【作者】 徐延昭；

【导师】 田子强；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 硕士

【摘要】 目的:食管癌是目前对人类健康和生命构成严重威胁的恶性肿瘤之一,其病死率在世界范围居恶性肿瘤的第七位。我国是食管癌的高发国家,在我国恶性肿瘤中其发病率居第四位,特别是河北南部、河南北部及山西的部分地区为高发地区。因此,深入研究食管癌的发病机制,以有效地对其进行预防和治疗有着非常重要的意义。研究表明,食管癌的发生发展并非单个分子事件,而是一个多因素、多阶段、长时间的多步骤过程,涉及多个癌基因和抑癌基因的改变以及细胞调控机制的异常。有关MT-1和MT-2基因的研究已有很多,有文献报道MT-1和MT-2基因在食管癌组织中呈高表达状态。而MT-3基因研究的较少,已知MT-3基因是细胞内一种金属硫蛋白的编码基因,不仅具有许多重要的生理功能,还可以抑制细胞增殖。我们的前期研究显示在食管癌组织中广泛存MT-3基因的超甲基化,而且这种超甲基化可导致MT-3表达水平的下调。基于以上研究,本实验采用基因转染技术,研究MT-3和MT-1E基因转染人食管鳞癌Eca-109和TE13细胞株后对其细胞周期的影响。方法:1选取食管鳞癌Eca-109和TE13细胞株,用RPMI1640培养基在六孔板中进行培养,在细胞汇合达70%后,准备进行细胞转染。2分别取含有EX-T3737-M03-MT-3、EX-T3737-M03、EX-T2598-M56- MT-1E及EX-T2598-M56四种质粒DNA的大肠杆菌菌株,经过夜培养后提取质粒DNA,并测定其浓度与纯度;然后配制质粒DNA/脂质体复合物,并设空白对照,进行细胞转染。3将以上质粒DNA/脂质体复合物与两种细胞株共同培养,分别在24h、48h、72h用荧光显微镜观察质粒DNA中荧光蛋白在细胞内的表达情况,并计算细胞转染效率,以确定质粒DNA在细胞内最佳转染时间。4在质粒DNA转染Eca-109和TE13细胞株48h后,分别收集六孔板中各转染组细胞及空白对照组细胞,应用流式细胞术检测细胞周期的变化情况。5所有计量资料用均值±标准差表示,两样本均数比较选择t检验或t’检验,以P<0.05为差异有统计学意义,统计过程由SPSS13.0统计软件完成。结果:1四种质粒DNA转染食管鳞癌Eca-109及TE13细胞株后,在24h、48h、72h荧光显微镜观察到各转染组均见有荧光标记。EX-T3737-M03-MT-3和其空载体EX-T3737-M03转染组呈绿色荧光;EX-T2598-M56-MT-1E和其空载体EX-T2598-M56转染组呈红色荧光。转染48h后细胞荧光表达强度最强,约占细胞总数的80%,提示转染成功,且效率较高。2 EX-T3737-M03-MT-3和EX-T3737-M03在转染Eca-109和TE13细胞株48h后,其G0/G1期的细胞比例分别为70.97±1.58%、53.20±2.13% (P<0.05),S期的细胞比例分别为14.32±1.23%、31.50±1.59%(P<0.05),G2/M期的细胞比例分别为14.72±1.08%、15.30±1.38%(P>0.05)。3 EX-T2598-M56-MT-1E和EX-T2598-M56在转染Eca-109和TE13细胞株48h后,其G0/G1期的细胞比例分别为54.58±2.38%、53.02±2.16% (P>0.05),S期的细胞比例分别为30.15±1.77%、31.90±1.71%(P>0.05),G2/M期的细胞比例分别为15.27±1.06%、15.08±0.78%(P>0.05)。4 EX-T3737-M03-MT-3和EX-T2598-M56-MT-1E在转染Eca-109和TE13细胞株48h后,其G0/G1期的细胞比例分别为70.97±1.58%、54.58±2.38%(P<0.05),S期的细胞比例分别为14.32±1.23%、30.15±1.77% (P<0.05),G2/M期的细胞比例分别为14.72±1.08%、15.27±1.06%(P>0.05)。结论:1 MT-3基因在转染食管鳞癌Eca-109和TE13细胞株后,使G0/G1期细胞比例明显增多,S期细胞比例明显减少,推测MT-3基因可能通过改变细胞周期来抑制食管癌细胞的增殖。2 MT-1E基因在转染食管鳞癌Eca-109和TE13细胞株后,对细胞周期影响不明显,推测MT-1E基因可能在食管癌细胞的增殖过程中作用不明显。更多还原

