目的：探讨细胞因子诱导杀伤(CIK)细胞联合贝伐单抗（bevacizuma）对肝癌HepG2细胞的体内外抗肿瘤活性及其作用机制。方法：提取健康供血者外周血的单个核细胞(PBMC)，加入多种细胞因子促进CIK细胞成熟，在流式细胞仪上进行CIK细胞的免疫表型分析。CIK细胞与贝伐单抗单独或联合作用于HepG2细胞后，利用CCK-8测定其对HepG2细胞体外增殖活性的影响；利用侵袭小室(Transwell)和划痕实验测定其对HepG2细胞侵袭迁移活性的影响；Western blotting检测HepG2细胞Akt和Erk信号通路相关蛋白磷酸化的变化。建立HepG2细胞裸鼠皮下移植瘤模型，并随机分为生理盐水组、CIK组、贝伐单抗组及CIK细胞联合贝伐单抗组，给药28 d后处死裸鼠，剥取瘤体，免疫组化法检测瘤组织CD31及Ki67蛋白的表达。结果：提取健康人PBMC诱导14 d后，CIK细胞表型分析显示CD3+CD56+细胞扩增达(36.33±2.58)%。与单独治疗组比较，联合组对HepG2细胞的抗肿瘤增殖活性显著增强(P<0.05)；CIK细胞和贝伐单抗两药联合比单独给药组对HepG2细胞的侵袭\[(75.6±9.53) vs (304.8±45.73)、(359.8±38.10)个，P<0.01\]和迁移\[(29.35±8.14)% vs (55.07±6.27)%、(60.50±9.73)%，P<0.05\]能力的抑制更强；CIK细胞和贝伐单抗两药单独及联合都能抑制Akt和Erk的磷酸化；CIK联合贝伐单抗组可显著抑制移植瘤生长以及移植瘤组织平均血管密度和Ki67表达，与单独治疗及对照组细胞比较差异有统计学意义(P<0.05)。结论： CIK联合贝伐单抗在体内外对HepG2细胞增殖、侵袭、迁移均有抑制作用，且优于单独治疗组。

Objective:To investigate the antitumor activity and the mechanism of cytokine-induced killer (CIK) cells combined with bevacizumab against hepatocellular carcinoma HepG2 cells in vitro and in vivo. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy blood donors were isolated and cultured in the presence of different cytokines to promote the maturation of CIK cells; and the immune phenotypes of CIK cells were analyzed by flow cytometry. The inhibitory effect on HepG2 cells of CIK cells combining with or without bevacizumab were analyzed by CCK-8 assay. Cell migration and invasion of HepG2 cells were detected by scratch assay and transwell assay. The phosphorylation levels of Erk/Akt signaling pathway related proteins were detected by Western blotting. HepG2 cell xenograft tumor model was established in nude mice. Tumor bearing mice were divided into normal saline control group, CIK group, bevacizumab group and CIK+bevacizumab group. On the 28th day after drug administration, the mice were sacrificed and tumors were isolated. Immunohistochemical staining was used to analyze the expressions of CD31 and Ki67. Results: Human PBMCs were stimulated for 14 days, and the CIK cell phenotype analysis showed that the amplification of CD3+CD56+ CIK cells reached (36.33±2.58)%. The inhibition rate of CIK cells combined with bevacizumab was significantly enhanced by comparing with CIK/bevacizumab alone groups (all P<0.05). CIK cells combined with bevacizumab showed a more intensive inhibition on cell migration(\[75.6±9.53\] vs \[304.8±45.73\], \[359.8±38.10\], P<0.01) and invasion (\[29.35±8.14\]% vs \[55.07±6.27\]%, \[60.50±9.73\]%, P<0.05\] by comparing with those two single treatment groups; CIK cell treatment, bevacizumab treatment and the combined treatment could all decrease the phosphorylation level of Erk and Akt; the combined treatment significantly inhibited the tumor growth, mean vessel density (MVD) and Ki67 expression, which had statistical significance when compared with the other groups (P<0.05). Conclusion: The current study suggested that CIK cells combined with bevacizumab inhibited HepG2 cells proliferation, invasion and migration both in vitro and in vivo, which significantly precede the treatment efficacy of single treatment groups.

国家自然科学基金资助项目 (No. 81273814)