目的 研究细胞因子诱导杀伤细胞(CIK)回输液中添加白蛋白和放置时间对其活性的影响,并建立简捷的CD3+CD56+细胞生成率及细胞死亡率的检测方法.方法 (1)抗CD3-FITC和抗CD56-PE同时标记CIK细胞,或FQ-AE染色CIK细胞,荧光显微镜观察并记录；(2)悬浮液中加或不加白蛋白,Annexin V-FITC标记CIK细胞,不同时间段取样,流式细胞仪检测.结果 (1)食道癌患者CIK细胞集落小,散在分布；胰腺癌患者CIK细胞集落大且集中；(2)显微镜可观察并计数CD3+CD56+细胞；(3)回输液中加与不加白蛋白的凋亡率及死亡率均无显著差异；(4)CIK细胞在培养箱外放置,0～90 min之间样品与150min样品之间凋亡细胞数差异显著,细胞死亡率与放置时间无显著差异.结论 (1)不同肿瘤患者CIK细胞的生长集落不同；(2)抗CD3和抗CD56标记可计数CD3+CD56+CIK细胞生成率,FQ-AE染色可计数死亡细胞比率,检测方法简便、快捷；(3)CIK细胞回输液中可不加白蛋白,对细胞存活率无影响；培养箱外放置时间90 min之内为宜,时间越短越好.%Objective To study the influence of albumin supplementation in the medium and the placement time outside the incubator on the viability of isolated cytokine-induced killer (CIK) cells.Methods CIK cells labeled with anti-CD3-FITC and anti-CD56-PE or with FQ-AE were observed under fluorescence microscope.The effect of albumin in the cell medium on the cell viability was analyzed using flow cytometry with Annexin V-FITC after different time lengths of placement.Results The clones of CIK cells from the patient with esophageal carcinoma were small and scattered in the medium,but the clones from the patients with pancreatic cancer were large and densely distributed.CD3+CD56+ cells could be detected under fluorescence microscope.The addition of albumin in the medium did not obviously affect cell apoptosis and death of CIK cells.CIK cells placed outside the incubator for less than 90 min showed a significant lower apoptosis rate than the cells placed for 150 min,whereas the cell death rate did not vary significantly with the placement time.Conclusion CIK cells from different cancer patients present with different growth pattern of the cells clones.Labeling with anti-CD3-FTTC and anti-CD56-PE allows convenient counting of the newly generated CD3+CD56+ CIK cells and FQ-AE labeling can be used for quantitative assessmentof cell death.Albumin is not necessary in the medium of CIK cells.Prolonged placement (for over 90 min) of CIK cells outsidethe incubator should be avoided,and the placement time should be shorten as much as possible.
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