本文關(guān)鍵詞：下調(diào)ZEB1表達(dá)抑制黑色素瘤干細(xì)胞的致瘤性和肺轉(zhuǎn)移研究　出處：《東南大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 惡性黑色素瘤 腫瘤干細(xì)胞 ZEB1 上皮間質(zhì)轉(zhuǎn)換 靶向治療

【摘要】：惡性黑色素瘤(malignant melanoma, MM)是來源于黑色素細(xì)胞的一種惡性腫瘤。在過去的20多年中,世界范圍內(nèi)惡性黑色素瘤的發(fā)病率呈逐步增高的趨勢(shì)。皮膚是惡性黑素色瘤的主要發(fā)病部位,占皮膚惡性腫瘤的死亡率約70%,也可起源于眼、鼻腔等處,其高度侵襲與轉(zhuǎn)移的能力是惡性黑色素瘤患者死亡的主要原因,早期可轉(zhuǎn)移至肺、腦等部位,晚期隨體循環(huán)擴(kuò)散至全身。而上皮-間充質(zhì)轉(zhuǎn)換(epithelialmesenchymal transition, EMT)在其中扮演著重要角色,EMT被認(rèn)為是啟動(dòng)腫瘤侵襲轉(zhuǎn)移的重要事件。研究表明,歷經(jīng)EMT的惡性黑色素瘤細(xì)胞能從腫瘤的原發(fā)灶逃離,侵襲到周圍其他組織,經(jīng)過血液或淋巴途徑到達(dá)更遠(yuǎn)處的器官形成轉(zhuǎn)移灶。新近研究還發(fā)現(xiàn),有EMT表型的惡性黑色素瘤細(xì)胞更具耐藥性、免疫抑制及類干細(xì)胞等特性。在EMT的發(fā)生發(fā)展過程中最主要的調(diào)控對(duì)象是E-鈣黏著蛋白(E-cadherin)。該蛋白是維持上皮細(xì)胞特性的重要分子,E-cadherin的丟失是EMT的一個(gè)重要特征,若E-cadherin表達(dá)下降,細(xì)胞則會(huì)呈現(xiàn)許出多非上皮細(xì)胞的特征,如細(xì)胞極性丟失,細(xì)胞間的粘附性降低,轉(zhuǎn)變成更易發(fā)生轉(zhuǎn)移的間質(zhì)細(xì)胞,而表達(dá)波形蛋白(Vimentin)上調(diào),這些蛋白表達(dá)改變是EMT的一個(gè)重要標(biāo)志。因此,探究EMT的發(fā)生和調(diào)控機(jī)制,對(duì)于尋找治療腫瘤細(xì)胞轉(zhuǎn)移的靶點(diǎn)有重要意義。隨著分子生物學(xué)的發(fā)展,研究者發(fā)現(xiàn)腫瘤組織中有極少數(shù)細(xì)胞具有類干細(xì)胞的特性：能無限增殖、不斷分化,對(duì)化療、放療不敏感,而這些細(xì)胞成為腫瘤發(fā)生、侵襲轉(zhuǎn)移和復(fù)發(fā)的關(guān)鍵細(xì)胞,即腫瘤干細(xì)胞(tumor stem cells, TSCs)或癌干細(xì)胞(cancer stem cells, CSCs)。近來對(duì)腫瘤發(fā)生發(fā)展過程的研究表明,鋅指結(jié)合蛋白(1 zinc finger E-box-binding protein 1, ZEB1)在腫瘤轉(zhuǎn)移過程中發(fā)揮著重要作用。ZEB1是鋅指結(jié)構(gòu)轉(zhuǎn)錄因子中的E盒結(jié)合鋅指蛋白,它不僅能調(diào)節(jié)EMT的發(fā)生和發(fā)展,還能通過調(diào)節(jié)細(xì)胞周期、細(xì)胞凋亡、衰老、侵襲轉(zhuǎn)移和間質(zhì)血管生成等,進(jìn)而調(diào)節(jié)腫瘤的進(jìn)展。本課題組前期實(shí)驗(yàn)結(jié)果顯示：降低B16F10細(xì)胞中ZEB1的表達(dá),可在一定程度上降低B16F10細(xì)胞在體外的克隆形成能力、增殖能力、耐藥性、侵襲轉(zhuǎn)移能力及體內(nèi)致瘤能力,但對(duì)B16F10細(xì)胞中CSCs的影響如何,期待進(jìn)一步探討。正因如此,本研究選擇小鼠黑色素瘤細(xì)胞系B16F10細(xì)胞中CD133+ CD44+CSCs作為研究對(duì)象,將其從B16F10細(xì)胞中分離,探討下調(diào)ZEB1的表達(dá)對(duì)CD44+CD133+CSCs的致瘤性和肺轉(zhuǎn)移的影響,從而為惡性黑色素瘤的治療提供新的思路。目的：探討下調(diào)黑色素瘤CSCs中ZEB1的表達(dá)對(duì)其致瘤性和肺轉(zhuǎn)移的影響,為尋找黑色素瘤新的治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。方法：1.將構(gòu)建好的靶向鼠ZEB1基因的pSUPER-EGFP1-ZEB1-shRNA干擾質(zhì)粒通過脂質(zhì)體轉(zhuǎn)染B16F10細(xì)胞,并通過G418篩選穩(wěn)定轉(zhuǎn)染細(xì)胞株。2.用免疫磁選技術(shù)從穩(wěn)定轉(zhuǎn)染pSUPER-EGFPl-ZEB1-shRNA的B16F10克隆中分離獲取ZEB1-shRNA2CD 133+CD44+CSCs以及scramble-shRNA CD133+ CD44+CSCs;從B16F10細(xì)胞中分選出CD133+CD44+CSCs。3.通過劃痕實(shí)驗(yàn)、侵襲性實(shí)驗(yàn),體內(nèi)致瘤性、荷瘤鼠生存實(shí)驗(yàn)及肺轉(zhuǎn)移實(shí)驗(yàn),綜合分析下調(diào)ZEB1表達(dá)后對(duì)B16F10 CD44+CD133+ CSCs的致瘤性和肺轉(zhuǎn)移的影響。4.用2×104B16F10 CD44+CD133+CSCs、ZEB1-shRNA CD44+CD133+CSCs、 scramble-pSUPER-EGFP CD44+CD133+CSCs分別攻擊C57BL/6小鼠,通Western Blotting實(shí)驗(yàn)和免疫組化實(shí)驗(yàn)分別檢測(cè)瘤組織中EMT相關(guān)蛋白ZEB1, E-cadherin N-cadherin及Vimentin的表達(dá)。結(jié)果：1.分離出經(jīng)G418篩選出穩(wěn)定轉(zhuǎn)染pSUPER-EGFP1-ZEB1-shRNA的B16F10細(xì)胞克�。�2.用免疫磁選技術(shù)成功分離出ZEB1-shRNA CD133+CD44+CSCs、scramble-pSUPER-EGFP CD133+CD44+CSCs以及B16F10 CD133+CD44+CSCs;劃痕實(shí)驗(yàn)、侵襲性實(shí)驗(yàn)、小鼠體內(nèi)致瘤性和肺轉(zhuǎn)移實(shí)驗(yàn)結(jié)果顯示：B16F10 CD44+CD133+ CSCs及scramble-pSUPER-EGFP CD44+CD133+CSCs的侵襲性和轉(zhuǎn)移性以及致瘤性均高于ZEB1-shRNA CD44+CD133+CSCs,差異有顯著性意義(P0.01)3. qRT-PCR法、Western Blotting及免疫組化檢測(cè)瘤組織中EMT相關(guān)蛋白顯示：ZEB1-shRNA CD44+CD133+CSCs的靶基因ZEB1表達(dá)減低,間質(zhì)型蛋白Vimentin和N-cadherin表達(dá)下降,而上皮型蛋白E-cadherin的表達(dá)升高；肺組織黑色素瘤轉(zhuǎn)移結(jié)節(jié)顯著減少,與B16F10 CD44+CD133+CSCs和scramble-pSUPER-EGFP CD44+CD133+CSCs組相比,差異有顯著性意義(P0.05或P0.01)。結(jié)論：下調(diào)黑色素瘤B16F10 CD44+CD133+CSCs中ZEB1的表達(dá),可在一定程度上降低其在體外的增殖能力、克隆形成能力,侵襲力及小鼠體內(nèi)致瘤性和抑制肺轉(zhuǎn)移能力,提示ZEB1可作為黑素瘤CSCs治療的新靶點(diǎn)。
