【作者】 尹晓玲；

【导师】 孙世良； 李少林；

【作者基本信息】 重庆医科大学 ， 肿瘤学， 2006， 硕士

【摘要】 目的:白介素-12(interleukin- 12, IL-12)是具有抗瘤活性的细胞因子,构建能在真核细胞内稳定表达小鼠白介素-12(murrine interleukin- 12 interleukin- 12, mIL-12)的质粒,建立稳定表达小鼠IL-12(mIL-12)的小鼠Lewis肺癌(Lewis lung carcinoma, LLC)细胞系LLC/ mIL-12,为进一步研究IL-12的免疫调节机制和抗瘤免疫反应奠定基础。方法:通过聚合酶链反应(PCR)扩增质粒pORF- mIL-12(Elasti,全长),双酶切后将目的片段mIL-12连接到真核表达载体pcDNA3.1(+)质粒上,mIL-12受pcDNA3.1(+)中mCMV启动子驱动,将mIL-12全长转录至同一mRNA上,对重组质粒进行鉴定后用脂质体转染小鼠Lewis肺癌细胞(LLC)测其表达。流式细胞仪(FCM)观察细胞周期和凋亡, G418筛选获得阳性克隆后大量培养并检测mIL-12活性。结果:PCR成功扩增出mIL-12片段,PCR鉴定、酶切鉴定及测序均证实pcDNA3.1(+)-mIL-12质粒构建成功, RT-PCR及ELISA证实重组质粒在mRNA及蛋白质水平均高表达mIL-12。mIL-12使LLC细胞周期重新分布,并促进其凋亡。LLC/ mIL-12细胞培养上清能明显引起刀豆蛋白A(ConA)激活的小鼠脾细胞增殖。结论:pcDNA3.1(+)-mIL-12真核表达质粒构建成功并能在LLC细胞中稳定表达,成功建立具有mIL-12生物学活性的LLC细胞系。更多还原

【Abstract】 Objective:Interleukin 12 (IL-12) is a proinflammatory cytokine with antitumor activity. To construct the plasmid that can express murine IL-12 gene stablely in eukaryotic cell, and establish the murine Lewis Lung Carcinoma cell(LLC) line which can express murine IL-12 gene stably through recombinant plasmid, provide basis for further study of its immunoregulatory mechanism and antitumor immune response with mIL-12.Methods:PORF- mIL-12(Elasti) plasmid was amplified through PCR ,the objective segment (mIL-12)was inserted into eukaryotic expression vector pcDNA3.1(+) after enzyme digestion with restriction enzyme HindⅢand EcoRⅠ, mIL-12 was drived by mCMV promoter of pcDNA3.1(+) and transcribed into the same mRNA,the recombinant plasmid was transfected into LLC cell line with lipofectin after identifying it and measured its expression, cells cycle and apoptosis was observed by FCM . Got positive clone cells after selected by G418 and cultured them largely, meantime the activity of mIL-12 was examined.Results The mIL-12 was amplified and inserted into eukaryotic expression vector pcDNA3.1(+) after enzyme digestion and interlink,更多还原