协和医学杂志

2012, (01)

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RNA干扰质粒对胰腺癌细胞系Panc-1原癌基因AKT2表达的影响
Employing RNAi to inhibit oncogenes AKT2 in pancreatic cancer cell line Panc-1

梁智勇;刘彤华;任新瑜;师晓华;吴焕文

摘要(Abstract):

目的 探讨RNA干扰质粒抑制胰腺癌细胞系Panc-1中原癌蛋白AKT2的表达对胰腺癌细胞细胞生长和凋亡情况的影响,并进行初步的机制探讨。方法 选择胰腺癌细胞系Panc-1,构建特异性抑制AKT2表达的RNA干扰质粒,瞬时和稳定转染胰腺癌细胞,采用MTT法及软琼脂克隆形成实验检测胰腺癌细胞的生长情况,通过Heochst染色及Annexin V-FITC/ PI染色法检测细胞凋亡情况,通过Western blot方法检测凋亡蛋白caspase-3表达情况。最后进行裸鼠移植瘤体内转染实验。结果 在胰腺癌细胞系Panc-1中,采用RNA干扰质粒,沉默原癌基因AKT2后,能够有效的抑制胰腺癌细胞Panc-1体外的生长能力、促进细胞的凋亡;诱导凋亡激酶caspase-3的表达;同时,动物体内试验结果显示,干扰质粒能够有效的抑制细胞Panc-1在动物体内的成瘤能力。结论RNA干扰质粒抑制原癌蛋白AKT2表达后,可以有效抑制胰腺癌细胞生长,促进凋亡,针对原癌蛋白AKT2的基因治疗在胰腺癌中具有重要的潜在应用价值。
Objective we used a plasmid-based RNAi system to evaluate the proliferation and apoptosis effect of silencing oncogenes AKT2 in pancreatic cancer cell line Panc-1, and discuss the possible mechanism. Methods we chose PANC-1 cell line as the target cancer cell line, constructed the RNAi plasmid targeting oncogene AKT2, and transfected cells transiently and stably. Using MTT and soft arga colony formation test to determine the proliferation status, using Heochst and Annexin V-FITC/ PI methods to determine the apotosis status of the cancer cells in vitro, using western blot to detect the protein level of AKT2 and caspase-3 kinase; finally, we evaluate the in vivo effect of the recombinant plamids on Panc-1 cells. Results RNAi inhibited oncogenes AKT2 can specifically downregulate mRNA and protein levels in Panc-1 cells; reduce cell proliferation and colony formation of Panc-1 cells; induced apoptosis in Panc-1 cells; increase the protein level of caspase-3 protein; inhibited tumor growth in vivo. Conclusions our research demonstrates oncogene AKT2 is a promising gene therapy target in pancreatic cancer.

关键词(KeyWords): |RNA干扰|胰腺癌|Panc-1|基因治疗|AKT2|
|RNA interference|Pancreatic cancer| Panc-1 cell line| gene therapy| oncogene AKT2|

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作者(Author): 梁智勇;刘彤华;任新瑜;师晓华;吴焕文

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