[Abstract]:Malignant melanoma (malignant melanoma MM) is a malignant tumor derived from melanoma cells. In the past 20 years, the worldwide incidence of malignant melanoma rate is gradually increased. The skin is the main pathogenesis of malignant tumor plain black, skin tumor mortality accounted for about 70% also, originated in the eyes, nasal cavity, the high ability of invasion and metastasis is the main cause of death in patients with malignant melanoma, early metastasis to the lung, brain and other parts, with advanced systemic spread to the whole body. The epithelial mesenchymal transition (epithelialmesenchymal transition EMT) plays an important role among them, EMT is considered to be an important event in tumor invasion and metastasis promoter. The results show that malignant melanoma after EMT from primary tumor escape, invasion to the surrounding tissues, blood or lymphatic pathway after arrival The more distant organ forming metastases. Recent studies also found that malignant melanoma EMT cells more resistant phenotype, characteristics of immune suppression and stem. In the development of EMT control is the most important E- cadherin (E-cadherin). The protein is an important molecule to maintain epithelial cells the characteristics of E-cadherin, the loss is an important feature of EMT, if E-cadherin was decreased and the cells are non epithelial cells Xu characteristics, such as loss of cell polarity, cell to cell adhesion decreased into more prone to metastasis mesenchymal cells, and up-regulated expression of vimentin (Vimentin), the the expression change is an important sign of EMT. Therefore, exploring the occurrence and regulation mechanism of EMT, is of great significance to target the tumor cell metastasis. With the development of molecular biology, the researchers found that tumor In a few cells have characteristics of stem cells: proliferation, differentiation, not sensitive to chemotherapy and radiotherapy, and these cells become cancer cells, key to metastasis and recurrence, tumor stem cells (tumor stem cells, TSCs) or cancer stem cells (cancer stem cells, CSCs). Recently the research of cancer occurrence and development process showed that the zinc finger binding protein (1 zinc finger E-box-binding protein 1, ZEB1) plays an important role in the process of tumor metastasis is.ZEB1 zinc finger transcription factor E box structure in combination with zinc finger protein, it can not only regulate the occurrence and development of EMT, but also through the adjustment cell cycle, apoptosis, senescence, invasion and angiogenesis, and regulate the progress of tumors. Our previous experimental results show that the decreased expression of ZEB1 in B16F10 cells, B16F10 can be reduced to a certain extent Cell formation ability in vitro clonal proliferation, drug resistance, invasion