本文選題：黑素瘤 + 二甲雙胍　； 參考：《承德醫(yī)學(xué)院》2017年碩士論文

【摘要】：黑素瘤是由黑素細(xì)胞惡性轉(zhuǎn)化形成的皮膚腫瘤。其轉(zhuǎn)移率高,且對(duì)傳統(tǒng)療法不敏感,一旦轉(zhuǎn)移,預(yù)后差。因其高轉(zhuǎn)移性,在皮膚腫瘤相關(guān)的死亡人數(shù)中黑素瘤占70%以上。目前,對(duì)轉(zhuǎn)移性黑素瘤尚無(wú)有效的治療方法,且患者5年生存率小于5%。如何阻斷黑素瘤的惡性進(jìn)展已成為全球關(guān)注的醫(yī)學(xué)難題。上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)是腫瘤惡性轉(zhuǎn)變的關(guān)鍵啟動(dòng)因素。其能夠使上皮樣腫瘤細(xì)胞轉(zhuǎn)化為更具能動(dòng)性、侵襲性及耐藥性的間質(zhì)樣細(xì)胞。EMT過(guò)程主要表現(xiàn)為上皮細(xì)胞標(biāo)志物,如E-cadherin等表達(dá)減少,間質(zhì)細(xì)胞標(biāo)志物,如N-cadherin等表達(dá)增多。轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factor-beta,TGF-β)被認(rèn)為是EMT的主要誘導(dǎo)因子,參與個(gè)體發(fā)育、纖維化及腫瘤進(jìn)展等生理病理過(guò)程。近年來(lái),二甲雙胍的抗腫瘤作用被廣泛證實(shí)。體內(nèi)外研究發(fā)現(xiàn)二甲雙胍能夠抑制黑素瘤細(xì)胞的生長(zhǎng)、侵襲及遠(yuǎn)處轉(zhuǎn)移。在前列腺癌、乳腺癌及肺癌研究中發(fā)現(xiàn),二甲雙胍能夠抑制TGF-β1誘導(dǎo)產(chǎn)生的EMT,從而影響腫瘤進(jìn)展,但在黑素瘤領(lǐng)域國(guó)內(nèi)外尚無(wú)相關(guān)報(bào)道。為此,本實(shí)驗(yàn)探討二甲雙胍對(duì)TGF-β1誘導(dǎo)下黑素瘤細(xì)胞EMT過(guò)程及侵襲的影響,為二甲雙胍抗黑素瘤作用機(jī)制提供新的理論依據(jù),也為黑素瘤的治療提供重要線索。目的:探究TGF-β1對(duì)黑素瘤細(xì)胞EMT過(guò)程及細(xì)胞侵襲的影響以及二甲雙胍對(duì)TGF-β1誘導(dǎo)下EMT過(guò)程及細(xì)胞侵襲的作用。方法:體外培養(yǎng)人黑素瘤細(xì)胞系1205Lu,根據(jù)實(shí)驗(yàn)?zāi)康姆譃?個(gè)處理組給予不同干預(yù)48h,即空白對(duì)照組、5ng/ml TGF-β1組、5ng/ml TGF-β1+1mM二甲雙胍組。1.顯微鏡下觀察藥物處理48h后各組細(xì)胞形態(tài)變化并采集圖像。2.Transwell侵襲實(shí)驗(yàn)檢測(cè)藥物處理48h后3組細(xì)胞的體外侵襲能力。3.Western-blot測(cè)定EMT過(guò)程的重要標(biāo)志分子,即轉(zhuǎn)錄因子Snail、上皮細(xì)胞標(biāo)志物claudin-1、間質(zhì)細(xì)胞標(biāo)志物n-cadherin的蛋白表達(dá)水平。4.rt-qpcr測(cè)定emt過(guò)程的重要標(biāo)志分子,即轉(zhuǎn)錄因子snail、上皮細(xì)胞標(biāo)志物claudin-1、間質(zhì)細(xì)胞標(biāo)志物n-cadherin的mrna表達(dá)水平。5.實(shí)驗(yàn)結(jié)果采用spss19.0統(tǒng)計(jì)軟件處理且以?x±s表示,多組比較采用單因素方差分析,兩兩比較采用lsd-t檢驗(yàn),p0.05被認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.二甲雙胍抑制tgf-β1誘導(dǎo)黑素瘤1205lu細(xì)胞侵襲transwell侵襲實(shí)驗(yàn)結(jié)果顯示:5ng/ml的tgf-β1作用48h后細(xì)胞穿過(guò)小室膜的數(shù)目(412.2±13.427)明顯多于對(duì)照組(194.1±8.295),差異有統(tǒng)計(jì)學(xué)意義(p0.05);聯(lián)合1mm二甲雙胍干預(yù)48h后細(xì)胞穿膜數(shù)目(175.3±8.693)與tgf-β1組相比顯著減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。2.二甲雙胍抑制tgf-β1誘導(dǎo)黑素瘤1205lu細(xì)胞emt過(guò)程2.1二甲雙胍逆轉(zhuǎn)tgf-β1誘導(dǎo)黑素瘤1205lu細(xì)胞emt樣形態(tài)改變顯微鏡下觀察各組細(xì)胞形態(tài)學(xué)差異發(fā)現(xiàn):對(duì)照組細(xì)胞生長(zhǎng)狀態(tài)良好,細(xì)胞呈多角形且分布均勻;tgf-β1誘導(dǎo)48h后細(xì)胞似成纖維細(xì)胞狀,且按平行、放射或旋渦狀排列分布,即由上皮細(xì)胞樣形態(tài)向間質(zhì)細(xì)胞樣形態(tài)轉(zhuǎn)變,同emt樣改變,說(shuō)明1205lu細(xì)胞在tgf-β1誘導(dǎo)下經(jīng)歷emt樣改變;聯(lián)合二甲雙胍干預(yù)后細(xì)胞失去原有的間質(zhì)細(xì)胞樣形態(tài),向?qū)φ战M細(xì)胞形態(tài)轉(zhuǎn)變,說(shuō)明tgf-β1誘導(dǎo)的emt樣形態(tài)改變?cè)诙纂p胍作用下發(fā)生逆轉(zhuǎn),即經(jīng)歷met樣改變。2.2二甲雙胍上調(diào)tgf-β1誘導(dǎo)1205lu細(xì)胞claudin-1蛋白及mrna表達(dá)western-blot及rt-qpcr結(jié)果顯示:與空白對(duì)照組相比,tgf-β1刺激后上皮細(xì)胞標(biāo)志分子claudin-1在蛋白及mrna水平的表達(dá)減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05);二甲雙胍能夠逆轉(zhuǎn)tgf-β1誘導(dǎo)的上述改變,即上調(diào)claudin-1蛋白及mrna的表達(dá),且具有顯著差異(p0.05)。2.3二甲雙胍下調(diào)tgf-β1誘導(dǎo)1205lu細(xì)胞snail、n-cadherin蛋白及mrna表達(dá)Western-blot及RT-q PCR結(jié)果顯示:與空白對(duì)照組相比,TGF-β1刺激后EMT轉(zhuǎn)錄因子Snail及間質(zhì)細(xì)胞標(biāo)志分子N-cadherin在蛋白及m RNA水平的表達(dá)增多,差異有統(tǒng)計(jì)學(xué)意義(P0.05);二甲雙胍能夠逆轉(zhuǎn)TGF-β1誘導(dǎo)的上述改變,即下調(diào)Snail及N-cadherin蛋白及mRNA的表達(dá),且具有顯著差異(P0.05)。結(jié)論:1.TGF-β1能夠誘導(dǎo)黑素瘤1205Lu細(xì)胞經(jīng)歷EMT過(guò)程,同時(shí)細(xì)胞侵襲力增強(qiáng)。2.二甲雙胍能夠逆轉(zhuǎn)TGF-β1誘導(dǎo)的黑素瘤1205Lu細(xì)胞EMT過(guò)程,并伴隨細(xì)胞侵襲力減弱。
