IL-2/抗IL-2抗体复合物体外诱导扩增外周血和骨髓CD56~+淋巴细胞的实验研究
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摘要
目的探索一种从人外周血单个核细胞(PBMC)和骨髓中高效扩增CD56~+细胞毒性淋巴细胞的方法。
方法以干细胞生长培养基(SCGM)为基础培养基,设计IL-2/抗IL-2抗体复合物(IL-2和抗IL-2抗体按质量比1:20混合)实验组、相应因子浓度的IL-2、抗IL-2抗体以及IL-2+IL-15三组对照组,分别从PBMC和骨髓单个核细胞中诱导扩增CD56~+淋巴细胞,流式细胞仪分别检测外周血来源细胞第7、10天和骨髓来源细胞第7、14天的CD3/CD56表达率。用四甲基氮唑蓝(MTT)法检测其对肿瘤细胞株K562的杀伤活性,并分别与对照组激活的杀伤细胞进行比较。
结果在以SCGM为基础培养基时,CD56~+淋巴细胞经IL-2/抗IL-2抗体作用获得大量增殖。在培养第7和第10天时,外周血实验组CD56~+细胞分别扩增了(24.67±3.14)和(31.63±5.01)倍;其中CD3~+CD56~+和CD3~-CD56~+细胞分别扩增(110.93±19.10)、(157.60±46.31)和(7.8±1.17)、(12.03±1.64)倍,与对照组相比,差别有统计学意义(P均＜0.05);在培养第7和第14天时,骨髓实验组CD56~+细胞分别扩增了(11.08±2.08)和(21.72±2.19)倍;其中CD3~+CD56~+和CD3CD56~+细胞分别扩增(30.30±5.14)、(60.18±6.68)和(6.05±1.05)、(10.75±1.51)倍,与对照组相比,差别有统计学意义(P＜均0.05);细胞效/靶比10:1时,外周血和骨髓实验组细胞对K562细胞的杀伤率分别为(83.46±1.56)%和(74.94±2.28)%;与同期对照组细胞相比具有更强的淋巴细胞毒性作用(P均＜0.05)。
结论在IL-2/抗IL-2抗体(IL-2/抗IL-2抗体质量比为1/20)符合物作用下,以SCGM为基础培养基,可获得大量以CD56~+淋巴细胞为主细胞毒性免疫效应细胞,为肿瘤过继免疫治疗提供了一种新的体外扩增CD56~+淋巴细胞的方法。
Objective:To explore an efficient method for inducing and expanding CD56~+ lymphocytes from mononuclear cells of human peripheral blood(PBMCs) and bone marrow in vitro.
Method:Mononuclear cells from human peripheral blood(PBMCs) and bone marrow were cultured in stem cell growth medium(SCGM) supplemented with IL-2/anti-IL-2 complex,IL-2,anti-IL-2 mAb and IL-2+IL-15 respectively to induce and expand CD56~+ lymphocytes,the immune phenotypes of cells from blood were analyzed by Flow Cytometry(FCM) on the 7th and 10th day after incubation separately,while those from bone marrow were analyzed on the 7th and 14th day.The cytotoxicity of cells from peripheral blood and bone marrow to K562 targets was detected by MTT assay after culturing for 10 days and 14 days separately.
Results:The CD56~+ cells from peripheral blood cultured with IL-2/anti-IL-2 complex got the best expansion and expanded to 24.67±3.14 folds in 7 days and 31.63±5.01 folds in 10 days after incubation;Among them,the expanded folds for CD3~+CD56~+ and CD3-CD56~+ were(110.93±19.10)、(7.8±1.17) after culturing for 7 days and(157.60±46.31)、(12.03±1.64) after 10 days respectively;while those from bone marrow expanded to11.08±2.08 folds at the 7~(th) day and 21.72±2.19 folds at the 14~(th) day, of which the expanded folds for CD3~+CD56~+ and CD3-CD56~+ were(30.30±5.14)、(6.05±1.05) after culturing for 7 days and(60.18±6.68)、(10.75±1.51) after 14 days respectively.At 10:1 of effecter/target ratio,the cytotoxicity of cells from peripheral blood and bone marrow cultured with IL-2/anti-IL-2 complex to K562 cells increased up to(83.46±1.56)%and(74.94±2.28)%respectively,significantly higher than the other control groups.
Conclusions:CD56~+ lymphocytes could be expanded effectively from PBMC and bone marrow using SCGM under the stimulation and induction of IL-2/anti-IL-2 complex.This work provided a novel and effective method of expanding human CD56~+ lymphocytes in vitro for the adoptive immunotherapy of malignant tumors.
