VCAM-1-shRNA慢病毒载体构建及其抑制口腔鳞癌HN12细胞株体外增殖的实验研究

发布时间：2018-04-08 17:24

  本文选题：VCAM-1　切入点：RNA干扰　出处：《滨州医学院》2014年硕士论文

【摘要】：目的：应用RNA干扰技术,通过构建针对人VCAM-1的慢病毒载体并转染人口腔鳞癌HN12细胞株,研究其对口腔鳞癌HN12细胞中VCAM-1基因表达的影响及抑制口腔鳞癌HN12细胞增殖情况,初步探讨下调VCAM-1基因抑制口腔鳞癌细胞增殖的分子机制,为口腔鳞癌的靶向基因治疗提供新途径。方法：1.构建四组人VCAM-1-shRNA慢病毒载体。针对人VCAM-1设计四组siRNA序列,BamHI和EcoRI进行载体质粒双酶切后连接入RNA干扰载体质粒pLVX-shRNA-GFP中,经PCR和DNA测序鉴定重组克隆的准确性。将正确的重组病毒质粒及辅助包装载体质粒共转染293T细胞,收集细胞培养上清液,浓缩后测定病毒滴度,并进行病毒包装。2. VCAM-1-shRNA慢病毒载体对HN12细胞体外增殖的抑制作用。实验设立空载体组为实验对照组(NC)和未转染组为空白对照组(CK),应用VCAM-1-shRNA慢病毒转染HN12细胞,荧光显微镜下观察并计算VCAM-1-shRNA;漫病毒转染效率,探索HN12细胞体外最佳转染复数。采用RT-PCR和Western-blot技术分别检测HN12中VCAM-1的mRNA和蛋白水平的敲减效率,CCK8法检测HN12细胞的生长情况。结果：1.RT-PCR和DNA测序鉴定重组克隆结果显示,成功构建了人VCAM-1-shRNA慢病毒载体：分别命名Target1、Target2、Target3、Target4,滴度分别为6.0×109 TU/ml、1.0×109 TU/ml、1.0×109 TU/ml、5.0×109 TU/ml。2.将各组VCAM-1-shRNA慢病毒转染HN12细胞,转染72小时后检测其VCAM-1mRNA抑制率分别为85.53%、70.57%、72.98%、34.03%;VCAM-1蛋白表达量亦明显下降,而内参B-action表达未受影响；CCK8检测表明细胞生长受到抑制。结论：本实验成功构建了靶向VCAM-1基因的重组慢病毒载体VCAM-1-RNAi,并利用其将VCAM-1小干扰RNA表达框架转导入HN12细胞,并在细胞内实现表达,实现了靶向VCAM-1基因的RNA干扰。通过本实验中VCAM-1-shRNA慢病毒载体有效地下了HN12中VCAM-1 mRNA及蛋白量的表达,从而抑制了HN12细胞的生长；进一步验证了VCAM-1在口腔鳞癌HN12细胞株的生长过程中可能起到肿瘤促进因子的作用,下调VCAM-1的表达可以成为治疗口腔鳞癌的治疗方法之一。
[Abstract]:Objective: to study the effect of lentivirus vector targeting human VCAM-1 on the expression of VCAM-1 gene in oral squamous cell carcinoma (HN12) cells and its inhibition on the proliferation of HN12 cells by using RNA interference technique and transfection into HN12 cell line of human oral squamous cell carcinoma (OSCC).To explore the molecular mechanism of down-regulation of VCAM-1 gene on the proliferation of oral squamous cell carcinoma cells, and to provide a new approach for targeted gene therapy of oral squamous cell carcinoma.Method 1: 1.Four groups of human VCAM-1-shRNA lentivirus vectors were constructed.Four groups of siRNA sequences, BamHI and EcoRI, were designed for human VCAM-1. The vector plasmids were digested into RNA interference vector pLVX-shRNA-GFP after double enzyme digestion. The accuracy of the recombinant clones was confirmed by PCR and DNA sequencing.The correct recombinant virus plasmid and auxiliary packaging vector plasmid were co-transfected into 293T cells. The supernatant of cell culture was collected, the titer of the virus was determined after concentration, and the virus packaging was carried out.Inhibitory effect of VCAM-1-shRNA lentivirus vector on proliferation of HN12 cells in vitro.The empty vector group was used as the experimental control group and the non-transfection group as the blank control group. The HN12 cells were transfected with VCAM-1-shRNA lentivirus, and VCAM-1-shRNAs were observed and calculated under fluorescence microscope, and the transfection efficiency of HN12 cells in vitro was explored.The mRNA and protein levels of VCAM-1 in HN12 were detected by RT-PCR and Western-blot. CCK8 was used to detect the growth of HN12 cells.Results 1. RT-PCR and DNA sequencing showed that the recombinant human VCAM-1-shRNA lentivirus vector was successfully constructed: Target1, Target2, Target3, Target4, with the titer of 1.0 脳 109TU / ml, 1.0 脳 109TUU / ml, 5.0 脳 109TUmlml.2.After transfection of VCAM-1-shRNA lentivirus into HN12 cells for 72 hours, the inhibition rate of VCAM-1mRNA was 85.53 and 70.57and 72.984.03. the expression of VCAM-1 protein was also significantly decreased, while the expression of B-action was not affected. The results of CCK8 assay showed that the cell growth was inhibited.Conclusion: the recombinant lentivirus vector VCAM-1-RNAi targeting VCAM-1 gene was successfully constructed in this experiment, and the VCAM-1 small interfering RNA expression frame was transformed into HN12 cells and expressed in the cells. RNA interference targeting VCAM-1 gene was realized.In this study, the expression of VCAM-1 mRNA and protein in HN12 was effectively down-regulated by VCAM-1-shRNA lentivirus vector, which inhibited the growth of HN12 cells, and further verified that VCAM-1 may play a role as a tumor promoter in the growth of HN12 cell line of oral squamous cell carcinoma.Down-regulation of VCAM-1 expression may be one of the therapeutic methods for oral squamous cell carcinoma.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.8
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本文编号：1722595