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目的:观察RNA干扰(RNAi)技术对人食管鳞癌细胞(Eca-109)VEGF表达的影响。方法:采用DNA载体细胞内表达小片段干扰RNA(siRNA)方法,选择3条针对人VEGFmRNA的特异性siRNA靶序列,设计合成其相应的双链DNA,分别将其定向克隆至BglⅡ和HindⅢ双酶切的空白质粒pSUPER,得到重组质粒psiRNA-VEGF1、psiRNA-VEGF2和psiRNA-VEGF3。在重组质粒psiRNA-VEGF1、psiRNA-VEGF2、psiRNA-VEGF3以及空白质粒pSUPER转染Eca-10924h后,RealTimePCR检测Eca-109的VEGFmRNA水平,ELISA测定培养液上清的VEGF蛋白浓度。结果:RealTimePCR显示,食管鳞癌细胞VEGFmR-NA分别下降了62.1%、30.3%、16.7%和0,P<0.01;ELISA显示培养液上清VEGF浓度分别下降了70.8%、36.5%、21.2%和5.1%,P<0.01。结论:RNAi技术可显著抑制人食管鳞癌细胞的VEGF表达,为抗血管生成治疗肿瘤的进一步研究开辟了新的方向。
Objective: To observe the effect of RNA interference (RNAi) technology on the expression of VEGF in human esophageal squamous carcinoma cell line (Eca-109). Methods: The specific siRNA targeting human VEGFmRNA was designed and synthesized by DNA vector expressing small interfering RNA (siRNA), and the corresponding double-stranded DNA was designed and synthesized. The specific double-stranded DNA was designed and cloned into BglⅡ and HindⅢ double enzyme The cut empty plasmid pSUPER resulted in recombinant plasmids psiRNA-VEGF1, psiRNA-VEGF2 and psiRNA-VEGF3. After transfection of Eca-10924h with recombinant plasmids psiRNA-VEGF1, psiRNA-VEGF2, psiRNA-VEGF3 and blank plasmid pSUPER, VEGF mRNA level of Eca-109 was detected by RealTime PCR and VEGF protein concentration of culture supernatant was measured by ELISA. Results: RealTimePCR showed that VEGFmR-NA of esophageal squamous cell carcinoma was decreased by 62.1%, 30.3%, 16.7% and 0 respectively (P <0.01). ELISA showed that VEGF concentration in culture supernatant decreased by 70.8%, 36.5%, 21.2% 5.1%, P <0.01. Conclusion: RNAi can significantly inhibit the expression of VEGF in human esophageal squamous cell carcinoma, which opens up a new direction for the further study of anti-angiogenesis in the treatment of cancer.