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目的通过研究RNA干扰(RNAi)对肝癌细胞激酶功能区受体(kinase domain receptor,KDR)基因表达的抑制,为肝癌基因治疗提供理论依据。方法应用逆转录聚合酶链反应(RT-PCR)技术检测KDR在肝癌细胞系HHCC、HepG2、Hep-6及正常肝细胞L-02的表达情况,筛选KDR高表达细胞株。设计构建针对KDR的靶向小干扰RNA(siRNA)质粒载体,通过脂质体转染高表达细胞株HHCC,观察其干扰效果。细胞计数法观察KDR-siRNA对HHCC细胞生长的影响。结果酶切鉴定、DNA测序分析证实重组质粒构建成功,分别命名为KDR-siRNA1、KDR-siRNA2和KDR-siRNA3。荧光定量PCR结果显示:3个KDR-siRNA转染的HHCC细胞株KDR的表达明显受到抑制,抑制率分别为68.86%、82.63%和64.47%,其中KDR-siRNA2抑制作用最为明显;转染阳性和阴性对照组载体的HHCC细胞和未转染的HHCC细胞生长趋势较为一致,且生长速度明显高于转染3种KDR-siRNA表达载体的HHCC。从接种第2天开始,KDR-siRNA转染组与对照组比较,细胞增殖明显受到抑制(P<0.01)。结论KDR靶向RNAi重组质粒载体能有效抑制肝癌HHCC细胞KDR基因的表达和细胞增殖,为探索KDR介导的肿瘤基因治疗的新途径奠定了实验基础。
Objective To study the inhibitory effect of RNAi on the gene expression of kinase domain receptor (KDR) in hepatocellular carcinoma cells and to provide a theoretical basis for gene therapy of hepatocellular carcinoma. Methods The expression of KDR in HHCC, HepG2, Hep-6 and normal liver cells L-02 was detected by reverse transcription-polymerase chain reaction (RT-PCR) The target small interfering RNA (siRNA) plasmid vector targeting to KDR was designed and constructed, and the high expression cell line HHCC was transfected by liposome. The interference effect was observed. Effect of KDR-siRNA on the growth of HHCC cells was observed by cell counting. Results Restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmids were successfully constructed and named KDR-siRNA1, KDR-siRNA2 and KDR-siRNA3, respectively. The results of real-time PCR showed that KDR-siRNA transfected HHCC cell line KDR significantly inhibited the expression of KDR, the inhibition rates were 68.86%, 82.63% and 64.47%, respectively, of which KDR-siRNA2 inhibition was the most obvious; The growth of HHCC cells and untransfected HHCC cells in negative control group were consistent and the growth rate was significantly higher than that of HHCC transfected with three KDR-siRNA expression vectors. From the second day after inoculation, cell proliferation was significantly inhibited in KDR-siRNA-transfected and control groups (P <0.01). Conclusion KDR targeting RNAi recombinant plasmid vector can effectively inhibit KDR gene expression and cell proliferation in HCC cells, which lays the foundation for further exploration of KDR-mediated gene therapy.