| 摘要: |
| 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GLI1 mRNA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GLI1 mRNA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。 |
| 关键词: 胰腺肿瘤 GLI1 小分子干扰RNA |
| DOI： |
| 投稿时间：2013-01-18修订日期：2013-05-08 |
| 基金项目:国家自然科学基金(81101575),上海市科委科技发展基金(114119a6700). |
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| Establishment of human pancreatic cancer cell line with stable knockdown of GLI1 gene |
| GUO Jie-fang,GAO Jun,LI Zhao-shen*,GONG Yan-fang,JIN Jing,WU Hong-yu,MAN Xiao-hua |
(Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To establish a human pancreatic cancer cell line stably transfected with siRNA expression vector targeting GLI1 gene and examine the interference efficiency. Methods The expression of GLI1 gene in five human pancreatic cancer cell lines was detected by quantitative real-time PCR (qRT-PCR); the one with the highest expression level of GLI1 was selected as the target cell line and was transfected with three recombinant plasmids pGCsi-U6-GLI1siRNA-1,-2,and -3. The positive clones were screened by G418, and the transfection rate was observed by fluorescence microscope. The expression of GLI1 mRNA and protein was analyzed by qRT-PCR and Western blotting analysis, respectively. Results Panc-1 cell line was found to have the highest GLI1 expression and was selected as the target cell line for transfection. Plasmids pGCsi-U6-GLI1siRNA-1, -2, and -3 were successfully transfected into Panc-1 cells separately. After 4 weeks of G418 screening, three stably transfected cell lines named Panc-1/GLI1siRNA-1, -2, and -3 were obtained, with the transfection rates all higher than 80%. qRT-PCR and Western blotting analysis showed that the expression levels of GLI1 in Panc-1/GLI1siRNA-1, -2, and -3 cells were all significantly lower than those in Panc-1/siControl cells and the blank control cells(P<0.05), with the lowest expression found in Panc-1/GLI1siRNA-1 cells. Conclusion We have successfully constructed a cell line Panc-1/GLI1siRNA-1 with GLI1 gene stably silenced, which paving a way for future research. |
| Key words: pancreatic neoplasms GLI1 small interfering RNA |
