兰州大学机构库 >学院待认领
载体介导的siRNA对LoVo细胞中VEGFR-3表达的抑制作用
Alternative TitlesiRNA vector silencing VEGFR-3 expression in LoVo cells
贾如江
Subtype硕士
Thesis Advisor张有成
2008-05-09
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordRNA干扰 siRNA VEGFR-3 表达载体 LoVo细胞
Abstract目的:构建靶向血管内皮生长因子受体3 (VEGFR-3)基因的小干扰RNA(siRNA)表达载体(psiRNA-VEGFR-3),转染至人结肠癌细胞中,检测这一表达载体是否能在人结肠癌细胞株LoVo细胞中引发RNA干扰(RNAi)效应,能否阻抑VEGFR-3基因表达,及阻抑VEGFR-3基因表达后肿瘤细胞生长和凋亡率变化等生物学效应的改变,探讨VEGFR-3在肿瘤细胞生长中的作用,期望为结肠癌基因治疗提供新的途径。 方法:GenBank中查找VEGFR-3 基因全序列,作为靶基因,按照siRNA设计、合成三对VEGFR-3编码基因的反向重复序列,中间间隔9个核苷酸序列,并设计、合成一对非特异性对比序列,退火形成短双链RNA,通过定向克隆至线形载体pSUPER,构建siRNA真核表达载体,酶切鉴定表达载体,利用Lipofeetamine2000将构建的psiRNA-VEGFR-3载体转入LoVo细胞中,应用半定量RT-PCR检测基因转染后各组细胞VEGFR-3 mRNA表达水平的变化,采用四甲基偶氮唑蓝显色法(MTT)观察各组细胞生长变化,并绘制细胞生长曲线,并应用流式细胞计数仪分析各组细胞凋亡的情况,统计学方法分析比较各组结果差异是否有统计学意义。 结果: 针对VEGFR-3基因的siRNA表达载体成功构建;psiRNA-VEGFR-3可显著抑制LoVo细胞中VEGFR-3基因的表达;转染psiRNA-VEGFR-3细胞生长明显抑制;转染psiRNA-VEGFR-3,可以诱导LoVo细胞出现凋亡。 结论: psiRNA-VEGFR-3载体能够在LoVo细胞中引发RNAi效应,下调VEGFR-3基因的表达可以抑制细胞增殖,诱导细胞凋亡,这种针对VEGFR-3的RNAi可能为肿瘤的基因治疗提供新途径。
Other AbstractObjective: Construction of targeting vascular endothelial growth factor receptor 3 (VEGFR-3) gene by small interfering RNA (siRNA) expression vector (psiRNA-VEGFR-3) and to investigate the effects on suppressing the expression of VEGFR-3 gene in human colon cancer cell line LoVo cells. Methods: One pair of 19 bp reverse repeated sequence targeting VEGFR-3 mRNA spaced by 9bp nucleotide were synthesized and inserted into plasmid pSUPER to generate siRNA eukaryotic expression vector. LoVo cells were transfected with psiRNA-VEGFR-3 vectors by lipofectamine 2000.The expressions of VEGFR-3 mRNA was detected by real-time PCR.MTT was used to measure cell growth and flow cytometric analysis was applied for analysis of cell apoptosis. Results: The VEGFR-3 siRNA expression vector was constructed successfully, psiRNA-VEGFR-3 significantly inhibited LoVo cell VEGFR-3 expression, transfection psiRNA-VEGFR-3 markedly inhibited cell growth, and transfection psiRNA-VEGFR-3 induced apoptosis in the cancer cells. Conclusion: psiRNA-VEGFR-3 vectors have the effects of RNA interference(RNAi) and lowering the expression of VEGFR-3 gene can inhibit the proliferation of LoVo cells and induce the apoptosis of LoVo cells. The RNAi of VEGFR-3 activity may be a new way to supress the development of tumor cell.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222272
Collection学院待认领
Affiliation
临床医学院
Recommended Citation
GB/T 7714
贾如江. 载体介导的siRNA对LoVo细胞中VEGFR-3表达的抑制作用[D]. 兰州. 兰州大学,2008.
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