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目的:利用滤纸挑选克隆方法简单、有效地筛选稳定表达VEGF-C shRNA的乳腺癌MCF-7细胞株。方法:用已构建的表达VEGF-C shRNA的重组质粒载体pSIREN-VEGF-C转染乳腺癌MCF-7细胞,利用嘌呤酶素筛选阳性克隆;以滤纸法挑选阳性克隆并扩大培养成稳定表达的细胞株;荧光定量PCR及蛋白质印迹法检测稳定表达的细胞株中VEGF-C的表达情况。结果:用滤纸法挑选并培养出的稳定表达细胞株中,VEGF-C的mRNA及蛋白表达均明显下调,抑制率分别为95%及100%,P<0.05。结论:利用滤纸挑选克隆方法成功筛选稳定表达VEGF-C shRNA的乳腺癌MCF-7细胞株,并将其命名为MCF-7/V-C。
OBJECTIVE: To screen the MCF-7 breast cancer cell line stably expressing VEGF-C shRNA by filter paper selection and cloning method. METHODS: Breast cancer MCF-7 cells were transfected with the constructed recombinant plasmid vector pSIREN-VEGF-C containing VEGF-C shRNA, and the positive clones were screened by purine enzyme. The positive clones were selected by filter paper method and expanded to be stably expressed Cell lines; Fluorescent quantitative PCR and Western blotting were used to detect the expression of VEGF-C in the stable cell lines. Results: The mRNA and protein expressions of VEGF-C were significantly down-regulated in the stable cell lines selected and cultured by filter paper method with the inhibition rates of 95% and 100%, respectively, P <0.05. Conclusion: The MCF-7 breast cancer cell line stably expressing VEGF-C shRNA was successfully screened by filter paper selection cloning method and named as MCF-7 / V-C.