【作者】 孙军祥；

【导师】 宋旭东；

【作者基本信息】 河北联合大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的:食管癌是消化系统一种常见的恶性肿瘤,占所有恶性肿瘤的2%,我国是食管癌的高发区,因食管癌死亡者仅次于胃癌而居第二位,但是其发生机制尚未完全清楚。肿瘤的发生是一个长期的并且是多因素,多步骤的复杂过程,包括细胞的增殖和分化、细胞的凋亡、细胞的粘附、规避机体免疫系统、新生血管的形成和肿瘤的浸润、转移等。Galectin-3蛋白作为半乳凝集素家族的一员,目前研究认为其广泛表达于正常组织和肿瘤组织,与肿瘤的发生、发展、转移和侵袭有着密切的关系。RNA干扰(RNA interference,RNAi)是一种核酸序列特异性阻止基因功能的有效工具,能高效并且特异抑制目标基因的表达。有研究发现用RNAi技术阻止肿瘤相关基因的表达可以有效地抑制肿瘤细胞的增殖。本实验通过设计合成针对Galectin-3蛋白特异性的小干扰RNA(short interfering RNA,siRNA)表达载体,然后采用Galectin-3 -siRNA干扰技术作用于食管癌细胞系Eca-109,从而来研究干扰前后Galectin-3、CyclinD1蛋白表达及细胞增殖和细胞周期的改变,进一步探求Galectin-3蛋白对食道癌细胞系Eca-109细胞增殖和细胞周期的影响以及与CyclinD1之间的关系,从而为食管癌的发生机制提供实验研究资料,为食管癌的临床治疗提供有价值的参考指标。方法:采用食管癌细胞系Eca-109为研究对象,设计合成针对Galectin-3的特异性siRNA并转染进食管癌Eca-109细胞。将实验分为转染组、空白对照组、阳性对照组、阴性对照组。采用MTT法检测各组食管癌细胞系Eca-109增殖情况,以检测的光密度值(OD值)代表各组的增殖情况,并计算各组的抑制率;采用免疫细胞化学方法检测实验各组食管癌细胞系Eca-109中Galectin-3、CyclinD1蛋白的表达,采用HIPAS-2000型计算机图像采集系统,在×200倍视野中采集图像,用Imageproplus5.0(IPP)测定阳性细胞的累积光密度值IOD,每张切片选5个视野并求其均值。应用流式细胞仪检测各组食管癌细胞系Eca-109的细胞周期变化,所得结果用细胞周期拟和软件ModFit分析。数据采用方差分析和双变量的相关性分析进行统计学分析,以p<0.05表示差异有统计学意义。结果:1.转染效率的检测:应用FAM标记的Negative siRNA转染食管癌细胞Eca-109 6h后用流式细胞仪检测转染效率,转染效率为69.60%。2.应用倒置相差显微镜进行细胞学观察,Galectin-3-siRNA转染后48h后细胞形态变化明显,转染组细胞与阴性对照组和空白对照组细胞相比细胞体积变小并趋向于均一,多数细胞由梭形趋向变圆,核浆比变小,瘤巨细胞减少,与空白对照组相比较细胞生长缓慢,部分细胞死亡并脱落。3.MTT检测结果显示:Galectin-3-siRNA转染进细胞48h以后,抑制了食管癌细胞系Eca-109的生长,转染组中各组与阳性对照组490nm波长处吸光度值分别为0.63±0.13、0.58±0.19、0.76±0.09、0.77±0.18,明显小于空白对照组(1.43±0.12)和阴性对照组(1.65±0.14),转染组、阳性对照组与空白对照组和阴性对照组相比较,差异均有显著统计学意义(p<0.05),转染各组细胞抑制率分别为55.85%、59.71%、46.17%。4.免疫细胞化学检测结果显示:应用Galectin-3-siRNA转染食管癌细胞系Eca-109后48h Galectin-3蛋白表达降低,免疫细胞化学染色明显变浅,应用IPP图像分析系统检测转染组中各组的IOD值分别为20.45±0.15、20.33±0.24、20.36±0.22,空白对照组IOD值为58.15±0.22,阴性对照组IOD值为57.65±0.43,阳性对照组IOD值为19.85±0.67;转染组、阳性对照组与空白对照组和阴性对照组相比较,差异均有显著统计学意义(p<0.05)。应用Galectin-3-siRNA转染食管癌细胞系Eca-109后48h CyclinD1蛋白表达降低,免疫细胞化学染色明显变浅,转染组中各组的IOD值分别为39.98±0.05、39.53±0.82、40.86±0.85,空白对照组IOD值为61.83±0.53,阴性对照组IOD值为62.28±0.57,阳性对照组IOD值为40.42±0.60;转染组、阳性对照组与空白对照组和阴性对照组相比较,差异均有显著统计学意义(p<0.05)。根据免疫细胞化学结果以IOD作为半定量分析数据,转染后48h转染各组和阳性对照组Galectin-3蛋白和CyclinD1蛋白的IOD值均降低,统计学相关性分析显示Galectin-3蛋白和CyclinD1蛋白存在相关性(r=0.806,p=0.000),随着Galectin-3蛋白的表达减少,CyclinD1蛋白也相应的降低。5.流式细胞仪检测结果显示:Galectin-3-siRNA转染食管癌细胞系Eca-109 48h后,转染组、阳性对照组的G1期细胞数百分比较空白对照组和阴性对照组明显升高,S期细胞数百分比较空白对照组和阴性对照组则明显减少。转染组、阳性对照组的G1期细胞百分比数分别为65.76±0.45、66.42±0.17、65.10±0.67、65.44±0.42,阴性对照组和空白对照组G1期细胞数分别为36.76±0.15、35.63±0.36,转染组、阳性对照组与空白对照组和阴性对照组相比较,差异均有统计学意义(p<0.05)。转染组、阳性对照组的S期细胞数百分比分别为23.13±1.34、23.19±1.28、24.40±2.17、24.32±1.73,阴性对照组和空白对照组S期细胞数分别为46.79±0.76、45.33±0.72,转染组、阳性对照组与空白对照组和阴性对照组相比较,差异均有统计学意义(p<0.05)。结论:将设计合成的针对Galectin-3的特异性siRNA转染进食管癌细胞系Eca-109 48h后,肿瘤细胞的生长受到抑制,明显降低了食管癌细胞系Eca-109中Galectin-3蛋白和Cyclin D1蛋白的表达,使细胞生长阻滞在G1期,转染组G1期细胞数明显高于非转染组和阴性对照组。说明Galectin-3在食管癌细胞系Eca-109增殖调控中与Cyclin D1蛋白的表达密切相关,而共同发挥作用,应用RNAi技术阻止肿瘤相关基因的表达可以有效地抑制肿瘤细胞的生长,有可能为肿瘤的诊断与治疗提供了一种新的重要手段。更多还原

【Abstract】 Objective Esophageal cancer is a common digestive system, accounting for 2% of all malignant tumors. Esophageal cancer is a high incidence in China, the death rate of which in the second, however, its mechanism remains unkonow. Tumor is a long-term multi-factor, multi-step complex process, including cell proliferation and differentiation, apoptosis, cell adhesion, circumventing the immune system, angiogenesis, tumor invasion, metastasis and so on. Galectin-3 protein as one of the lectin family of galactose is suggested that widely expressed in normal