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目的:研究慢病毒介导的PLK1(Polo-like kinase1)基因RNAi对食管鳞癌细胞侵袭转移的影响.方法:利用RT-PCR和Western blot检测不同食管鳞癌细胞中PLK1的表达.根据人PLK1mRNA序列设计干扰片段,Western blot检测干扰效率;利用划痕愈合实验及Transwell实验研究靶向PLK1的RNAi对食管鳞癌细胞体外侵袭转移能力的影响.将有效干扰序列构建至慢病毒干扰载体pGLV/H1/GFP+Puro中,测序鉴定.重组慢病毒载体与包装质粒共转染293T细胞包装病毒,制备的重组病毒感染食管鳞癌细胞,利用荧光定量PCR和Western blot检测干扰效率;利用裸鼠肺转移模型实验研究慢病毒介导的PLK1基因RNAi对食管鳞癌细胞在体内侵袭转移能力的影响.结果:筛选出一种高表达PLK1的食管鳞癌细胞株TE-8用于实验研究;筛选出有效的干扰片段,并成功构建靶向干扰PLK1基因的慢病毒载体,制备重组病毒颗粒用于感染食管鳞癌细胞;靶向PLK1的RNAi在体内外均能明显抑制食管鳞癌细胞的侵袭与转移.结论:靶向干扰PLK1基因的重组慢病毒能够抑制食管鳞癌细胞的侵袭与转移,PLK1基因影响着食管鳞癌的恶性进展.
Objective: To study the effect of lentivirus-mediated RNAi of PLK1 gene on the invasion and metastasis of esophageal squamous cell carcinoma.Methods: The expression of PLK1 in different esophageal squamous cell carcinoma tissues was detected by RT-PCR and Western blot.According to the expression of human PLK1 mRNA Sequence interference fragment was designed and Western blot was used to detect the interference efficiency.With Scratch healing assay and Transwell experiment, the effect of RNAi targeting PLK1 on invasion and metastasis of esophageal squamous carcinoma cells in vitro was studied.Effective interference sequences were constructed into lentiviral vector pGLV / H1 / GFP + Puro, and identified by sequencing. Recombinant lentiviral vector and packaging plasmid were cotransfected into 293T cell packaging virus and the recombinant virus was transfected into esophageal squamous carcinoma cells. Fluorescent quantitative PCR and Western blot were used to detect the interference efficiency. Model experiment to study lentivirus-mediated PLK1 gene RNAi on esophageal squamous cell carcinoma invasion and metastasis in vivo.Results: A high expression of PLK1 esophageal squamous cell carcinoma cell line TE-8 for experimental study; screening effective And successfully constructed a lentiviral vector targeting to interfere with the PLK1 gene to prepare recombinant virus particles for infecting esophageal squamous cell carcinoma cells and targeting PLK1 RNAi can significantly inhibit the invasion and metastasis of esophageal squamous cell carcinoma in vitro and in vivo.Conclusion: Recombinant lentivirus targeting PLK1 gene can inhibit the invasion and metastasis of esophageal squamous cell carcinoma, PLK1 gene affects the malignant progression of esophageal squamous cell carcinoma.