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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Hep G2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Hep G2细胞后,利用CCK-8测定其对Hep G2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Hep G2细胞侵袭迁移活性的影响;Western blotting检测Hep G2细胞Akt和Erk信号通路相关蛋白磷酸化的变化。建立Hep G2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Hep G2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Hep G2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Hep G2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 mechanism of cytokine-induced killer (CIK) cells combined with bevacizuma on Hep G2 cells in vitro and in vivo. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy blood donors were extracted, CIK cells were matured by adding various cytokines, and immunophenotype analysis of CIK cells was performed on flow cytometer. After treated with CIK cells and bevacizumab alone or in combination with Hep G2 cells, the effect of CCK-8 on the proliferation of Hep G2 cells was determined by CCK-8 assay. The invasion of Hep G2 cells was detected by Transwell assay and scratch assay Migration. The changes of phosphorylation of Akt and Erk signaling pathway-related proteins in Hep G2 cells were detected by Western blotting. A subcutaneous xenograft tumor model of Hep G2 cells in nude mice was established and randomly divided into normal saline group, CIK group, bevacizumab group and CIK cells combined with bevacizumab group. After 28 days of administration, nude mice were killed and tumors Immunohistochemistry was used to detect the expression of CD31 and Ki67 in tumor tissues. Results: After PBMCs were extracted from healthy donors for 14 days, phenotypic analysis of CIK cells showed that the number of CD3 + CD56 + cells was (36.33 ± 2.58)%. Compared with the untreated group, the anti-tumor activity of Hep G2 cells in the combination group was significantly increased (P <0.05); the invasion of Hep G2 cells by CIK cells and bevacizumab alone was significantly higher than that in Hep G2 cells [(75.6 ± (P <0.05)], (P <0.01) and [(29.35 ± 8.14)% vs (55.07 ± 6.27)%, (60.50 ± 9.73)% vs (304.8 ± 45.73) vs (359.8 ± 38.10) CIK cells and bevacizumab alone and in combination can inhibit Akt and Erk phosphorylation; CIK combined with bevacizumab can significantly inhibit the growth of xenografts and the mean tumor density and Ki67 expression in xenograft tumors, Compared with the single treatment and control group, the difference was statistically significant (P <0.05). Conclusion: CIK combined with bevacizumab inhibits the proliferation, invasion and migration of Hep G2 cells in vitro and in vivo, and is superior to the single treatment group.