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目的:通过体内外实验探讨阿糖胞苷对抗CD3/抗CD19双功能抗体介导的T淋巴细胞杀伤人急性B淋巴细胞白血病(B acute lymphoblastic leukemia,B-ALL)细胞的协同作用。方法:采用流式细胞术检测经阿糖胞苷(0.062 5、0.125、0.25、0.5μmol/L)刺激的Nalm-6细胞(1×106)及5例B-ALL患者临床标本CD80和CD86分子的表达;Ficoll-Hypaque法分选健康志愿者外周血T淋巴细胞,以4×105 Nalm-6细胞(经过或未经Ara-C刺激)为靶细胞,建立T细胞体外杀伤实验并分组加入各种抗体(PBS、抗CD3/抗CD19双抗、抗CD3scFv、抗CD19scFv、抗CD3scFv+抗CD19scFv和抗CD3/抗Pgp双抗组),通过实时定量PCR测定各组激活的T细胞细胞因子IL-3、IFN-γ和TNF-α的表达,利用流式细胞术检测各组T细胞穿孔素和颗粒酶B的释放。以1×107 Nalm-6细胞建立NOD/SCID鼠移植瘤模型,依次注射阿糖胞苷,T细胞及各种或各浓度抗体,比较各组体内靶向杀伤效果。建立临床标本的动物模型,重复上述分组,并进一步验证双抗联合阿糖胞苷介导的体内杀伤效果。结果:Nalm-6经阿糖胞苷刺激后,细胞CD80的表达量MFI为13.25±3.93,0.062 5μmol/L为20.58±3.94,P=0.015;0.125μmol/L为38.6±5.37,P=0.011;0.25μmol/L为62.56±6.24,P=0.007;0.5μmol/L为75.04±4.42,P=0.005,呈浓度依赖性。抗CD3/抗CD19组T淋巴细胞穿孔素(MFI为10.61±2.43,P=0.024)和颗粒酶B(MFI为20.60±2.61,P=0.037)的释放较其余各实验组明显升高,Ara-C刺激Nalm-6细胞组,穿孔素(MFI为23.11±2.90,P=0.015)和颗粒酶B(MFI为31.71±3.08,P=0.008),较未经Ara-C刺激组表达量高。抗CD3/抗CD19组T淋巴细胞IL-3(100.60±8.32,P=0.017)、IFN-γ(67.60±7.41,P=0.025)和TNF-α(47.60±3.24,P=0.031)表达对照T细胞组明显升高,并且Ara-C刺激Nalm-6细胞组IL-3(132.55±5.56)、IFN-γ(109.60±6.50)和TNF-α(64.30±8.55)较未经Ara-C刺激组表达量更高,P值均<0.05。在NOD/SCID鼠移植瘤模型的生物治疗实验中,阿糖胞苷联合双功能抗体组抑制移植瘤的生长效果较对照组明显增强,P<0.05。5例临床标本阿糖胞苷刺激后有2例CD80表达上调,体内实验中该2例标本体内移植瘤生长受到明显抑制,P<0.05。结论:阿糖胞苷通过上调Nalm-6细胞和部分B-ALL患者临床标本共刺激分子CD80的表达,可有效激活T细胞,并增强双功能抗体介导T细胞体内杀伤细胞的作用。
OBJECTIVE: To investigate the synergistic effect of cytarabine on anti-CD3 / anti-CD19 dual-function antibody-mediated T lymphocyte killing in B acute lymphoblastic leukemia (B-ALL) cells in vitro and in vivo. Methods: Flow cytometry was used to detect the expression of CD80 and CD86 in Nalm-6 cells (1 × 106) and 5 cases of B-ALL stimulated by cytarabine (0.062 5,0.125,0.25,0.5μmol / L) T cells in peripheral blood of healthy volunteers were sorted by Ficoll-Hypaque method, and T cells were killed in vitro by 4 × 105 Nalm-6 cells (with or without Ara-C stimulation) The anti-CD3 / anti-CD19 double antibody, anti-CD3scFv, anti-CD19scFv, anti-CD3scFv + anti-CD19scFv and anti-CD3 / anti-Pgp group were detected by real- , IFN-γ and TNF-α, and the release of perforin and granzyme B in T cells of each group was detected by flow cytometry. The model of NOD / SCID mouse xenografts was established with 1 × 107 Nalm-6 cells, followed by injection of cytarabine, T cells and various antibodies at various concentrations. The targeted killing effect in each group was compared. The establishment of animal specimens of clinical specimens, repeat the above group, and further verification of dual anti-cytarabine mediated in vivo killing effect. Results: After stimulated by cytarabine, the expression of CD80 in the cells of Nalm-6 was 13.25 ± 3.93,0.062 and 5μmol / L was 20.58 ± 3.94, P = 0.015, and 0.125μmol / L was 38.6 ± 5.37, P = 0.011. 0.25μmol / L was 62.56 ± 6.24, P = 0.007; 0.5μmol / L was 75.04 ± 4.42, P = 0.005, in a concentration-dependent manner. The release of perforin (MFI of 10.61 ± 2.43, P = 0.024) and granzyme B (MFI of 20.60 ± 2.61, P = 0.037) in T lymphocyte of anti-CD3 / anti-CD19 group were significantly higher than those in the other experimental groups. Ara- Peritone (MFI was 23.11 ± 2.90, P = 0.015) and granzyme B (MFI was 31.71 ± 3.08, P = 0.008) were significantly higher in Nalm-6 group than those in C group. IL-3 (100.60 ± 8.32, P = 0.017), IFN-γ (67.60 ± 7.41, P = 0.025) and TNF-α (47.60 ± 3.24, Cell group was significantly increased, and Ara-C stimulation of IL-3 (132.55 ± 5.56), IFN-γ (109.60 ± 6.50) and TNF-α (64.30 ± 8.55) in Nalm- The expression level was higher, P <0.05. In the biological therapy experiment of NOD / SCID mouse xenograft model, the cytotoxicity of xenograft combined with bifunctional antibody group was significantly higher than that of the control group (P <0.05) The expression of CD80 in 2 cases was up-regulated. The in vivo experiments showed that the growth of xenografts in 2 cases was significantly inhibited (P <0.05). CONCLUSIONS: Cytarabine can effectively activate T cells and up-regulate the killer cells in T cells by up-regulating the expression of costimulatory molecule CD80 in clinical specimens of Nalm-6 cells and some B-ALL patients.