目的：应用慢病毒介导的RNA干扰技术，靶向沉默人胰腺癌CFPAC-1细胞中的 EphB2 基因的表达，观察 EphB2 对CFPAC-1细胞增殖和凋亡的影响。 方法： 构建靶向 EphB2 基因的siRNA干扰质粒，通过LipofectamineTM 2000将干扰质粒转染进293T细胞，筛选出能有效抑制EphB2蛋白表达的干扰序列。构建含有效序列的慢病毒载体pLentiGFP-EphB2，感染CFPAC-1细胞，筛选出稳定干扰EphB2蛋白表达的CFPAC-1细胞系。定量PCR、Western blotting检测CFPAC-1细胞中 EphB2 mRNA和蛋白的表达，CCK8法和流式细胞术检测 EphB2 下调对CFPAC-1细胞增殖和凋亡的影响。 结果： 成功构建了EphB2慢病毒干扰载体pLentiGFP-EphB2，获得稳定转染pLentiGFP-EphB2的CFPAC-1细胞(CFPAC-1 EphB2 RNAi)。CFPAC-1 EphB2 RNAi细胞中 EphB2 mRNA和蛋白表达较空载体组显著降低， EphB2 mRNA的沉默效率为63%。与空载体组和未转染组相比，pLentiGFP-EphB2感染显著促进CFPAC-1细胞的增殖(1.89±0.17 vs 1.63±0.13、1.71±022，P<0.05)，抑制细胞的凋亡\[(7.02±1.27)% vs (13.37±1.89)%、(15.71±2.35)%，P<0.05\]。 结论： pLentiGFP-EphB2可有效下调胰腺癌CFPAC-1细胞中 EphB2 的表达，促进CFPAC-1细胞增殖和抑制其凋亡。

Objective : To investigate the effect of EphB2 on proliferation and apoptosis of human pancreatic cancer CFPAC-1 cells by target-silencing EphB2 expression using lentivirus-mediated RNA interference technique. Methods: The recombinant interfering plasmid targeting EphB2 gene was constructed and transfected into 293T cells by LipofectamineTM 2000, and the most effective interfering sequence was screened from inhibiting expression of EphB2 protein. The effective interference sequence was cloned into the pLentiGFP vector to construct lentivirus pLentiGFP-EphB2, and then transfected into CFPAC-1 cells. Stable cell line with down-regulated expression of EphB2 protein was obtained. Real-time PCR and Western blotting analysis were performed to determine the expression of EphB2 at mRNA and protein levels. Moreover, the effect of downregulation of EphB2 on proliferation and apoptosis of CFPAC-1 cells was detected by CCK8 and flow cytometry assay, respectively. Results: Recombinant lentivirus pLentiGFP-EphB2 was successfully constructed and stably transfected CFPAC-1 cells (CFPAC-1 EphB2 RNAi cells) were obtained. EphB2 expression at mRNA and protein levels in CFPAC-1 EphB2 RNAi cells were significantly lower than those at blank group, with silencing rate of EphB2 was 63% in the pLentiGFP-EphB2 group. In addition, the proliferation of CFPAC-1 EphB2 RNAi cells was increased more than those of the other two groups (1.89±0.17 vs 1.63±0.13, 1.71±0.22, P<0.05) and apoptosis was decreased \[(7.02±0.01)% vs (13.37±0.02)%, (15.71±0.02)%, P<0.05). Conclusion: pLentiGFP-EphB2 can effectively silence EphB2 expression in pancreatic cancer CFPAC-1 cells and then promote the proliferation and inhibit cell apoptosis.

天津市自然科学基金项目资助（No. 09JCZDJC21800）