tissue and tumor tissue, and tumor development, metastasis and invasion are closely relatedRNA interference (RNA interference, RNAi) is an effective tool to prevent nucleic acid sequence-specific gene function that can efficiently and specifically inhibits the expression of target genes. Studies have found that using RNAi technology to prevent tumor-related gene expression can effectively inhibit tumor cell proliferation. We design and synthesis small interfering RNA (short interfering RNA, siRNA) expression vector specifically on Galectin-3 gene, and then make Galectin-3-siRNA to interfer on esophageal cancer cell line Eca-109, which in order to study the interference on Galectin-3, CyclinD1 protein expression, cell proliferation and cell cycle, and to explore the effection of the Galectin-3 protein on the esophageal cancer cell line Eca-109 cell proliferation and cell cycle kinetics and the relationship with the CyclinD1, so as to provide data for the mechanism and clinical treatment of esophageal cancer.Methods Galectin-3 siRNA was transfected into esophageal cancer Eca-109 cells. The experiment was divided into transfection group, blank control group, positive control group and negative control group. MTT assay used in each group of esophageal cancer cell line Eca-109 proliferation, to detect the optical density (OD) on behalf of the group, proliferation, and inhibition rate of each group; detected by immunocytochemistry in each group esophageal cancer cell line Eca-109 in the Galectin-3, CyclinD1 protein expression, the use of HIPAS-2000-based computer image acquisition system,×200 magnification in the images in the collection, use Imageproplus5.0 (IPP) measured integrated optical density of positive cells value of the IOD, each slice select five horizons and find their average. Were detected by flow cytometry esophageal cancer cell line Eca-109 cell cycle, the results to be used and the software ModFit cell cycle analysis. All data between groups by analysis of variance compared mean to p <0.05 indicated significant difference. Results 1. Transfection efficiency of detection: application of FAM labeled N-egative siRNA transfected esophageal cancer cell Eca-109 6h detected by flow cytometry after the transfection efficiency, transfection efficiency was 69.60%.2.Inverted phase contrast microscope for cytological observation, G- alectin-3-siRNA transfected cells after 48h obvious morphological changes, cells transfe cted with negative control group and blank control group were smaller than the cell volume and tend to uniformity, the majority of cells tr- end from the spindle an d round, smaller than the nuclear plasma, tumor gi ant cells decreased compared with the control group cells grew slowly, so me cells die and fall off.3. MTT results showed that: Galectin-3-siRNA into the cells 48h after transfection, inhibition of the esophageal cancer Eca-109 cell line growth in the group transfected with the positive control group absorbance at 490 nm wave length values were 0.63±0.13, 0.58±0.19, 0.76±0.09, 0.77±0.18, significantly less than control group 1.43±0.12 and the negative control group 1.65±0.14, transfection, positive control group and blank con trol group and negative control group, the difference are There was signifi cant (P <0.05), the cells were transfected with the inhibitory rates were 5 5.85%, 59.71%, 46.17%. 