【作者】 刘玉含；

【导师】 刘红春； 贺付成；

【作者基本信息】 郑州大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 食管癌是世界上最常见的消化系统恶性肿瘤之一,而我国是世界上食管癌发病率和死亡率最高的国家,其中,鳞癌是最多见的组织学类型,占90%以上。食管癌的发生和发展涉及多基因多阶段的改变,研究这些变化对阐明食管癌发病机制有重要意义。RNA干扰(RNA interference, RNAi)技术,是指双链RNA(dsRNA)通过转录后的加工,特异性抑制目的基因mRNA生成,并导致特异性蛋白合成降低,以此来诱导细胞内同源序列的基因表达降低的现象,因此又被称作转录后的基因沉默(posttranscriptional gene silencing, PTGS)。RNA干扰的基本原理是：通过特异性dsRNA核酸酶裂解dsRNA,形成若干个长度为21-25bp的核苷酸,形成小的siRNA作为介导子,引起同源序列的特异性mRNA降解。siRNA技术不仅能够有效地使特异性的基因发生沉默,甚至可以替代基因敲除技术,它是目前研究基因调控功能的一个重要手段。RNA干扰的相关技术可用于抑制肿瘤细胞中异常基因的表达,使发生突变的基因恢复正常的生理功能,增加肿瘤细胞对现有治疗手段的敏感性,同时,我们还能通过研究各种作用因子的功能,从而筛选出治疗恶性肿瘤的有效的基因治疗的靶点。TPX2 (targeting protein for Xklp2)基因是近年来发现的一个候选的癌基因。国内外发表的期刊和杂志中已有多种体内、外实验均表明了,TPX2过表达可以导致细胞内中心体的扩增,出现DNA异倍体甚至多倍体,干扰细胞核正常分裂的完成并抑制其自身激活的生理途径,因而TPX2在细胞中的异常表达可能在细胞癌变以及恶性肿瘤发生发展中发挥重要的作用。已有外文文献证明TPX2基因在人类多种恶性肿瘤中如肺鳞状细胞癌、唾液腺癌、胰腺癌和卵巢癌中呈现高表达,但关于TPX2在食管鳞癌细胞中表达情况的报道却较为少见。本研究拟从以下几个方面进行研究：1、通过体外设计合成三种针对人类TPX2靶基因的siRNA(TPX2 siRNA988,TPX2 siRNA1042,TPX2 siRNA1725),分别转染高表达TPX2基因的食管癌EC9706细胞,探讨对食管癌EC9706细胞mRNA干扰效果最佳的有效转染位点和转染浓度；2、应用细胞增殖抑制试验法观察TPX2 siRNA对食管鳞癌EC9706细胞的增殖抑制影响,通过RT-PCR和Western blot技术检测对TPX2 mRNA和蛋白质表达的影响,探讨在不同的时间点干扰效果的差异。一材料和方法1.食管鳞癌EC9706细胞的培养：将食管鳞癌EC9706细胞置于含10%胎牛血清的1640培养基中,在37℃,5% C02,饱和湿度的细胞培养箱内进行贴壁培养。2.三条TPX2 siRNA的设计与合成：三种TPX2 siRNA(TPX2 siRNA-988,TPX2 siRNA-1042,TPX2 siRNA-1725)由中国上海吉玛制药技术有限公司设计并合成。3.转染：应用LipofectamineTM2000 Transfection Reagent将TPX2 siRNA转染到处于对数生长期的食管鳞癌EC9706细胞。4.最佳TPX2 siRNA的筛选：采用300 nM的转染浓度将三种siRNA转染到EC9706细胞48 h,分别设空白对照组和阴性对照组,用RT-PCR法检测转染后各组EC9706细胞中的TPX2 mRNA的表达情况。5.最佳siRNA浓度的筛选：采用终浓度为100 nM,200 nM和300 nM的TPX2siRNA-1725,转染细胞48 h,分别设空白对照组和阴性对照组,用RT-PCR法检测转染后各组EC9706细胞中的TPX2 mRNA的表达情况。6.最佳转染时间的筛选：采用终浓度为300 nM的TPX2siRNA-1725转染EC9706细胞24 h,48 h,72 h和96 h,设空白和阴性对照组,分别用RT-PCR法和Western blot法检测转染后各组EC9706细胞中的TPX2 mRNA和蛋白的表达情况,用MTT法检测各组细胞增殖抑制情况。7.统计方法：应用SPSS15.0统计软件进行统计学处理,资料数据均以均数±标准差(X±S)来表示,多样本均数比较采用单因素方差分析,方差不齐时先进行变量转换。α=0.05为显著性标准。二结果1.最佳TPX2 siRNA的筛选结果：三种TPX2siRNA以300 nM的浓度转染细胞48 h后,与空白对照组和阴性对照组相比,TPX2 siRNA-1725组的TPX2mRNA的表达水平下降明显,具有统计学意义(P<0.05)。因此,TPX2siRNA-1725为筛选出的最佳的TPX2 siRNA。2.最佳TPX2 siRNA-1725转染剂量的筛选结果：以终浓度为100 nM,200 nM和300 nM TPX2 siRNA1725分别转染细胞48 h,与空白对照组和阴性对照组(200 nM)相比,TPX2 siRNA-1725在转染终浓度为300 nM时,TPX2mRNA的表达水平下降最为明显,且具有统计学意义(P<0.05)。因此,筛选出的TPX2 siRNA-1725最佳的转染浓度为300 nM。3.最佳转染时间的筛选结果：以终浓度为300 nM的TPX2 siRNA-1725转染细胞24 h,48 h,72 h和96 h后,与对照组相比,TPX2 mRNA的表达在24 h开始受到抑制,至72 h时TPX2 mRNA表达最低(P<0.05)；与各对照组相比,EC9706细胞在转染后24 h其TPX2蛋白表达水平开始受到抑制,至72h时TPX2蛋白表达最低(P<0.05)；与各对照组相比,细胞在转染后48 h其增殖特性开始明显受到抑制,至72 h时抑制作用最为明显,生长抑制率为35.28%。4.TPX2 siRNA-1725体外瞬时转染食管癌EC9706细胞24 h,48 h,72 h和96 h后,其对TPX2蛋白表达的抑制效应和对TPX2 mRNA表达的抑制效应,呈正相关关系。三结论1.对化学合成的TPX2 siRNA采用RT-PCR和Western blot技术进行最有效干扰筛选,结果发现TPX2 siRNA-1725特异性的抑制TPX2表达的效果最佳。2.TPX2 siRNA-1725转染EC9706细胞后72 h,可使食管鳞癌EC9706细胞中TPX2 mRNA和蛋白的表达量明显降低,MTT实验表明转染TPX2siRNA-1725后可抑制食管癌细胞的增殖,提示TPX2特异性siRNA干扰可作为食管癌靶向治疗的手段。更多还原