[Abstract]:Melanoma is a malignant tumor of the skin formed by malignant melanocytes. It has a high metastasis rate and is insensitive to traditional therapy. Once transferred, the prognosis is poor. Because of its high metastasis, more than 70% of the melanoma associated with the death of the skin tumor. There is no effective treatment for metastatic melanoma, and the 5 year survival rate of patients is less than 5%. such as How to block the malignant progress of melanoma has become a global medical problem. Epithelial-mesenchymal transition (EMT) is the key promoter of malignant transformation of the tumor. The.EMT process that can transform the epithelioid tumor cells into more active, invasive and drug-resistant mesenchymal like cells is mainly manifested in the epithelium. The expression of cell markers, such as E-cadherin, and the increasing expression of interstitial cell markers such as N-cadherin. Transforming growth factor beta (transforming growth factor-beta, TGF- beta) is considered to be the main inducer of EMT and is involved in the physiological and pathological processes such as ontogenesis, fibrosis and tumor progression. In recent years, the antitumor effect of metformin It is widely confirmed that metformin in vivo and in vivo have found that metformin can inhibit the growth, invasion and distant metastasis of melanoma cells. In the study of prostate cancer, breast cancer and lung cancer, metformin can inhibit the EMT induced by TGF- beta 1 and affect the progression of the tumor. However, there are no related reports in the field of melanoma. To explore the effect of metformin on the EMT process and invasion of melanoma cells induced by TGF- beta 1, provide a new theoretical basis for the mechanism of metformin resistance to melanoma and provide important clues for the treatment of melanoma. Objective: To explore the effect of TGF- beta 1 on the EMT process and cell invasion of melanoma cells and the effect of metformin on EMT induced by TGF- beta 1. Process and the effect of cell invasion. Methods: human melanoma cell line 1205Lu was cultured in vitro. According to the experimental purpose, 3 treatment groups were divided into different intervention groups, that is, blank control group, 5ng/ml TGF- beta 1 group, 5ng/ml TGF- beta 1+1mM metformin group.1. microscope to observe the morphological changes of each cell after 48h, and collect the image.2.Transwell. The invasion ability of 3 groups of cells after the invasive test of 48h,.3.Western-blot, an important marker for the determination of EMT processes, the transcription factor Snail, the epithelial marker claudin-1, the protein expression level of the interstitial cell marker N-cadherin, the important marker for the determination of the EMT process, namely, the transcription factor snail, and the epithelium. Cell marker claudin-1, mRNA expression level of interstitial cell marker N-cadherin,.5. experimental results