4. Immunocytochemistry showed that: The Galectin-3-siRNA transfe cted esophageal cancer cell line Eca-109 protein after 48h Galectin-3 expr ession decreased significantly lighter immunocytochemistry, application IPP image analysis system in the detection of the transfected group IOD values were 20.45±0.15, 20.33±0.24, 20.36±0.22, control group IOD valu e of 58.15±0.22, IOD value of the negative control group 57.65±0.43, po- sitive control group IOD value of 19.85±0.67; transfection group, positive control group and blank control group and negative control group compare d to the differences were statistically significant (p <0.05).Application of Galectin-3-siRNA transfected esophageal cancer cell line Eca-109 protein after 48h cyclinD1 decreased significantly lighter imm unocytochemistry, transfected IOD values of each group were 39.98±0.05, 39.53±0.82, 40.86±0.85, control group IOD value of 61.83±0.53, IOD value of the negative control group 62.28±0.57, positive control group IO- D value of 40.42±0.60; transfection, positive control group and blank control group and negative control group compared The difference was statistically significant (p <0.05).According to the results of immunohistochemistry analysis to IOD as a semi-quantitative data, 48h after transfection, transfection of the group and the positive control group CyclinD1 Galectin-3 and the IOD values were decreased, statistical correlation analysis showed that Galectin-3 and the correlation CyclinD1 Resistance (r = 0.806, p = 0.000), with Galectin-3 expression is reduced, CyclinD1 reduced accordingly.5.Flow cytometry showed that: Galectin-3-siRNA transfected esophageal cancer cell line Eca-109 48h after transfection group in each group, positive control group sub-G1 phase cells compared hundreds of blank control group and negative control group increased, Sphase cells compared hundreds of hours the control group and negative control group were significantly reduced. Transfected in each group, the positive control group the percentage of G1 phase cells, respectively 65.76±0.45, 66.42±0.17, 65.10±0.67, 65.44±0.42, negative control group and blank control group G1 phase cells were 36.76±0.15, 35.63±0.36, transfection, positive control group and blank control group and negative control group, the difference was significant (p<0.05). Transfected in each group, the positive control group the percentage of S phase cells were 23.13±1.34, 23.19±1.28, 24.40±2.17, 24.32±1.73, negative control group and blank control group the number of S phase cells were 46.79±0.76 , 45.33±0.72, transfection, positive control group and blank control group and negative control group, the difference was significant (p <0.05).Conclusions Being transfected specific Galectin-3 siRNA into esophageal cancer cell line Eca-109, after 48h the cell growth was significantly inhibited; expression of Galectin-3 protein and Cyclin D1 protein in esophageal cancer cell line Eca-109 reduced; the cell growth was arrested in G1; the number of cells in transfection group was significantly higher than that of non-transfection group and negative control group. Galectin-3 was closely related with Cyclin D1 protein in the proliferation of esophageal cancer cell line Eca-109. Using RNAi technology can prevent tumor-related gene expression and effectively inhibit tumor cell growth, which possibly provides an important method in diagnosis and treatment of tumors.更多还原