DC及CIK共培养对肝癌细胞杀伤活性探究.docVIP

DC及CIK共培养对肝癌细胞杀伤活性探究 　 作者：陈宝安，李曼，孙载阳，李翠萍，高冲，孙耘玉 【摘要】 本研究的目的是观察细胞因子诱导的杀伤细胞（CIK）与同源树突状细胞（DC）共培养后DC-CIK细胞的增殖活性、表型的变化，及其对肝癌细胞细胞毒作用的影响。采集健康供者的外周血单个核细胞（MNC），置于37℃，5% CO2培养箱培养2小时，收集非贴壁细胞用于诱导培养CIK细胞，贴壁细胞诱导分化出成熟DC，将成熟DC和CIK细胞按1∶5的比例混合培养3天，用MTT法检测DC-CIK共培养细胞杀伤SMMC-7721肝癌细胞株的活性。结果显示： DC与CIK细胞共培养后，DC-CIK细胞群的增殖活性和杀伤活性较单纯的CIK细胞更高。结论： DC与CIK共培养细胞是一种增殖活性和细胞毒活性均高于CIK细胞的免疫活性细胞。 【关键词】 肝癌细胞 　　Killing Activity in DC and CIK Co-culture against Hepatocar-cinoma Cells 　　Abstract This study was aimed to investigate the proliferation activities and phenotype changes of DC，CIK and DC-CIK，and their cytotoxicity against hepatocarcinoma cells in co-culture of DC with CIK. Peripheral blood mononuclear cells(PBMNC) were isolated from heathy adult donors. After incubation of PBMNC for 2 hours，DCs were induced from adherent cells by some cytokines and CIKs were generated from non-adherent cells. Mature DCs were harvested after incubation for 9 days，and then were co-cultured with CIK at ratio of 1:5 for 3 days. The cytotoxicity activity against SMMC-7721 hepatocellular carcinoma cell line was detected by MTT assay. The results showed that CIK cells were able to lyse SMMC-7721 hepatocellular carcinoma cells at low ratios of effector to target. This effect was significantly enhanced by co-culture with DCs. It is concluded that CIK cells have high lytic activity against 7721 hepatocellular carcinoma cell line，which can be enhanced by co-culture with DC. DC-CIK cells are highly effective immune cells. 　　Key word Dendritic cells; cytokine induced killer cells; Immune therapy 　　近年来随着细胞免疫学和分子生物学的发展，恶性肿瘤的细胞免疫治疗已经开始在临床应用，并取得一定疗效。细胞因子诱导的杀伤细胞（cytokine induced killer cell，CIK）是一种高效、新型免疫活性细胞，是恶性肿瘤过继免疫治疗的重要手段之一。树突状细胞（dendritic cells，DC）是目前发现的功能最强的抗原呈递细胞（APC），它通过处理、呈递抗原，介导机体免疫应答。由于许多肿瘤宿主缺乏功能性DC而不能诱发抗原特异性T细胞应答，故在体外诱导功能性DC用于主动免疫治疗具有重要的临床应用价值。本实验将成熟DC和CIK细胞共培养，观察DC-CIK细胞群的增殖活性和表型变化，及其对肝癌细胞的体外杀伤作用。 　　材料和方法 　　主要试剂和仪器 　　IMDM完全培养基为Gibco公司产品，10%AB灭活血清为南京红十字血液中心产品，AIM-V无血清培养基为Gibco公司产品，淋巴细胞分离液购自上海第二制药厂，CD3单克隆抗体购自Ortho Biot