摘要
目的观察Plk-1基因过表达对gemcitabine诱导胰腺癌细胞AsPC-1化疗敏感性的影响。方法①将携带有人外源性Plk-1基因的真核表达质粒转染AsPC-1细胞系设为处理组,转染空载体设为对照组,无处理组设为空白组,转染24 h后,提取细胞总RNA和总蛋白,利用RT-PCR法及Western blot鉴定AsPC-1细胞内Plk-1基因和蛋白表达水平。②采用流式细胞术检测转染组细胞凋亡的改变。③不同浓度gemcitabine作用于转染细胞,MTT法测定转染48 h后细胞增殖能力。结果①测序结果表明成功的从AsPC-1细胞总RNA中扩增Plk-1基因。RT-PCR结果表明:未转染组AsPC-1细胞组、转染空载体pcDNA3.1组及pcDNA3.1/Plk-1组24 h各组细胞内Plk-1 mRNA相对量分别为(2.14±0.16)、(2.18±0.15)、(2.58±0.18),转染pcDNA3.1/Plk-1组与其他各组相比,差异有高度统计学意义(P<0.01)。Western blot结果显示:未转染组AsPC-1细胞组、转染空载体pcDNA3.1及pcDNA3.1/Plk-1组24 h各组细胞内Plk-1基因的蛋白表达水平为(0.989±0.018)、(1.022±0.021)、(1.243±0.143),转染pcDNA3.1/Plk-1组与其他各组相比,差异有高度统计学意义(P<0.01)。②在gemcitabine作用下,pcDNA3.1/Plk-1转染组细胞凋亡率明显低于未转染组及空载体转染组,差异有高度统计学意义(P<0.01)。③AsPC-1/Plk-1转染组细胞的生长抑制率亦明显高于未转染组及空载体转染组,差异有高度统计学意义(P<0.01)。结论胞浆过表达Plk-1活性分子可明显降低gemcitabine诱导的AsPC-1细胞凋亡,增强其对化疗药物的耐受性。
Objective To investigate the effects of PlK-1 gene on the chemotherapeutic sensitivity of pancreatic carcinoma cell line AsPC-1 response to gemcitabine. Methods ①AsPC-1 cells transfected with Plk-1 overexpression plasmid（pcDNA/ Plk-1） was selected as treatment group,and empty vector（pcDNA3.1） was selected as control group,total RNA and total protein were extracted,AsPC-1 cells without any treatment was selected as blank group. The RT-PCR and western blotting assay were performed to detect the expression of Plk-1 gene and protein level in AsPC-1 cells after transfection with pcDNA/Plk-1 for 24 hours. ②Flow cytometry assay was used to detect the cell apoptosis change after treatment. ③MTT assays were used to test the effect of different concentration of gemcitabine on proliferation of transfected AsPC-1 cells. Results ①The expression level of Plk-1 mRNA in non-transfection AsPC-1 group,empty vector pcDNA3.1 transfection group and pcDNA3.1/Plk-1 transfection group were（2.14±0.16）,（2.18±0.15）,（2.58±0.18）; the Plk-1 mRNA in pcDNA3.1/Plk-1 transfection group was higher than that in other groups,the differences were statistically significant（P 0.01）. According to Western blot results,the protein expression level of Plk-1 in non-transfection AsPC-1 group,empty vector pcDNA3.1 transfection group and pcDNA3.1/Plk-1 transfection group were（0.989±0.018）,（1.022±0.021）,（1.243±0.143）,the protein expression level of Plk-1 in pcDNA3.1/Plk-1 transfection group was higher than that in other groups,the differences were statistically significant（P 0.01）. ②Under the function of Gemcitabine,apoptosis rate in pcDNA3.1/Plk-1 transfection group was lower than that in nontransfection group and empty vector transfection group,the differences were statistically significant（P 0.01）. ③The inhibition proliferation rate of AsPC-1 cells of pcDNA3.1/ Plk-1 transfection group was higher than that of non-transfection group and empty vector transfection group,the differences was statistically significant（P 0.01）. Conclusion Overexpression of Plk-1 gene in AsPC-1 cells can significantly decrease gemcitabine induced apoptosis and increase its chemotherapeutic resistentivity.
出处
《中国医药导报》
CAS
2013年第36期4-7,11,共5页
China Medical Herald
基金
国家自然科学基金面上项目(编号30972910
81172269)
中国博士后基金科学基金资助项目(编号20060390294)
江苏省自然科学基金项目(编号BK2011858)
