)胞樣細(xì)胞上皮向間質(zhì)轉(zhuǎn)變的調(diào)控[A];2011年浙江省婦產(chǎn)科學(xué)學(xué)術(shù)年會(huì)暨“婦產(chǎn)科常見(jiàn)疾病的臨床研究新進(jìn)展”學(xué)習(xí)班論文匯編[C];2011年

3 劉冰潔;李小平;王建六;魏麗惠;;子宮內(nèi)膜癌干細(xì)胞樣細(xì)胞特性初步研究[A];中華醫(yī)學(xué)會(huì)第十次全國(guó)婦產(chǎn)科學(xué)術(shù)會(huì)議婦科腫瘤會(huì)場(chǎng)（婦科腫瘤學(xué)組、婦科病理學(xué)組）論文匯編[C];2012年

4 賴(lài)東梅;劉特;黃勇;王麗華;程蔚蔚;;原代卵巢癌干細(xì)胞樣細(xì)胞的分離、培養(yǎng)及鑒定[A];第四屆長(zhǎng)三角婦產(chǎn)科學(xué)術(shù)論壇暨浙江省2009年婦產(chǎn)科學(xué)術(shù)年會(huì)論文匯編[C];2009年

相關(guān)博士學(xué)位論文 前5條

1 楊棟;川芎嗪、丹參酮ⅡA對(duì)PG干細(xì)胞樣細(xì)胞惡性生物學(xué)行為影響的研究[D];中國(guó)中醫(yī)科學(xué)院;2015年

2 張燁;上皮間質(zhì)轉(zhuǎn)化與胰腺癌腫瘤干細(xì)胞樣細(xì)胞的相關(guān)性研究[D];南京醫(yī)科大學(xué);2012年

3 沈明;負(fù)性共刺激分子B7-H1在A2B5陽(yáng)性人腦膠質(zhì)瘤干細(xì)胞樣細(xì)胞中的負(fù)性調(diào)控作用探討[D];復(fù)旦大學(xué);2011年

4 王新宇;卵巢癌干細(xì)胞樣細(xì)胞與耐紫杉醇細(xì)胞的差異表達(dá)蛋白譜特征及其臨床意義[D];浙江大學(xué);2014年

5 阮潔;化療富集卵巢癌腫瘤干細(xì)胞樣細(xì)胞及對(duì)相關(guān)干性基因表達(dá)影響的研究[D];北京協(xié)和醫(yī)學(xué)院;2009年

相關(guān)碩士學(xué)位論文 前8條

1 曹恒;人肝癌細(xì)胞系SMMC-7721中肝癌干細(xì)胞樣細(xì)胞的初步探討[D];河北醫(yī)科大學(xué);2015年

2 習(xí)臻暢;Casticin選擇性的增強(qiáng)TRAIL誘導(dǎo)H446干細(xì)胞凋亡[D];湖南師范大學(xué);2015年

3 胡慧;人絨癌干細(xì)胞樣細(xì)胞的體內(nèi)鑒定及其化療耐藥特性的初步研究[D];中南大學(xué);2014年

4 燕麗;5Fu對(duì)乳腺癌細(xì)胞向腫瘤干細(xì)胞樣細(xì)胞逆向分化的作用[D];鄭州大學(xué);2011年

5 趙清霞;肝癌、結(jié)腸癌干細(xì)胞樣細(xì)胞新型培養(yǎng)體系及其細(xì)胞系建立的研究[D];浙江理工大學(xué);2010年

6 徐銘竹;CXCR4在舌鱗癌組織及癌干細(xì)胞樣細(xì)胞中的生物學(xué)表達(dá)與調(diào)控[D];廣西醫(yī)科大學(xué);2010年

7 仇志坤;膠質(zhì)瘤干細(xì)胞樣細(xì)胞中MGMT表達(dá)以及與替莫唑胺耐藥相關(guān)機(jī)制研究[D];廣東藥學(xué)院;2011年

8 張小麗;肝癌干細(xì)胞樣細(xì)胞的分離及其耐藥性受Akt通路調(diào)節(jié)[D];重慶醫(yī)科大學(xué);2013年

，

本文編號(hào)：2493400