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目的: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mIL-12的免疫调节机制及其抗肿瘤作用奠定基础。方法:构建真核表达质粒pcDNA3.1(+)-mIL-12,脂质体转染LLC细胞并测其表达,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:pcDNA3.1(+)-mIL-12质粒构建正确并成功转导入LLC细胞,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:成功构建pcDNA3.1(+)-mIL-12真核表达质粒,建立具有mIL-12生物学活性的LLC细胞系。
OBJECTIVE: To establish a mouse Lewis lung carcinoma (LLC) cell line LLC / mIL-12 stably expressing mouse IL-12 (mIL-12). To further investigate the immunoregulatory mechanism of mIL-12 and its anti-tumor effect Lay the foundation. Methods: The eukaryotic expression plasmid pcDNA3.1 (+) - mIL-12 was constructed and transfected into LLC cells by lipofectamine. The cell cycle and apoptosis were observed by flow cytometry (FCM). Positive clones were screened by G418 screening The mIL-12 activity was extensively cultured and tested. Results: The recombinant plasmid pcDNA3.1 (+) - mIL-12 was constructed successfully and successfully transfected into LLC cells. The recombinant plasmid mIL-12 overexpressed at both mRNA and protein levels, and mIL-12 re-distributed the LLC cell cycle and promoted its apoptosis Death. The LLC / mIL-12 cell culture supernatant significantly induced splenocyte A (ConA) -activated mouse splenocyte proliferation. Conclusion: The pcDNA3.1 (+) - mIL-12 eukaryotic expression plasmid was successfully constructed and the LLC cell line with mIL-12 biological activity was established.