NK细胞培养体系的优化及对T47D细胞杀伤力的比较
发布时间：2019-04-30 17:27
【摘要】：目的:优化NK细胞培养体系,提高NK细胞在体外培养的效率,检测不同培养体系NK细胞对乳腺癌细胞T47D体外杀伤效果。本文为NK细胞在肿瘤过继细胞免疫治疗的临床应用提供了理论与实践依据。方法:采集健康人静脉全血,分离出外周血单个核细胞,通过添加不同的细胞因子与添加ErbB2抗体的培养体系进行NK细胞培养,实验分为NK1组(培养基中含IL-2)、NK2组(培养基中含IL-2,IL-15和IL-21)、和NK3组(ErbB2抗体包被后培养基中含IL-2)三组。分别将培养第0天、7天和第14天的NK细胞进行流式分析检测CD3、CD4、CD8和CD56分子表型,检测不同培养时期NK细胞的比率。将培养14天后的三组培养体系的NK细胞分别以效靶比5:1、10:1、20:1分别和T47D细胞共同培养,用CCK-8法进行体外肿瘤细胞杀伤实验。结果:第7天时流式结果显示:NK3组与NK1及NK2组细胞相比,NK细胞比率开始有显著升高(P0.05),NK1与NK2组相比无显著性差异(P0.05);第14天时三组细胞相比,NK3组中NK细胞比率与NK1及NK2相比升高,有显著性差异(P0.05)。经离心收集NK细胞并调整细胞浓度,分别以效靶比5:1、10:1、20:1与T47D共培养24小时后,以CCK-8法检测细胞存活率,NK3组NK细胞对T47D的细胞的杀伤活性最高,与NK1及NK2组相比均有显著性差异(P0.05)。结论:经不同的方案诱导培养NK细胞,结果显示添加了ErbB2抗体的培养组,NK细胞的比率显著高于其它培养体系培养的细胞;并且添加了ErbB2抗体诱导培养体系培养出的NK细胞对乳腺癌细胞T47D的体外杀伤效果显著优于其它各组。
[Abstract]:Aim: to optimize the NK cell culture system, improve the efficiency of NK cell culture in vitro, and detect the killing effect of NK cells on breast cancer cell line T47D in vitro. This study provides a theoretical and practical basis for the clinical application of NK cells in tumor adoptive cellular immunotherapy. Methods: peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy people. NK cells were cultured by adding different cytokines and ErbB2 antibody. The experiment was divided into NK1 group (containing IL-2 in culture medium). NK2 group (containing IL-2,IL-15 and IL-21 in culture medium) and NK3 group (containing IL-2 in ErbB2 antibody coated medium). NK cells were cultured on day 0, day 7 and day 14 respectively. Flow cytometry was used to detect the phenotypes of CD3,CD4,CD8 and CD56, and the ratio of NK cells at different culture stages was detected. After 14 days of culture, NK cells were co-cultured with T47D cells with the effect-target ratio of 5, 10 and 20, respectively. The killing effect of T47D cells in vitro was tested by CCK- 8 method. Results: the results of flow cytometry on the 7th day showed that the ratio of NK cells in NK3 group was significantly higher than that in NK1 and NK2 groups (P0.05), but there was no significant difference between NK1 group and NK2 group (P0.05). On the 14th day, the ratio of NK cells in NK3 group was significantly higher than that in NK1 and NK2 groups (P0.05). After centrifugation, NK cells were collected and the cell concentration was adjusted. After 24 hours of co-culture with T47D with the effect-target ratio of 5, 10, 20, respectively, the survival rate of T47D cells was measured by CCK-8 assay. The cytotoxicity of NK cells to T47D cells in NK3 group was the highest. Compared with NK1 and NK2 group, there was significant difference (P0.05). Conclusion: NK cells were induced by different methods. The results showed that the ratio of NK cells in the culture group added with ErbB2 antibody was significantly higher than that in other culture systems. In addition, the killing effect of NK cells induced by ErbB2 antibody on breast cancer cell line T47D in vitro was significantly better than that of other groups.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q813.11;R730.51
本文编号：2468904
[Abstract]:Aim: to optimize the NK cell culture system, improve the efficiency of NK cell culture in vitro, and detect the killing effect of NK cells on breast cancer cell line T47D in vitro. This study provides a theoretical and practical basis for the clinical application of NK cells in tumor adoptive cellular immunotherapy. Methods: peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy people. NK cells were cultured by adding different cytokines and ErbB2 antibody. The experiment was divided into NK1 group (containing IL-2 in culture medium). NK2 group (containing IL-2,IL-15 and IL-21 in culture medium) and NK3 group (containing IL-2 in ErbB2 antibody coated medium). NK cells were cultured on day 0, day 7 and day 14 respectively. Flow cytometry was used to detect the phenotypes of CD3,CD4,CD8 and CD56, and the ratio of NK cells at different culture stages was detected. After 14 days of culture, NK cells were co-cultured with T47D cells with the effect-target ratio of 5, 10 and 20, respectively. The killing effect of T47D cells in vitro was tested by CCK- 8 method. Results: the results of flow cytometry on the 7th day showed that the ratio of NK cells in NK3 group was significantly higher than that in NK1 and NK2 groups (P0.05), but there was no significant difference between NK1 group and NK2 group (P0.05). On the 14th day, the ratio of NK cells in NK3 group was significantly higher than that in NK1 and NK2 groups (P0.05). After centrifugation, NK cells were collected and the cell concentration was adjusted. After 24 hours of co-culture with T47D with the effect-target ratio of 5, 10, 20, respectively, the survival rate of T47D cells was measured by CCK-8 assay. The cytotoxicity of NK cells to T47D cells in NK3 group was the highest. Compared with NK1 and NK2 group, there was significant difference (P0.05). Conclusion: NK cells were induced by different methods. The results showed that the ratio of NK cells in the culture group added with ErbB2 antibody was significantly higher than that in other culture systems. In addition, the killing effect of NK cells induced by ErbB2 antibody on breast cancer cell line T47D in vitro was significantly better than that of other groups.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q813.11;R730.51
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1 张金莉;NK细胞培养体系的优化及对T47D细胞杀伤力的比较[D];遵义医学院;2017年
，本文编号：2468904
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