论文部分内容阅读
目的建立稳定转染GLI1siRNA表达质粒的人胰腺癌细胞株,并鉴定其干扰效率。方法采用荧光定量PCR法(qRT-PCR)检测5株人胰腺癌细胞中GLI1基因的表达,筛选出GLI1基因表达量最高的细胞作为目标转染细胞。脂质体转染法将构建好的3个干扰质粒pGCsi-U6-GLI1siRNA-1、pGCsi-U6-GLI1siRNA-2、pGCsi-U6-GLI1siRNA-3分别转染入目标细胞,G418抗性筛选阳性克隆,荧光显微镜下观察转染效率。采用qRT-PCR法和蛋白质印迹法检测各组稳定转染细胞中GLI1mRNA及蛋白的表达水平,鉴定干扰效率。结果筛选出GLI1基因表达量最高的Panc-1细胞株作为目标转染细胞;3个干扰质粒均成功转染Panc-1细胞,G418筛选后获得稳定转染细胞Panc-1/GLI1siRNA-1、Panc-1/GLI1siRNA-2、Panc-1/GLI1siRNA-3,荧光显微镜下可见3个质粒的转染效率均在80%以上;qRT-PCR及蛋白质印迹检测证实Panc-1/GLI1siRNA-1、Panc-1/GLI1siRNA-2、Panc-1/GLI1siRNA-3细胞中GLI1mRNA和蛋白的表达水平均显著低于阴性对照质粒(Panc-1/siControl)转染细胞及空白对照细胞(P<0.05),其中Panc-1/GLI1siRNA-1细胞表达量最低。结论成功构建了稳定沉默GLI1基因表达的胰腺癌细胞株Panc-1/GLI1siRNA-1,为后续研究奠定了基础。
OBJECTIVE: To establish a human pancreatic cancer cell line stably transfected with GLI1 siRNA expression plasmid and identify its interference efficiency. Methods The expression of GLI1 gene in 5 human pancreatic cancer cells was detected by qRT-PCR, and the cells with the highest expression of GLI1 gene were screened out as target cells. The plasmids pGCsi-U6-GLI1siRNA-1, pGCsi-U6-GLI1siRNA-2 and pGCsi-U6-GLI1siRNA-3 were transfected into the target cells respectively by lipofection. The positive clones were screened by G418 resistance The transfection efficiency was observed under a fluorescence microscope. The qRT-PCR and Western blotting were used to detect the expression of GLI1mRNA and protein in each stable transfected group, and the interference efficiency was identified. Results Panc-1 cells with the highest expression of GLI1 gene were screened out as the target cells. Panc-1 / GLI1siRNA-1 was transfected into Panc-1 cells by three interference plasmids. Panc-1 / GLI1siRNA- 1 / GLI1siRNA-2, Panc-1 / GLI1siRNA-3. The transfection efficiency of the three plasmids was above 80% under the fluorescence microscope. The results of qRT-PCR and Western blot showed that the transfection efficiency of Panc-1 / GLI1siRNA- 1 / GLI1siRNA-2, Panc-1 / GLI1siRNA-3 cells were significantly lower than that of the negative control plasmid (Panc-1 / siControl) transfected cells and blank control cells (P <0.05) -1 / GLI1siRNA-1 cells were the lowest. Conclusion Panc-1 / GLI1siRNA-1, a pancreatic cancer cell line stably silenced by GLI1 gene, was successfully constructed and laid the foundation for further study.