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目的探讨通过RNAi下调非小细胞肺癌局部粘着斑激酶(FAK)的活性后对吉西他滨药理的影响。方法靶向FAK的shRNA重组质粒转染并下调细胞FAK蛋白表达,用Western Blot检测转染细胞中FAK的下调;利用MTT检测吉西他滨在不同浓度下对转染细胞增殖的影响;用流式细胞术、PI荧光染色检测吉西他滨作用下转染细胞的凋亡;Caspase与Akt活性分别用Apo-ONETM均质Caspase-3/7检测系统、Western blot检测。结果在吉西他滨不参与的情况下,FAK RNAi不能影响细胞的增殖和凋亡,但FAK RNAi明显增加了吉西他滨对肿瘤细胞的灵敏度,Akt活性的下调与这一现象有关。结论靶向FAK的shRNA重组质粒下调细胞FAK蛋白表达能够增加吉西他滨对非小细胞肺癌的细胞毒性。
Objective To investigate the effect of gemcitabine on the pharmacological activities of focal adhesion kinase (FAK) in non-small cell lung cancer (NSCLC) by RNAi. Methods FAK shRNA targeting recombinant plasmid was transfected and down-regulated the expression of FAK protein. Western Blot was used to detect the down-regulation of FAK in transfected cells. MTT assay was used to detect the effect of gemcitabine on the proliferation of transfected cells. Flow cytometry PI staining was used to detect the apoptosis of transfected cells. The activity of Caspase and Akt was detected by Apo-ONETM homogeneous Caspase-3/7 system and Western blot respectively. Results In the absence of gemcitabine, FAK RNAi did not affect cell proliferation and apoptosis, but FAK RNAi significantly increased the sensitivity of gemcitabine to tumor cells. The down-regulation of Akt activity was associated with this phenomenon. Conclusion shRNA recombinant plasmid targeting FAK down-regulates the expression of FAK protein in cells, which can increase the cytotoxicity of gemcitabine on non-small cell lung cancer.