【Abstract】 In the worldwide, esophageal carcinoma is one of the most popular malignant tumors in digestive system. And China is the country which has the highest incidence and mortality of Esophageal Carcinoma in the world. Squamous cell carcinoma is the most popular histological type, which accounts for 90%. Malignant transformation of cells is not the result of a single genetic mutation; it is caused by several genes and multistage changes. It has important significance to search the changes for mechanism of esophageal carcinoma.RNA interference (RNAi) technique, it inhibits the production of the target gene mRNA through the processing of transcription, and it results in the reduction of special proteins. After all, RNAi induce the phenomenon of gene expression’s decrease, so it is called posttranscriptional gene silencing (PTGS). The fundamental principle of RNAi is that dsRNA was splitted by nucleicacidase into small ribonucleotide with the length of 21-25bp, and the small ribonucleotide as the mediator can effectively revoke the degradation of homologous sequences special mRNA. The cross-correlation technology of RNA interference could silence the special genes, it also can replace the technology of gene knockout, and it is a important method to research the function of gene regulation. RNAi may be used to inhibit the expression of abnormal gene in tumor cell, then the cells resume the normal physiological function of the mutational gene, meanwhile, it enhanced the sensitivity of the drugs to humor cells, at the same time, according the research of the functions of all kinds of cytokines, we may could screen the effective targeting gene of the gene therapy to cure the tumors. Recently, TPX2 is considered as a new candidate oncogene by many researchers. The results of multiple experiments which were published in domestic and abroad journals showed that overexpression of TPX2 could induce the amplification of centrosome which lies in cells, we may found heteroploid even polyploids DNA in the cells, and it interferes with the nucleolus’s normal disruption, and inhibits the self-activated physiological pathway. In many abroad documents, it is over-expressed in many cancer tissues, for examples:lung squamous cell carcinoma, salivary-gland carcinoma, pancreatic cancer, ovarial cancer, but it is low-expression in the respective normal tissues. And we found that the report of the TPX2 expression of esophageal carcinoma cells is rarely founded.So our research was covered with those aspects.1. We designed and synthesised three kinds of TPX2 siRNA (TPX2 siRNA988, TPX2 siRNA1042 and TPX2 siRNA1725), then we transfected them into EC9706 cells in which TPX2 was highly expressed, and we investigated the effective transfection site and density to inhibit the expression of TPX2; 2. The method of cellar proliferation was used to observe the effect of siRNA inhibition of EC9706. RT-PCR and Western blot were used to detect the expressive influence of TPX2 mRNA and protein; it could tell us the distinction in different time points of transfection.Materials and Methods1 The culture of the esophageal carcinoma EC9706 cells:The EC9706 cells were adherent-cultured with 1640culture fluid of 10% fetal bovine serum under the condition of 37℃and 5%CO2.2 Design and synthesis.of three kinds of TPX2 siRNA:three nucleotide sequence of siRNA (TPX2 siRNA-988, TPX2 siRNA-1042, TPX2 siRNA-1725) corresponding to different sites of TPX2 gene were designed and synthesized by Shanghai GenePharma Co. Ltd.3 Transfecion:TPX2 siRNA was transfected by LipofectamineTM2000Transfection Reagent into esophageal carcinoma EC9706 cells which were in logarithmic growth phase. 