本文選題：VCAM-1　切入點(diǎn)：RNA干擾　出處：《濱州醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：應(yīng)用RNA干擾技術(shù),通過構(gòu)建針對(duì)人VCAM-1的慢病毒載體并轉(zhuǎn)染人口腔鱗癌HN12細(xì)胞株,研究其對(duì)口腔鱗癌HN12細(xì)胞中VCAM-1基因表達(dá)的影響及抑制口腔鱗癌HN12細(xì)胞增殖情況,初步探討下調(diào)VCAM-1基因抑制口腔鱗癌細(xì)胞增殖的分子機(jī)制,為口腔鱗癌的靶向基因治療提供新途徑。方法：1.構(gòu)建四組人VCAM-1-shRNA慢病毒載體。針對(duì)人VCAM-1設(shè)計(jì)四組siRNA序列,BamHI和EcoRI進(jìn)行載體質(zhì)粒雙酶切后連接入RNA干擾載體質(zhì)粒pLVX-shRNA-GFP中,經(jīng)PCR和DNA測(cè)序鑒定重組克隆的準(zhǔn)確性。將正確的重組病毒質(zhì)粒及輔助包裝載體質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,收集細(xì)胞培養(yǎng)上清液,濃縮后測(cè)定病毒滴度,并進(jìn)行病毒包裝。2. VCAM-1-shRNA慢病毒載體對(duì)HN12細(xì)胞體外增殖的抑制作用。實(shí)驗(yàn)設(shè)立空載體組為實(shí)驗(yàn)對(duì)照組(NC)和未轉(zhuǎn)染組為空白對(duì)照組(CK),應(yīng)用VCAM-1-shRNA慢病毒轉(zhuǎn)染HN12細(xì)胞,熒光顯微鏡下觀察并計(jì)算VCAM-1-shRNA;漫病毒轉(zhuǎn)染效率,探索HN12細(xì)胞體外最佳轉(zhuǎn)染復(fù)數(shù)。采用RT-PCR和Western-blot技術(shù)分別檢測(cè)HN12中VCAM-1的mRNA和蛋白水平的敲減效率,CCK8法檢測(cè)HN12細(xì)胞的生長(zhǎng)情況。結(jié)果：1.RT-PCR和DNA測(cè)序鑒定重組克隆結(jié)果顯示,成功構(gòu)建了人VCAM-1-shRNA慢病毒載體：分別命名Target1、Target2、Target3、Target4,滴度分別為6.0×109 TU/ml、1.0×109 TU/ml、1.0×109 TU/ml、5.0×109 TU/ml。2.將各組VCAM-1-shRNA慢病毒轉(zhuǎn)染HN12細(xì)胞,轉(zhuǎn)染72小時(shí)后檢測(cè)其VCAM-1mRNA抑制率分別為85.53%、70.57%、72.98%、34.03%;VCAM-1蛋白表達(dá)量亦明顯下降,而內(nèi)參B-action表達(dá)未受影響；CCK8檢測(cè)表明細(xì)胞生長(zhǎng)受到抑制。結(jié)論：本實(shí)驗(yàn)成功構(gòu)建了靶向VCAM-1基因的重組慢病毒載體VCAM-1-RNAi,并利用其將VCAM-1小干擾RNA表達(dá)框架轉(zhuǎn)導(dǎo)入HN12細(xì)胞,并在細(xì)胞內(nèi)實(shí)現(xiàn)表達(dá),實(shí)現(xiàn)了靶向VCAM-1基因的RNA干擾。通過本實(shí)驗(yàn)中VCAM-1-shRNA慢病毒載體有效地下了HN12中VCAM-1 mRNA及蛋白量的表達(dá),從而抑制了HN12細(xì)胞的生長(zhǎng)；進(jìn)一步驗(yàn)證了VCAM-1在口腔鱗癌HN12細(xì)胞株的生長(zhǎng)過程中可能起到腫瘤促進(jìn)因子的作用,下調(diào)VCAM-1的表達(dá)可以成為治療口腔鱗癌的治療方法之一。
[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.
【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.8
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本文編號(hào)：1722595