and tumorigenicity in vivo, but the effect of CSCs in B16F10 cells to look forward to further discussion. Because of this, this study selected CD133+ B16F10 cells in CD44+CSCs mouse melanoma cell line as the research object, separating it from B16F10 cells in the study of CD44+CD133+CSCs induced down-regulation of ZEB1 expression in vitro and lung metastasis, thus provide a new way of thinking for the treatment of malignant melanoma. Objective: To investigate the effect of down-regulation of ZEB1 melanoma in CSCs on the expression of the tumorigenic effect and lung metastasis, and provide experimental basis for the search for new treatment targets melanoma. Methods: 1. constructed pSUPER-EGFP1-ZEB1-shRNA interference targeting ZEB1 gene plasmid were transfected into B16F10 cells with liposome and screened by G418 stable transfected cell line.2. with immune Magnetic separation technology to obtain ZEB1-shRNA2CD CD44+CSCs isolated from CD133+ scramble-shRNA 133+CD44+CSCs and B16F10 pSUPER-EGFPl-ZEB1-shRNA in the stably transfected clones were selected by CD133+CD44+CSCs.3.; the scratch test from the B16F10 cells, the invasive experiments in vivo tumorigenicity of tumor bearing mice survival experiment and lung metastasis experiment, comprehensive analysis of expression of ZEB1 on B16F10 CD44+CD133+ CSCs induced.4. tumor and the lung metastasis with 2 * 104B16F10 CD44+CD133+CSCs, ZEB1-shRNA CD44+CD133+CSCs, scramble-pSUPER-EGFP CD44+CD133+CSCs were challenged C57BL/6 mice, Western Blotting assay and immunohistochemical experiments were detected in tumor tissues of EMT related protein ZEB1, expression of E-cadherin N-cadherin and Vimentin. Results: 1. isolated screened with G418 B16F10 cell clones stably transfected with pSUPER-EGFP1-ZEB1-shRNA; 2. by magnetic separation technology of immune The successful isolation of ZEB1-shRNA CD133+CD44+CSCs, scramble-pSUPER-EGFP CD133+CD44+CSCs and B16F10 CD133+CD44+CSCs; scratch test, invasive experiment, mice tumorigenicity and metastasis experimental results show that B16F10 CD44+CD133+ CSCs and scramble-pSUPER-EGFP CD44+CD133 +CSCs invasion and metastasis and tumorigenicity were higher than that of ZEB1-shRNA CD44+CD133+CSCs, there were significant differences (P0.01) 3. qRT-PCR Western, Blotting and immunohistochemistry in tumor tissues of EMT associated protein showed that the target gene ZEB1 ZEB1-shRNA expression of CD44+CD133+CSCs decreased and decreased interstitial protein Vimentin and N-cadherin expression, and the expression of E-cadherin protein E-cadherin increased; lung metastasis of melanoma nodules was significantly reduced, compared with B16F10 CD44+CD133+CSCs and scramble-pSUPER-EGFP CD44+CD133+CSCs group, a significant differences (P0.05 or P 0.01). Conclusion: the expression of ZEB1 in melanoma B16F10 CD44+CD133+CSCs, to a certain extent, reduce the ability of in vitro, colony forming ability, and inhibit lung metastasis of tumor invasion and in vivo in mice, suggesting that ZEB1 can be used as a new target for treatment of CSCs melanoma.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.5

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