were treated with spss19.0 statistical software and X + s, multiple groups were compared by single factor analysis of variance, 22 compared with LSD-t test, P0.05 was considered to be statistically significant. Results: 1. metformin inhibited tgf- beta 1 induced melanin. The results of the invasion of Transwell by tumor 1205lu cells showed that the number of cells passing through tgf- beta 1 after 48h (412.2 + 13.427) was significantly more than that of the control group (194.1 + 8.295), and the difference was statistically significant (P0.05). The number of membrane cells (175.3 + 8.693) after the intervention of 1mm metformin (175.3 + 8.693) decreased significantly compared with the tgf- beta 1 group. Statistical significance (P0.05).2. metformin inhibited tgf- beta 1 induced melanoma 1205lu cells EMT process 2.1 metformin reverse tgf- beta 1 induced melanoma 1205lu cell EMT like morphological changes under microscope to observe the morphological differences of each cell observed under microscope: the cells in the control group grew well, the cells were polygonal and distributed evenly; tgf- beta 1 induced 48h. The posterior cells appear to be fibroblast like, arranged in parallel, radiating or vortex like distribution, that is, from the epithelial cell like morphology to the interstitial cell like form, with EMT like changes, indicating that the 1205lu cells undergo EMT like changes under the induction of tgf- beta 1. Transformation, indicating that tgf- beta 1 induced EMT like morphologic changes were reversed under the action of metformin, that is, met like changes in.2.2 metformin up regulation of tgf- beta 1 induced 1205lu cells claudin-1 protein and mRNA expression Western-blot and RT-qPCR results: compared with the blank control group, tgf- beta 1 stimulated epithelial cell marker molecules claudin-1 in protein And the expression of mRNA level decreased, the difference was statistically significant (P0.05); metformin can reverse the above changes induced by tgf- beta 1, that is, up regulation of claudin-1 protein and mRNA expression, and there is a significant difference (P0.05).2.3 metformin downregulation tgf- beta 1 to induce 1205lu cell snail, N-cadherin protein and mRNA expression Compared with the blank control group, the expression of EMT transcription factor Snail and interstitial cell marker N-cadherin increased in protein and m RNA levels after TGF- beta 1, and the difference was statistically significant (P0.05); metformin could reverse the above changes induced by TGF- beta 1, namely, down regulation of Snail and N-cadherin protein and mRNA expression (P), and there were significant differences (P) 0.05). Conclusion: 1.TGF- beta 1 can induce the EMT process of melanoma 1205Lu cells, and the cell invasiveness enhanced.2. metformin can reverse the EMT process of TGF- beta 1 induced melanoma 1205Lu cells and attenuated the cell invasiveness.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Metformin induces apoptosis of pancreatic cancer cells[J];World Journal of Gastroenterology;2008年47期

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本文編號(hào)：1959657