γδ T细胞对多发性骨髓瘤RPMI 8226细胞杀伤作用的实验研究

Experimental Study of γδ T Cells Cytotoxic Against Multiple Myeloma Cells

目的探讨体外诱导培养的γδT细胞对多发性骨髓瘤(MM)细胞的杀伤作用。方法采用健康志愿者外周血分离获得外周血单个核细胞(PBMNC),并在1μmol/L唑来膦酸(Zol)和200IU/mL重组人白细胞介素-2(IL-2)条件下扩增获得γδT细胞。采用流式细胞术检测γδT细胞表型,然后与RPMI 8226细胞共培养。采用CCK-8检测γδT细胞对RPMI 8226细胞增殖的抑制作用,乳酸脱氢酶(LDH)法检测γδT细胞对RPMI 8226细胞的杀伤作用,流式细胞术检测RPMI 8226细胞的凋亡率改变,并分别用抗人γδTCR mAb、抗人NKG2D mAb、抗人CD3mAb和同型对照IgG mAb封闭γδT细胞后,作用于RPMI 8226细胞,LDH法检测不同抗体封闭后对RPMI 8226细胞杀伤作用的影响。结果体外条件下可成功高效扩增获得γδT细胞,可抑制RPMI 8226细胞株的增殖并呈一定的量效关系;不同效靶比(1∶1,5∶1和10∶1)的γδT细胞作用于RPMI 8226细胞的杀伤率分别为(12.23±1.75)%,(31.11±6.12)%和(43.56±3.29)%;效靶比为1∶1和10∶1时,γδT细胞诱导RPMI8226细胞凋亡率分别为(38.21±1.98)%和(68.18±2.16)%,明显高于RPMI 8226单独培养组(13.32±1.55)%,差别有统计学意义(P<0.05);抗人γδTCR mAb、抗人NKG2D mAb、抗人CD3mAb和同型对照IgG mAb封闭γδT细胞后按10∶1的效靶比与RPMI 8226混合培养,各组杀伤率分别为(21.54±1.52)%,(29.25±1.51)%,(32.68±0.78)%和(43.0±2.91)%。结论体外利用Zol+IL-2刺激培养可高效扩增γδT细胞,并具有杀伤RPMI 8226细胞的作用,为MM的细胞免疫治疗提供实验依据。

Objective To investigate the cytotoxicity ofγδT cells on human multiple myeloma cells in vitro. Methods Peripheral blood mononuclear cells(PBMNC)were isolated from healthy volunteers and stimulated with 1μmol/L zoledronic acid(Zol)in combination with 200 IU/mL rhIL-2. The phenotype ofγδT cells was detected by flow cytometry and co-cultured with multiple myeloma RPMI 8226 cells directly. Then the inhibitory effect and cytotoxic effect ofγδT cells were detected using CCK-8 and lactate dehydrogenase(LDH)release assay kit. The apoptosis rate of RPMI 8226 cells was detected using flow cytometry. After theγδT cells were treated with Anti-humanγδTCR monoclonal antibody(mAb),Anti-human NKG2 DmAb,Anti-human CD3 mAb and Anti-mouse IgG mAb,LDH release assay kit was repeated to analyze the cytotoxity ofγδT cells. Results γδT cells could be expanded with Zol and IL-2 in vitro and could inhibit the proliferation of RPMI 8226 cell line in the manner of effector target ratio dependence. Co-culturedγδT cells with RPMI 8226 cells at 1∶1,5∶1,and 10∶1 effector target ratios,the corresponding cytotoxic rates were(12.23 ± 1.75)%,(31.11 ± 6.12)%,and(43.56±3.29)%,respectively. At effect/target ratio of 1∶1 and 10∶1,the apoptosis rate of RPMI 8226 cells induced byγδT cells were(38.21±1.98)%and(68.18±2.16)%,respectively. Both were significantly higher than the pure culture of RPMI 8226(13.32±1.55)%(P<0.05). AfterγδT cells were treated with anti-humanγδTCR mAb,Anti-human NKG2 D mAb,Anti-human CD3 mAb,and Antimouse IgG mAb,co-cultured with RPMI 8226 cells at 10∶1 ratio,the corresponding cytotoxic rates were(21.54±1.52)%,(29.25±1.51)%,(32.68±0.78)%,and(43.0±2.91)%,respectively. Conclusion γδT cells can be efficiently expanded with Zol and IL-2 in vitro and have higher cytotoxic activity against RPMI 8226 cells therefore,which can provide experimental evidence for cellular immunotherapy of MM.