Xiao Wei-Ling, Liang Shu-Juan, Mu Dong-Zhen, Wang Xue-Jing, Wu Hui-Na
Key Laboratory of Molecular Immunology, Weifang Medical College, Weifang 261042, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1130-2.
To investigate the effect of the expression of recombinant IL-1beta in H22 hepatoma cells on its response to NK cell mediated cytotoxicity.
BALB/c mouse was stimulated by 6% of starch. Total RNA was prepared from peripheral blood monocytes (PBMCs). IL-1beta gene (843 bp) was obtained by RT-PCR. The purified PCR product digested by Xho I and EcoR I was cloned into pIRES2-EGFP to construct the recombinant pIRES2-EGFP-mIL-1beta expression vector which was verified by PCR, restriction enzyme assay (Xho I and EcoR I) and DNA sequencing. Then the purified pIRES2-EGFP-mIL-1beta plasmid was transfected into H22 hepatoma cells by jetPEI. The expression level of recombinant IL-1beta was detected by RT-PCR and confocal microscopy. The cytotoxicity of wild-type spleenic NK cells against H22 cells was assessed by MTT assay.
After the total RNA isolated from the starch stimulated BALB/c mouse PBMC, 843 bp IL-1beta gene in length was prepared by RT-PCR. The purified PCR product digested by EcoR I and Xho I was ligated by pIRES2-EGFP to create pIRES2-EGFP-mIL-1beta expression plasmid which was verified by PCR, restriction enzyme assay and DNA sequencing. Then pIRES2-EGFP-mIL-1beta was transfected into H22 hepatoma cells by jetPEI. RT-PCR and confocal microscopy assay showed these cells expressed high level of recombinant IL-1beta expression vector. In a 4-hour based MTT assay, IL-1beta in H22 cells was more resistant to NK92 cell mediated cytotoxicity compared with the cells transfected with pIRES2-EGFP. Meanwhile, the cytolytic capacity of the spleenic NK cells separated from wild-type mouse decreased about 10% when the ratio of effector to target was 40:1.
The expression of proinflammatory cytokine IL-1beta can significantly down-regulate the cytolytic activity of NK cells against H22 hepatoma cells. It plays a crucial role in the immune escape of hepatoma from NK cell mediated innate immunity.
研究重组白细胞介素-1β（IL-1β）在H22肝癌细胞中的表达对其对自然杀伤（NK）细胞介导的细胞毒性反应的影响。
用6%淀粉刺激BALB/c小鼠。从外周血单核细胞（PBMC）中提取总RNA。通过逆转录聚合酶链反应（RT-PCR）获得IL-1β基因（843 bp）。经Xho I和EcoR I酶切纯化的PCR产物克隆到pIRES2-EGFP中，构建重组pIRES2-EGFP-mIL-1β表达载体，经PCR、酶切鉴定（Xho I和EcoR I）及DNA测序验证。然后将纯化的pIRES2-EGFP-mIL-1β质粒通过jetPEI转染至H22肝癌细胞。通过RT-PCR和共聚焦显微镜检测重组IL-1β的表达水平。采用MTT法评估野生型脾NK细胞对H22细胞的细胞毒性。
从经淀粉刺激的BALB/c小鼠PBMC中分离出总RNA后，通过RT-PCR制备了长度为843 bp的IL-1β基因。经EcoR I和Xho I酶切纯化的PCR产物与pIRES2-EGFP连接，构建pIRES2-EGFP-mIL-1β表达质粒，经PCR、酶切鉴定及DNA测序验证。然后通过jetPEI将pIRES2-EGFP-mIL-1β转染至H22肝癌细胞。RT-PCR和共聚焦显微镜检测显示这些细胞高水平表达重组IL-1β表达载体。在基于4小时的MTT试验中，与转染pIRES2-EGFP的细胞相比，H22细胞中的IL-1β对NK92细胞介导的细胞毒性更具抗性。同时，当效应细胞与靶细胞比例为40:1时，从野生型小鼠分离的脾NK细胞的溶细胞能力下降约10%。
促炎细胞因子IL-1β的表达可显著下调NK细胞对H22肝癌细胞的溶细胞活性。它在肝癌逃避NK细胞介导的固有免疫的免疫逃逸中起关键作用。