【Abstract】 Objective:Esophageal cancer pose a serious threat to human health and to life as one of the malignant tumors. The mortality of esophageal cancer in the world ranks the seventh of malignant tumors .China is a country of high incidence of esophageal cancer which the incidence ranks the fourth place, especially in the south of Hebei, the north of Henan and parts of Shanxi as the high-risk area.Therefore,in-depth study of the pathogenesis of esophageal cancer in order to effectively prevent and treat them have very important significance.Studies have shown that the incidence and development of esophageal cancer is non-single molecular events,but a multi-factor,multi- stage,long multi-step process,involving multiple oncogenes and tumor suppressor genes and cell regulatory mechanism to change the exception.There are many reports about the MT-1 and MT-2 genes,which exists in esophageal carcinoma with overexpression condition.But,there are little reports about the MT-3 gene,It is well-known that MT-3 gene encodes metallothionein in cells,which not only has many important physiological functions, but also inhibits cells proliferation.The hypermethyla- tion of the MT-3 gene extensively exists in esophageal carcinoma in our early day,s study,moreover this hypermethylation results in the expression down regulation of the MT-3 gene.Based on the above studies,this study was designed to research effects of transfections with MT-3 and MT-1E genes on cell cycle in cell lines of squamous cell carcinoma of the esophagus.Methods: 1 The Eca-109 and TE13 cells of squamous cell carcinoma of the esophagus were cultured in six-well plate with RPMI1640 medium,in cell confluence of 70%,preparation for cell transfection.2 Four kinds of plasmid DNA of E.coli strains which contain EX-T3737-M03-MT-3, EX-T3737-M03, EX-T2598-M56-MT-1E and EX- T2598-M56 were obtained,through one night,extracted plasmid DNA and determined its concentration and purity; then prepared plasmid DNA/liposome complexes, and established a blank control, carried out cell transfection.3 The above plasmid DNA/liposome complexes and the two kinds of cell lines were cultured, and fluorescent protein expression of plasmid DNA were abserved in the cells with the fluorescence microscope in 24h, 48h and 72h ,respectively ,and calculated cells transfection efficiency in order to determine the optimal time of transfection of plasmid DNA in the cells.4 After the Eca-109 and TE13 cells which were transfected by plasmid DNA for 48h, the cells of the transfection groups and control groups were collected in six-well plate and observed the change of cell cycle by flow cytometry.5 All measurement data were demonstrated with the mean±standard deviation,and two samples mean were selected t test or t, test ,P<0.05 as statistically significant difference, and statistical process was completed by the SPSS13.0 statistical software.Results:1 After transfected the Eca-109 and TE13 cells of squamous cell carcinoma of the esophagus by four kinds of plasmid DNA,fluorescent marker were observed in all groups by fluorescence microscope in 24h,48h and 72h.The transfection groups of EX-T3737-M03-MT-3 and EX-T3737-M03 were green fluorescent.The transfection groups of EX-T2598-M56-MT-1E and EX-T2598-M56 were red fluorescent. the fluorescence intensity was the strongest after transfection cells for 48h,accounting for 80% of the total number of cells, suggesting that transfection was successful and efficient.2 After transfected the Eca-109 and TE13 cells by EX-T3737-M03-MT-3 and EX-T3737-M03 for 48h,its proportion of G0/G1 phase cells ratios were 70.97±1.58% vs 53.20±2.13 %(P<0.05),S phase cell ratios were 14.32±1.23% vs 31.50±1.59%(P<0.05), G2/M phase cells ratios were 14.72±1.08% vs 15.30±1.38%(P>0.05).3 After transfected the Eca-109 and TE13 cells by EX-T2598-M56-MT- 1E and EX-T2598-M56 for 48h,its proportion of G0/G1 phase cells ratios were 54.58±2.38% vs 53.02±2.16%(P>0.05),S phase cell ratios were 30.15±1.77% vs 31.90±1.71%(P>0.05),G2/M phase cells ratios were 15.27±1.06% vs 15.08±0.78 %(P>0.05).4 After transfected the Eca-109 and TE13 cells by EX-T3737-M03-MT-3 and EX-T2598-M56-MT-1E for 48h,its proportion of G0/G1 phase cells ratios were 70.97±1.58% vs 54.58±2.38%(P<0.05),S phase cell ratios were 14.32±1.23% vs 30.15±1.77%(P<0.05),G2/M phase cells ratios were 14.72±1.08% vs 15.27±1.06 %(P>0.05).Conclusion:1 After transfection with the MT-3 gene in Eca-109 and TE13 cells of squamous cell carcinoma of the esophagus, the proportion of G0/G1 phase cell ratios were significantly increased,and the S phase cell ratios were significantly reduced,so the gene of MT-3 was speculated to inhibit the cell growing of esophageal carcinoma through changing the cell cycle.2 After transfection with the MT-1E gene in Eca-109 and TE13 cell of squamous cell carcinoma of the esophagus, the proportion of the cell cycle ratios were no difference, so the gene of MT-1E was speculated that it may be no effect on the cell growing of esophageal carcinoma.更多还原