二甲双胍对TGF-β1诱导黑素瘤细胞EMT及侵袭的影响

发布时间：2018-05-31 12:20

本文选题：黑素瘤 + 二甲双胍　；参考：《承德医学院》2017年硕士论文

【摘要】：黑素瘤是由黑素细胞恶性转化形成的皮肤肿瘤。其转移率高,且对传统疗法不敏感,一旦转移,预后差。因其高转移性,在皮肤肿瘤相关的死亡人数中黑素瘤占70%以上。目前,对转移性黑素瘤尚无有效的治疗方法,且患者5年生存率小于5%。如何阻断黑素瘤的恶性进展已成为全球关注的医学难题。上皮-间质转化(epithelial-mesenchymal transition,EMT)是肿瘤恶性转变的关键启动因素。其能够使上皮样肿瘤细胞转化为更具能动性、侵袭性及耐药性的间质样细胞。EMT过程主要表现为上皮细胞标志物,如E-cadherin等表达减少,间质细胞标志物,如N-cadherin等表达增多。转化生长因子β(transforming growth factor-beta,TGF-β)被认为是EMT的主要诱导因子,参与个体发育、纤维化及肿瘤进展等生理病理过程。近年来,二甲双胍的抗肿瘤作用被广泛证实。体内外研究发现二甲双胍能够抑制黑素瘤细胞的生长、侵袭及远处转移。在前列腺癌、乳腺癌及肺癌研究中发现,二甲双胍能够抑制TGF-β1诱导产生的EMT,从而影响肿瘤进展,但在黑素瘤领域国内外尚无相关报道。为此,本实验探讨二甲双胍对TGF-β1诱导下黑素瘤细胞EMT过程及侵袭的影响,为二甲双胍抗黑素瘤作用机制提供新的理论依据,也为黑素瘤的治疗提供重要线索。目的:探究TGF-β1对黑素瘤细胞EMT过程及细胞侵袭的影响以及二甲双胍对TGF-β1诱导下EMT过程及细胞侵袭的作用。方法:体外培养人黑素瘤细胞系1205Lu,根据实验目的分为3个处理组给予不同干预48h,即空白对照组、5ng/ml TGF-β1组、5ng/ml TGF-β1+1mM二甲双胍组。1.显微镜下观察药物处理48h后各组细胞形态变化并采集图像。2.Transwell侵袭实验检测药物处理48h后3组细胞的体外侵袭能力。3.Western-blot测定EMT过程的重要标志分子,即转录因子Snail、上皮细胞标志物claudin-1、间质细胞标志物n-cadherin的蛋白表达水平。4.rt-qpcr测定emt过程的重要标志分子,即转录因子snail、上皮细胞标志物claudin-1、间质细胞标志物n-cadherin的mrna表达水平。5.实验结果采用spss19.0统计软件处理且以?x±s表示,多组比较采用单因素方差分析,两两比较采用lsd-t检验,p0.05被认为差异具有统计学意义。结果:1.二甲双胍抑制tgf-β1诱导黑素瘤1205lu细胞侵袭transwell侵袭实验结果显示:5ng/ml的tgf-β1作用48h后细胞穿过小室膜的数目(412.2±13.427)明显多于对照组(194.1±8.295),差异有统计学意义(p0.05);联合1mm二甲双胍干预48h后细胞穿膜数目(175.3±8.693)与tgf-β1组相比显著减少,差异有统计学意义(p0.05)。2.二甲双胍抑制tgf-β1诱导黑素瘤1205lu细胞emt过程2.1二甲双胍逆转tgf-β1诱导黑素瘤1205lu细胞emt样形态改变显微镜下观察各组细胞形态学差异发现:对照组细胞生长状态良好,细胞呈多角形且分布均匀;tgf-β1诱导48h后细胞似成纤维细胞状,且按平行、放射或旋涡状排列分布,即由上皮细胞样形态向间质细胞样形态转变,同emt样改变,说明1205lu细胞在tgf-β1诱导下经历emt样改变;联合二甲双胍干预后细胞失去原有的间质细胞样形态,向对照组细胞形态转变,说明tgf-β1诱导的emt样形态改变在二甲双胍作用下发生逆转,即经历met样改变。2.2二甲双胍上调tgf-β1诱导1205lu细胞claudin-1蛋白及mrna表达western-blot及rt-qpcr结果显示:与空白对照组相比,tgf-β1刺激后上皮细胞标志分子claudin-1在蛋白及mrna水平的表达减少,差异有统计学意义(p0.05);二甲双胍能够逆转tgf-β1诱导的上述改变,即上调claudin-1蛋白及mrna的表达,且具有显著差异(p0.05)。2.3二甲双胍下调tgf-β1诱导1205lu细胞snail、n-cadherin蛋白及mrna表达Western-blot及RT-q PCR结果显示:与空白对照组相比,TGF-β1刺激后EMT转录因子Snail及间质细胞标志分子N-cadherin在蛋白及m RNA水平的表达增多,差异有统计学意义(P0.05);二甲双胍能够逆转TGF-β1诱导的上述改变,即下调Snail及N-cadherin蛋白及mRNA的表达,且具有显著差异(P0.05)。结论:1.TGF-β1能够诱导黑素瘤1205Lu细胞经历EMT过程,同时细胞侵袭力增强。2.二甲双胍能够逆转TGF-β1诱导的黑素瘤1205Lu细胞EMT过程,并伴随细胞侵袭力减弱。
[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.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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