方法以干细胞生长培养基(SCGM)为基础培养基,设计IL-2/抗IL-2抗体复合物(IL-2和抗IL-2抗体按质量比1:20混合)实验组、相应因子浓度的IL-2、抗IL-2抗体以及IL-2+IL-15三组对照组,分别从PBMC和骨髓单个核细胞中诱导扩增CD56~+淋巴细胞,流式细胞仪分别检测外周血来源细胞第7、10天和骨髓来源细胞第7、14天的CD3/CD56表达率。用四甲基氮唑蓝(MTT)法检测其对肿瘤细胞株K562的杀伤活性,并分别与对照组激活的杀伤细胞进行比较。
结果在以SCGM为基础培养基时,CD56~+淋巴细胞经IL-2/抗IL-2抗体作用获得大量增殖。在培养第7和第10天时,外周血实验组CD56~+细胞分别扩增了(24.67±3.14)和(31.63±5.01)倍;其中CD3~+CD56~+和CD3~-CD56~+细胞分别扩增(110.93±19.10)、(157.60±46.31)和(7.8±1.17)、(12.03±1.64)倍,与对照组相比,差别有统计学意义(P均＜0.05);在培养第7和第14天时,骨髓实验组CD56~+细胞分别扩增了(11.08±2.08)和(21.72±2.19)倍;其中CD3~+CD56~+和CD3CD56~+细胞分别扩增(30.30±5.14)、(60.18±6.68)和(6.05±1.05)、(10.75±1.51)倍,与对照组相比,差别有统计学意义(P＜均0.05);细胞效/靶比10:1时,外周血和骨髓实验组细胞对K562细胞的杀伤率分别为(83.46±1.56)%和(74.94±2.28)%;与同期对照组细胞相比具有更强的淋巴细胞毒性作用(P均＜0.05)。
结论在IL-2/抗IL-2抗体(IL-2/抗IL-2抗体质量比为1/20)符合物作用下,以SCGM为基础培养基,可获得大量以CD56~+淋巴细胞为主细胞毒性免疫效应细胞,为肿瘤过继免疫治疗提供了一种新的体外扩增CD56~+淋巴细胞的方法。
Objective:To explore an efficient method for inducing and expanding CD56~+ lymphocytes from mononuclear cells of human peripheral blood(PBMCs) and bone marrow in vitro.
Method:Mononuclear cells from human peripheral blood(PBMCs) and bone marrow were cultured in stem cell growth medium(SCGM) supplemented with IL-2/anti-IL-2 complex,IL-2,anti-IL-2 mAb and IL-2+IL-15 respectively to induce and expand CD56~+ lymphocytes,the immune phenotypes of cells from blood were analyzed by Flow Cytometry(FCM) on the 7th and 10th day after incubation separately,while those from bone marrow were analyzed on the 7th and 14th day.The cytotoxicity of cells from peripheral blood and bone marrow to K562 targets was detected by MTT assay after culturing for 10 days and 14 days separately.
Results:The CD56~+ cells from peripheral blood cultured with IL-2/anti-IL-2 complex got the best expansion and expanded to 24.67±3.14 folds in 7 days and 31.63±5.01 folds in 10 days after incubation;Among them,the expanded folds for CD3~+CD56~+ and CD3-CD56~+ were(110.93±19.10)、(7.8±1.17) after culturing for 7 days and(157.60±46.31)、(12.03±1.64) after 10 days respectively;while those from bone marrow expanded to11.08±2.08 folds at the 7~(th) day and 21.72±2.19 folds at the 14~(th) day, of which the expanded folds for CD3~+CD56~+ and CD3-CD56~+ were(30.30±5.14)、(6.05±1.05) after culturing for 7 days and(60.18±6.68)、(10.75±1.51) after 14 days respectively.At 10:1 of effecter/target ratio,the cytotoxicity of cells from peripheral blood and bone marrow cultured with IL-2/anti-IL-2 complex to K562 cells increased up to(83.46±1.56)%and(74.94±2.28)%respectively,significantly higher than the other control groups.
Conclusions:CD56~+ lymphocytes could be expanded effectively from PBMC and bone marrow using SCGM under the stimulation and induction of IL-2/anti-IL-2 complex.This work provided a novel and effective method of expanding human CD56~+ lymphocytes in vitro for the adoptive immunotherapy of malignant tumors.
引文
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