呼肠孤病毒感染体外扩增的CIK及NK细胞后联合西妥昔单抗的抗结直肠癌效应的初步研究

发布时间：2018-04-28 15:37

本文选题：NK细胞 + CIK细胞　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:研究体外扩增的自然杀伤细胞(NK)及CIK细胞(CIK)在联合西妥昔单抗(cetuximab)后对人EGFR阳性的结直肠癌细胞株DLD-1及Caco-2的杀伤效应,以及呼肠孤病毒(reovirus)能否进一步增强CIK及NK细胞与cetuximab的联合抗肿瘤效应,为临床结直肠癌的治疗提供新的治疗策略。方法:从健康献血者的外周血分离出单个核细胞(PBMC),分别常规诱导出CIK细胞及NK细胞,收集培养第14天的CIK细胞及NK细胞,ELISA实验检测CIK细胞培养上清中IFN-γ及TGF-β的水平,通过流式细胞分析仪分析CIK细胞及NK表型及结直肠癌细胞DLD-1和Caco-2的EGFR受体的表达水平。分别采用RTCA(RTCA)及乳酸脱氢酶(LDH)释放实验检测NK细胞的细胞毒作用。利用TCID50法和甲基纤维素改良的噬斑实验测定reovirus病毒的滴度,使用不同滴度的reovirus病毒感染CIK细胞及NK细胞。采用实时无标记分析仪分别动态监测CIK及NK细胞在不同滴度reovirus感染后联合cetuximab对DLD-1及Caco-2细胞株的杀伤效应。结果:流式细胞仪检测CIK细胞亚群可见培养后CD8+T(67.5±16.5%)、CD3+CD56+NKT(20.1±13.7%)及CD16+CD56+NK细胞比例(7.0±6.6%)均较培养前CD8+T(26.7±9.5%)、CD3+CD56+NKT(3.1±1.8%)及CD16+CD56+NK(2.6±2.7%)显著增高;CIK细胞培养培养上清中细胞因子浓度IFN-γ水平由34.8±35.4 ng/L增高到2041.8±697.9ng/L(P0.01),而同时TGF-β的水平由1293.8±633.9ng/L降为116.7±26.8ng/L(P0.01);经过14天体外扩增培养的NK细胞纯度可达86.2%;DLD-1及Caco-2肿瘤细胞表面均高表达EGFR,分别为81.6%及82.9%;LDH释放实验结果与RTCA检测所显示的杀伤趋势基本一致;TCID50法及噬斑实验求得病毒滴度分别为2.53×109pfu/L及1.36×109 pfu/L。RTCA检测发现NK细胞联合西妥昔单抗对结直肠癌细胞株杀伤作用明显高于单一的NK细胞组;CIK细胞对DLD-1及Caco-2细胞株的杀伤率分别为16.04±0.87%及27.67±3.10%,而当联合使用cetuximab后杀伤率分别增高到31.56±3.55%及35.96±1.73%,且用10pfu和100pfu滴度reovirus病毒感染CIK细胞及NK细胞后联合cetuximab对肿瘤细胞杀伤效应更明显。结论:CIK细胞及NK细胞联合cetuximab的杀伤效果明显增强,对KRAS野生型DLD-1细胞及KRAS突变型Caco-2细胞均有效,reovirus能进一步增强CIK细胞和NK细胞与cetuximab的联合抗肿瘤效应,为今后的临床研究奠定实验基础。
[Abstract]:Objective: to study the killing effect of natural killer cells (NKK) and CIK cells (CIK) on EGFR positive colorectal cancer cell lines DLD-1 and Caco-2 after combined with cetuximab. And whether reovirus can further enhance the anti-tumor effect of CIK and NK cells combined with cetuximab, and provide a new treatment strategy for clinical colorectal cancer. Methods: mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy blood donors. CIK cells and NK cells were induced by routine method. The levels of IFN- 纬 and TGF- 尾 in the supernatant of CIK cell culture were detected by Elisa of CIK cells and NK cells on the 14th day of culture. Flow cytometry was used to analyze the expression of DLD-1 and Caco-2 EGFR receptors in CIK cells and NK cells. The cytotoxicity of NK cells was detected by RTCA and LDH release assay. The titer of reovirus virus was determined by TCID50 assay and modified plaque assay of methylcellulose. CIK cells and NK cells were infected with different titers of reovirus virus. The killing effects of CIK and NK cells combined with cetuximab on DLD-1 and Caco-2 cell lines after reovirus infection with different titers were dynamically monitored by a real-time unlabeled analyzer. Results: flow cytometry analysis of CIK cell subsets showed that the levels of IFN- 纬 in the supernatant of CD8 T cell culture were significantly higher than those of CD8 T _ (26.7 卤9.5 卤9.5T) and CD16 CD56 NK(2.6 卤2.7 ~ (th) after culture. The ratio of CD3 CD56 NKT(20.1 卤13.7T and CD16 CD56 NK cells were significantly higher than that of CD8 T _ (26.7 卤9.5 卤1.8T) and CD16 CD56 NK(2.6 卤2.7T (P < 0.05). 34.8 卤35.4 ng/L increased to 2041.8 卤697.9 ng / L P 0.01g / L, while the level of TGF- 尾 decreased from 1293.8 卤633.9ng/L to 116.7 卤26.8ng / L P 0.01.The purity of NK cells cultured in vitro for 14 days was 86.2ng-1 and 82.9ng / L, respectively, which were 81.6% and 82.9%, respectively. The cytotoxic effect of NK cells combined with cetuximab on colorectal cancer cell line was significantly higher than that of single NK cell group. TCID50 assay and plaque assay showed that the titer of virus was 2.53 脳 109pfu/L and 1.36 脳 10 ~ 9 pfu/L.RTCA, respectively. The cytotoxicity of NK cells combined with cetuximab to DLD-1 was higher than that of single NK cell group. The killing rate of Caco-2 cell line was 16.04 卤0.87% and 27.67 卤3.10%, respectively, but the killing rate was increased to 31.56 卤3.55% and 35.96 卤1.73% respectively after combined use of cetuximab, and the killing effect of cetuximab combined with 10pfu and 100pfu titer of reovirus virus on CIK cells and NK cells was more obvious. Conclusion the killing effect of cetuximab combined with cetuximab was significantly enhanced by cetuximab, and both KRAS wild-type DLD-1 cells and KRAS mutant Caco-2 cells could be effectively treated with reovirus. The combined anti-tumor effect of CIK cells and NK cells with cetuximab was further enhanced. [WT5HZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ] [WT5BZ] To lay the experimental foundation for the clinical research in the future.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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