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目的:探讨不同细胞培养基及血清的培养体系对细胞因子诱导杀伤(cytokine-induced killer,CIK)细胞的增殖和功能的影响。方法:采集6例肺癌患者外周血,分离获得单个核细胞,按照不同血清与培养基分为8组:GT-T551培养基+患者自体血清组、GT-T551培养基+健康人血清组、GT-T551培养基+FBS组、GT-T551培养基组、RPMI 1640培养基+患者自体血清组、RPMI 1640培养基+健康人血清组、RPMI 1640培养基+FBS组及RPMI 1640培养基组。采用CFSE染色法检测细胞增殖能力,流式细胞术检测CIK细胞CD3+CD8+T细胞及CD3+CD4+T细胞颗粒酶B、IFN-γ、穿孔素的分泌情况及以白血病NB4、K562细胞为靶细胞时CD3+CD56+T细胞、CD3+CD4+T细胞及CD3+CD8+T细胞表面CD107a的表达。结果:GTT551培养基加入患者自体血清组CIK细胞的增殖指数显著高于其他各组(P<0.05)。GT-T551培养基或RPMI 1640培养基中加入患者自体血清组CD4+T细胞颗粒酶B的分泌水平均显著高于加健康人血清组[(22.85±3.50)%vs(13.28±1.75)%,(22.57±3.45)%vs(15.37±4.08)%,均P<0.01],而且GT-T551+患者自体血清组CD8+T细胞分泌IFN-γ的能力显著高于加健康人血清组(P<0.05)。以白血病细胞系NB4和K562细胞作为靶细胞时,检测CD3+CD56+NKT细胞表面CD107a的表达,GT-T551培养基中加入患者自体血清组优于加健康人血清组[(7.10±1.94)%vs(2.73±0.79)%,(8.00±1.82)%vs(3.03±0.78)%,P<0.01],同时优于RPMI1640+患者自体血清组[(4.45±1.96)%、(3.30±1.47)%,P<0.01]。结论:GT-T551培养基加患者自体血清的培养体系更有利于CIK细胞的增殖、细胞因子分泌及发挥杀伤功能,可推荐作为CIK细胞最佳培养体系。
OBJECTIVE: To investigate the effects of different cell culture medium and serum culture system on the proliferation and function of cytokine-induced killer (CIK) cells. Methods: Peripheral blood was collected from 6 patients with lung cancer. Mononuclear cells were isolated and divided into 8 groups according to different serum and medium: GT-T551 medium + patients’ autologous serum group, GT-T551 medium + healthy human serum group, GT -T551 medium + FBS group, GT-T551 medium group, RPMI 1640 medium + patient autologous serum group, RPMI 1640 medium + healthy human serum group, RPMI 1640 medium + FBS group and RPMI 1640 medium group. CFSE staining was used to detect cell proliferation. Flow cytometry was used to detect the secretion of granzyme B, IFN-γ and perforin in CD3 + CD8 + T cells and CD3 + CD4 + T cells in CIK cells, and the leukemic cells NB4 and K562 were The expression of CD107a on the surface of CD3 + CD56 + T cells, CD3 + CD4 + T cells and CD3 + CD8 + T cells on target cells. Results: Proliferation index of CIK cells in GTT551 medium was significantly higher than that in other groups (P <0.05). The secretion of granzyme B of CD4 + T cells in GT-T551 or RPMI 1640 medium was significantly higher than that in healthy human serum [(22.85 ± 3.50)% vs (13.28 ± 1.75)%, (22.57 ± 3.45)% vs (15.37 ± 4.08)% respectively, all P <0.01]. Moreover, the ability of secreting IFN-γ from CD8 + T cells of GT-T551 + patients was significantly higher than that of healthy volunteers ). When leukemia cell lines NB4 and K562 were used as target cells, CD107a expression on CD3 + CD56 + NKT cells was detected. Compared with healthy human serum group (7.10 ± 1.94)%, GT-T551 medium was superior to healthy human serum group (4.73 ± 0.79)%, (8.00 ± 1.82)% vs (3.03 ± 0.78)%, P <0.01, respectively, but also higher than those in RPMI1640 + patients (4.45 ± 1.96% vs 3.30 ± 1.47% P <0.01]. CONCLUSION: The culture system of GT-T551 plus autologous serum is more conducive to the proliferation of CIK cells, the secretion of cytokines and the exertion of cytotoxicity and can be recommended as the best culture system for CIK cells.