双硫仑抑制TGF-β诱导的口腔癌KB细胞上皮—间充质转化

发布时间：2018-03-10 02:12

本文选题：双硫仑　切入点：上皮-间充质转化　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:口腔癌是世界第六位最常见的癌症,它的转移、复发以及化疗耐受是导致肿瘤治疗失败和患者死亡的主要原因。然而针对以上问题仍缺乏有效的治疗手段。上皮-间充质转化(epithelial-mesenchymal transition,EMT)在肿瘤的转移、复发中起重要作用。而转化生长因子-β(transforming growth factor-β,TGF-β)是进行EMT相关研究的主要诱导剂。双硫仑(disulfiram,DSF)是一种有效治疗酗酒的药物。近年来,人们发现DSF具有促进肿瘤细胞凋亡、抑制侵袭转移并逆转肿瘤细胞耐药的特征。本实验利用TGF-β诱导口腔癌细胞株KB发生EMT,探究DSF是否能逆转这一过程,进而达到抑制口腔癌侵袭迁移并逆转耐药的效果。研究方法:本实验以口腔癌细胞株(KB)及口腔癌细胞耐药株(KBV)为研究对象。首先,采用不同浓度(1μM、5μM、10μM、20μM和40μM)DSF及10 ng/ml TGF-β对细胞预处理24 h。通过MTT法检测细胞增殖能力;通过细胞克隆形成实验测定细胞克隆形成能力和增殖能力;通过Annexin V/PI双染色法检测细胞的凋亡和坏死;通过transwell实验和细胞划痕实验检测细胞的侵袭和迁移能力;通过实时定量RT-PCR方法检测上皮和间充质标志物(E-钙粘蛋白、波形蛋白)、干细胞标志基因(SOX2及OCT4)的m RNA表达水平。所有实验结果至少重复3次。统计学分析采用SPSS17.0统计软件进行t检验。研究结果:1.TGF-β(10 ng/ml)能有效诱导KB由多角形变为长梭形,并促进其侵袭和迁移。随着DSF浓度的增加,KB细胞的形态逐渐恢复为TGF-β诱导前的形态。DSF浓度≥5μM时,表现出对TGF-β诱导的KB侵袭、迁移的明显抑制(P0.05),并呈浓度依赖性。2.TGF-β能促进KB细胞间充质标志物(波形蛋白)的表达,降低上皮标志物(E-钙粘蛋白)的表达。这表明TGF-β能诱导KB细胞发生EMT。DSF能显著降低波形蛋白的表达,在高剂量时增加E-钙粘蛋白的表达。这表明DSF能抑制TGF-β诱导的KB细胞EMT。3.当DSF浓度≥5μM时,能显著抑制KB细胞的增殖(P0.05)。当DSF浓度≥10μM时,能显著抑制KB细胞形成克隆的数量和直径(P0.05)。当DSF浓度≥20μM时,能明显促进KB细胞的凋亡(P0.05)。4.KBV细胞对肿瘤化疗药物长春新碱显示出耐受性。当联合5μMDSF时,KBV细胞对长春新碱的耐受性降低。5.DSF能有效降低KB细胞干细胞标志基因SOX2和OCT4的m RNA表达水平(P0.05)。研究结论:DSF不仅能在体外抑制TGF-β诱导的EMT,抑制口腔癌细胞的侵袭和迁移,还能体外抑制肿瘤细胞的增殖、克隆形成,促进凋亡。此外,DSF能降低口腔癌耐药株的耐药性,并降低口腔癌细胞的干性。
[Abstract]:Objective: oral cancer is the sixth most common cancer and its metastasis, recurrence and chemoresistance is a major cause of treatment failure and death of the tumor patients. However, in view of the above problems is the lack of effective therapy. Epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) in tumor metastasis, play an important the role of recurrence and transforming growth factor beta (transforming beta growth factor-, TGF- beta) is the main inducer of EMT related research. Disulfiram (disulfiram, DSF) is an effective treatment for alcoholism drugs. In recent years, it is found that DSF can promote tumor cell apoptosis, inhibit the invasion and metastasis characteristics reversal of drug resistance of tumor cells. This experiment using TGF- beta induced oral cancer cell line KB EMT, to explore whether DSF can reverse the process, and then to inhibit oral cancer invasion and the effect of reversing drug resistance Research methods: in this experiment, oral cancer cell line (KB) and oral cancer cells resistant strains (KBV) as the research object. Firstly, with different concentrations (1 M, 5 M, 10 M, 20 M and 40 M) and 10 DSF ng/ml TGF- 24 beta on cell pretreatment the H. cell proliferation was evaluated by MTT assay; the cell clone experimental determination of cell colony formation and proliferation ability formation; through apoptosis and necrosis of Annexin cells was determined by V/PI double staining method; the invasion and migration of Transwell experiment and cell scratch assay; through quantitative real-time RT-PCR method for detection of epithelial and mesenchymal markers (E-, E-cadherin, vimentin), stem cell marker genes (SOX2 and OCT4) the expression level of M RNA. All experiments were repeated at least 3 times for statistical analysis. T test using SPSS17.0 statistical software. Results: 1.TGF- beta (10 ng/ml) can effectively induce KB from polygonal. To fusiform and promote its invasion and migration. With the increase of DSF concentration, the morphology of KB cells gradually restored to form the.DSF concentration is induced by TGF- before 5 M, showed the invasion of KB induced by TGF- beta, inhibit migration (P0.05), and.2.TGF- in a concentration dependent manner. Beta cells can promote KB mesenchymal markers (vimentin) expression, reduced epithelial marker (E- cadherin) expression. This indicates that TGF- beta induced EMT.DSF significantly reduced the expression of vimentin in KB cells, increase the expression of E- cadherin in high dose. This indicates that KB EMT.3. DSF can inhibit TGF- beta cells induced when the DSF concentration is 5 M, can significantly inhibit the proliferation of KB cells (P0.05). When the DSF concentration is 10 M, can significantly inhibit the formation of KB cells. The diameter and number of clones (P0.05). When the DSF concentration is 20 M, can significantly promote the apoptosis of KB cells (P0.05.4.KBV cell) Of chemotherapy vincristine showed tolerance. When combined with 5 MDSF, the tolerance of KBV cells to vincristine decreased.5.DSF can effectively reduce the KB cell stem cell marker M RNA gene SOX2 and OCT4 expression level (P0.05). Conclusion: DSF can inhibit TGF- induced EMT in vitro, inhibition of invasion the migration and oral cancer cells, inhibit the proliferation of tumor cells, but also in vitro clone formation, promote apoptosis. Moreover, DSF can reduce the drug resistant strains of oral cancer, oral cancer cells and reduce the dry.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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