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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。
To construct the prokaryotic expression system of bicistronic vector targeting human IL-18-EGF tumor targeting molecule and to study the effect of recombinant human IL-18-EGF fusion protein on the expression of natural killer cell (NK) and human hepatoma cell line SMMC- 7721). Recombinant human IL-18-EGF prokaryotic bicistronic expression system pET28a (+) - proIL-18-EGF-Caspase-4 / BL21 was constructed. The purified recombinant protein was applied to NK cells. CCK-8 and ELISA Kits were detected NK cell proliferation and IFN-γ secretion. The Cy3 fluorescent labeled IL-18-EGF was used to detect the binding of the fusion protein to EGFR on the surface of tumor cells. After incubation with IL-18-EGF and NK cells for 24 h, the culture supernatant was applied to human hepatocellular carcinoma cell line SMMC-7721. The cell scratch assay and Transwell chamber assay were used to detect the migration and invasion of hepatoma cells The impact of ability. The experimental results showed that recombinant human IL-18-EGF can accelerate the proliferation of NK cells and promote the secretion of IFN-γ by NK cells. IL-18-EGF can specifically bind EGFR on the surface of tumor cells. In the cell scratch assay, recombinant human IL In the transwell chamber assay, the number of cells in the IL-18-EGF group was 94.6 ± 2.9,101.8 ± 4.0 and 116.2 ± 12, respectively, at 12, 24 and 48 h 4.5, all of which were significantly lower than the corresponding time control group (128.6 ± 8.5,133.0 ± 7.5 and 138.8 ± 5.4 respectively) (P <0.05). The above results indicate that IL-18-EGF can significantly inhibit the migration and invasion of human hepatocellular carcinoma cell SMMC-7721, increase the immunity of the human body, and may be used as an auxiliary drug in the treatment of liver cancer.