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目的观察仅转染无启动子绿色荧光蛋白(GFP)的 cDNA 全长片段能否使整合有 GFP基因的胰腺癌细胞内 GFP 表达降低。方法用 pEGFP-C1质粒转染人胰腺癌细胞株 PANC-1,G418筛选及流式细胞术分选建立 GFP 蛋白高表达的稳定细胞株。用 PCR 方法从 pEGFP-C1扩增出 GFP基因全长 cDNA 并将其克隆至无启动子的复制型质粒 PUC19,得到重组质粒即 PUC-GFP。实验分为4组:空白对照组、空质粒组(PUC 组)、GFP 重组质粒干扰组(PUC-GFP 组)、GFP 小 RNA 干扰组(siGFP 组)。分别用稳定表达 GFP 和未经处理 PANC-1细胞进行实验。用 Western 印迹、流式细胞术与倒置荧光显微镜检测重组质粒对细胞内稳定表达的 GFP 的影响及对 GFP 阴性细胞 pEGFPC1质粒瞬时转染后绿色荧光表达强度的影响。结果 PUC-GFP 可使稳定细胞株内的 GFP 表达下降,并且呈剂量依赖性。PUC-GFP 组绿色荧光强度减弱程度和空白对照及空质粒组差异有统计学意义(P<0.05)和 siGFP 组差异无统计学意义(P>0.05)。转染后第4天 GFP 表达降低并可持续至第6天,第4天后 PUC-GFP 组和 siGFP 组对 GFP 的抑制差异无统计学意义(P>0.05)。PUC-GFP 与 pEGFP-C1共转染 GFP 阴性 PANC-1细胞株可使 pEGPC1的瞬时转染效率降低。这两种情况下,荧光降低程度均和转染 PUC-GFP 的量呈正相关。结论仅转染无启动子的某个特定基因的全长 cDNA 能使哺乳细胞内相应同源性基因的表达降低。DNA 干扰可用于哺乳动物细胞。
Objective To observe whether transfection of full-length cDNA fragment without promoter GFP could decrease the expression of GFP in pancreatic cancer cells with GFP gene. Methods The human pancreatic cancer cell lines PANC-1 and G418 were transfected with pEGFP-C1 plasmid and the stable cell lines with high expression of GFP protein were established by flow cytometry. The full length cDNA of GFP gene was amplified by PCR from pEGFP-C1 and cloned into the promoterless replicative plasmid PUC19 to obtain the recombinant plasmid PUC-GFP. The experiment was divided into 4 groups: blank control group, empty plasmid group (PUC group), GFP recombinant plasmid interference group (PUC-GFP group), GFP small RNA interference group (siGFP group). Experiments were performed with stable expression of GFP and untreated PANC-1 cells, respectively. Western blotting, flow cytometry and inverted fluorescence microscopy were used to detect the effect of the recombinant plasmid on the stable expression of GFP in cells and the effect on the expression of green fluorescence after transient transfection of pEGFPC1 plasmid in GFP negative cells. Results PUC-GFP decreased the expression of GFP in stable cell lines in a dose-dependent manner. The decrease of green fluorescence intensity in PUC-GFP group was significantly different from that in blank control group and empty plasmid group (P <0.05) and that of siGFP group was not statistically significant (P> 0.05). GFP expression decreased on the 4th day after transfection and lasted for 6 days. There was no significant difference in the inhibition of GFP between the PUC-GFP group and the siGFP group after 4 days (P> 0.05). Cotransfection of GFP-negative PANC-1 cell lines with PUC-GFP and pEGFP-C1 reduced the transient transfection efficiency of pEGPC1. In both cases, the degree of fluorescence reduction was positively correlated with the amount of transfected PUC-GFP. Conclusion Transfection of full-length cDNA of a specific gene without promoter can reduce the expression of corresponding homologous genes in mammalian cells. DNA interference can be used in mammalian cells.