4 Screening of optimum TPX2siRNA:the experiment was designed in 300 nM transfecting concentration after 48 h transfection. Blank control and negative control were set. The technique of RT-PCR was used for detection of TPX2mRNA in different groups.5 Screening of the optimum dosage TPX2siRNA-1725:the experiment was designed in 100 nM,200 nM and 300 nM transfecting concentrations with TPX2 siRNA-1725 after 48 h transfection. Blank control and negative control were set. The technique of RT-PCR was used for detection of TPX2mRNA in different groups.6 Screening of the optimum time point of TPX2siRNA-1725:300 nM of TPX2 siRNA were transfected into EC9706 cells. Then they were cultured 24 h,48 h,72 h and 96 h, we set blank control and negative control. RT-PCR and Western blot were used to detect the expression of TPX2mRNA and protein, and cellar proliferation was detected by MTT assay.7 Statistical analyses:The SPSS 15.0 statistical software package was used for all analyses. The data were expressed by mean±standard deviation(X±S)and analyzed using the ANVOA. The level of significant difference isα=0.05Results1 The screening results of optimum TPX2siRNA:three kinds of TPX2siRNA were transfected into EC9706cells for 48 h, the expression level of TPX2 mRNA in TPX2 siRNA-1725 group decreased, and it has the statistical significance(P< 0.05). So we got the result that TPX2 siRNA-1725 is the optimum TPX2siRNA.2 The screening results of optimum dosage of TPX2 siRNA-1725:TPX2siRNA-1725 were transfected into EC9706 cells with the density of 100 nM,200 nM and 300 nM for 48 h, the expression of TPX2 mRNA in 300 nM groups were suppressed evidently compared with control groups, and it has the statistical significance(P<0.05). So we got the result that the optimum dosage of TPX2 siRNA-1725 is 300 nM.3 The screening results of optimum transfection time point:EC9706 cells were transfected at the density of 300 nM TPX2siRNA for 24 h,48 h,72 h and 96 h, the expression quality of TPX2mRNA and protein were lower in experimental groups compared with control groups after transfecting for 48 h,72 h, especially 72 h. There was significant difference at different transfecting times (P<0.05). And the cellar proliferation was inhibited after 48 h, the inhibitory effect was mostly obvious after 72 h, and the rate of growth suppression reached 35.28%.4 There was a correlation between the expression of TPX2mRNA and the expression of protein after the transient transfection of TPX2siRNA at 24 h,48 h,72 h and 96 h.Conclusions1 The foundation of TPX2siRNA could research the functions of TPX2 conveniently; TPX2 siRNA can specially inhibit the expression of TPX2.2 Specific siRNA1725 of TPX2 can inhibit the expression of TPX2 gene and TPX2 Protein in the esophageal carcinoma EC9706 cells. It indicates that TPX2 siRNA can inhibit the proliferation of esophageal carcinoma by inhibiting expression of TPX2 gene.更多还原