重组人IL-18-EGF的双顺反子表达及其对NK细胞和肝癌细胞的影响

构建重组人IL-18-EGF肿瘤靶向分子双顺反子原核表达系统,研究重组人IL-18-EGF融合蛋白对人自然杀伤细胞（natural killer cell,NK细胞）和人肝癌细胞（SMMC-7721）的影响。构建重组人IL-18-EGF原核双顺反子表达系统pET28a（＋）-proIL-18-EGF-Caspase-4/BL21,重组蛋白经纯化后,作用于NK细胞,应用CCK-8法和ELISA试剂盒分别检测NK细胞的增殖情况和IFN-γ的分泌量。Cy3荧光标记IL-18-EGF检测融合蛋白与肿瘤细胞表面EGFR的结合情况。IL-18-EGF与NK细胞共同孵育24 h后,取培养上清液作用于人肝癌细胞SMMC-7721,分别使用细胞划痕实验和Transwell小室实验检测IL-18-EGF对肝癌细胞迁移和侵袭能力的影响。实验结果显示：重组人IL-18-EGF能加快NK细胞的增殖,促进NK细胞分泌IFN-γ;IL-18-EGF能与肿瘤细胞表面EGFR特异性结合;细胞划痕实验中,重组人IL-18-EGF组空白区域的抗填充能力高于对照组;Transwell小室实验中,12,24,48 h时IL-18-EGF组细胞穿膜数分别为94.6±2.9、101.8±4.0和116.2±4.5,均显著低于相应时间的对照组（分别为128.6±8.5、133.0±7.5和138.8±5.4）（P〈0.05）。以上结果表明,IL-18-EGF对人肝癌细胞SMMC-7721的迁移和侵袭能力有明显的抑制作用,能提高机体的免疫能力,有可能作为辅助药物运用于肝癌的治疗。

Bicistronical Expression of the Recombinant Human IL-18-EGF and Its Effects on NK Cell and the Human Hepatoma Cells

Recombinant human IL-18-EGF tumor targeting molecular was due to prokaryotic bicistronical expression system, and the effects of this fusion protein on human natural killer cell （NK cell） and hepatocellular carcinnoma cell line SMMC-772 l were investigated. A bicistronical expression system pET28a（＋）-prolL-18-EGF- Caspase-4/BL21 was constructed to express recombinant human IL-18-EGF. The recombinant protein was purified and its effect on human natural killer cell was studied. CCK-8 method was used to observe NK cell proliferation ability stimulated by IL-18-EGF and the IFN-7 secreted by NK cell was determined by ELISA. IL-18-EGF was labeled by Cy3 dye to observe its ability of binding with the tumor cell surface EGFR. After IL-18-EGF and NK cells were incubated for 24 h, the culture super-natant was took to hepatocellular carcinoma cell SMMC-7721. Then wound healing assay and Transwell assay were used to study the suppressions of migration and invasion of hepatocellular carcinoma cell, respectively. The experimental results showed that the recombinant human IL-18-EGF can promote NK cell proliferation and increase the IFN-7 secreted by NK cell. IL-18-EGF can specially bind to tumor cell surface EGFR. Wound healing assay indicated that the capacity of filling blank area in the IL-18-EGF group was higher than that in the control group. Cell invasion assay results showed that the average amount of invading cells in the IL-18-EGF group （94.6±2.9, 101.8±4.0 and 116.2±4.5 after 12, 24, 48 h, respectively） was significantly lower than the control group （128.6±8.5, 133.0±7.5 and 138.8±5.4 after 12, 24, 48 h, respectively, P〈0.05）. The results suggest that IL-18-EGF can inhibit migration and invasion of hepatocellular carcinoma cell and improve human immune ability. It may be used in the treatment of liver cancer as a auxiliary drug.