DC-CIK对K562/A多药耐药基因mdr1表达的影响

The Expression Effect of DC-CIK on K562/A Multidrug Resistance Gene mdr1

目的:体外观察树突状细胞(dendritic cell,DC)联合细胞因子诱导的杀伤细胞(cytokine inducedkiller,CIK)对K562/A细胞株多药耐药基因mdr1表达的影响。方法:采集健康人的外周血,分离出单个核细胞(peripheral blood mononuclear cell,PBMC),在体外加入多种细胞因子经诱导生成DC及CIK细胞,以流式细胞仪检测其表面标志,将DC细胞内加入K562/A细胞裂解物致敏后,再与CIK细胞混合培养48小时。将致敏后的DC-CIK细胞与K562/A及K562分组培养后以荧光定量PCR检测其mdr1基因表达的情况,PBMC作为对照组。结果:RT-PCR中可见K562/A+DC-CIK组中mdr1 mRNA表达较K562/A明显降低,经荧光定量PCR观察到K562/A内mdr1 mRNA表达为K562的10.27倍、K562/A/PBMC略低于未处理的K562/A(P>0.05),K562/A/DC-CIK细胞中mdr1 mRNA含量较K562/A、K562/A/PBMC少(P<0.05)。DC-CIK细胞与细胞株混合培养后,mdr1基因表达较混合培养前明显降低。结论:实验数据显示DC-CIK可使耐药细胞株内mdr1基因表达下调。但K562与DC-CIK混合培养后该基因降低不明显,提示该基因在细胞中存在着基础表达,意义在于维持细胞内稳态。目前针对逆转白血病耐药的研究较少,需要多进行相关研究以拓宽细胞免疫治疗在逆转耐药领域的应用。DC-CIK是具有发展潜力的抗肿瘤方法。本实验将为下一阶段研究逆转耐药的机制提供依据,DC-CIK细胞免疫疗法有望成为逆转肿瘤耐药的新方法。

Objective： Observe the expression effect of dendritic cells（DC） combined with cytokine-induced killer cells（CIK） on the multidrug resistance gene mdr1 of K562/A cell line in vitro. Methods： Peripheral blood mononuclear cells（PBMC） isolated from peripheral blood of healthy people were collected, and induced them to DC and CIK by mixing in a variety of cytokines,then detected their surface markers by flow cytometry. Added cell lysates of K562/A to DC, and then made the sensitized DC culturing with CIK for 48 hours. Detected the expression of mdr1 gene by Quantitative PCR after sensitized DC-CIK were cultured with K562/A and K562 respectively, used PBMC as a control group. Results：The mdr1 mRNA in K562 / A ＋ DC-CIK group was significantly lower than K562 / A group by RT-PCR. Through Quantitative PCR we observed that the expression of mdr1 mRNA in K562 / A group was 10.27 times than K562 group, K562/A/PBMC group was slightly lower than the untreated K562/A group（P〈0.05）, and K562/A/DC-CIK group was lower than K562/A、K562/A/PBMC group（P〈0.05）. The expression of mdr1 gene after mixed culture was significantly lower than DC-CIK mixed culture with two cell lines before. Conclusion： The experimental datas showed that mdr1 gene in resistant cell lines can be downregulated by DC-CIK. But this gene was not reduced significantly after K562 mixed culture with DC-CIK, and it suggesting that there is basal gene expression in cells to maintain the intracellular homeostasis. The research about reversing resistance against leukemia is few currently, need to conduct more research in order to broaden the immunotherapy field of application in reversing drug resistance. DC-CIK is a anti-tumor approach with development potential. This experiment will provide a basis for the study of reversing drug resistance mechanisms next phase, and DC-CIK cellular immunotherapy is expected to become a new way reversing tumor resistance. The mothod is expected to become the leader in cancer treatment, it has broad prospects for development.