女性外周血来源γδ T淋巴细胞对子宫内膜癌细胞的杀伤效果

Efficacy of γδ T lymphocytes derived from peripheral blood on killing endometrial cancer cells

目的 观察女性外周血来源γδT淋巴细胞体外扩增能力,探讨其对子宫内膜癌细胞的杀伤效果。方法 青年女性3例为青年组,围绝经期女性4例为围绝经期组,采集2组肘静脉血4 mL,采用固相抗体包被法联合白细胞介素-2体外扩增外周血γδT淋巴细胞。扩增14 d,记录γδT淋巴细胞数量、扩增倍数,采用流式细胞术检测γδ及Vδ1、Vδ2 T淋巴细胞比率。取子宫内膜癌HEC-1A、Ishikawa细胞,分别以11、51、101、201和401效靶比加入青年组及围绝经期组γδT淋巴细胞处理24 h,采用CCK-8法检测细胞存活率。取HEC-1A、Ishikawa细胞,分为卡铂组、γδT淋巴细胞组、联合组,卡铂组使用卡铂处理72 h,γδT淋巴细胞组使用20∶1效靶比青年组γδT淋巴细胞处理24 h,联合组使用卡铂处理72 h后加入201效靶比青年组γδT淋巴细胞继续处理24 h,采用CCK-8法检测细胞存活率。结果 (1)扩增14 d,青年组γδT淋巴细胞计数[(5.76±0.43)×10^(6)/mL]多于围绝经期组[(3.19±0.48)×10^(6)/mL](t=3.819,P=0.012),扩增倍数(213.50±15.10)大于围绝经期组(95.87±11.98)(t=5.288,P=0.003),γδT淋巴细胞比率[(92.77±0.69)%]、Vδ2 T淋巴细胞比率[(94.23±0.77)%]均高于围绝经期组[(70.40±3.26)%、(67.43±2.30)%](t=5.743,P=0.002;t=9.589,P=0.002),Vδ1 T淋巴细胞比率[(0.72±0.49)%]与围绝经期组[(1.94±1.32)%]比较差异无统计学意义(t=0.867,P=0.435)。(2)效靶比11、51、101、201、401的青年组和围绝经期γδT淋巴细胞处理24 h, HEC-1A、Ishikawa细胞存活率比较差异均有统计学意义(P<0.05),细胞存活率均随γδT淋巴细胞效靶比升高而降低(P<0.05)。效靶比为101、201、401的青年组γδT淋巴细胞处理24 h时HEC-1A细胞存活率[(73.71±3.07)%、(50.14±3.02)%、(37.36±0.86)%]分别低于围绝经期组γδT淋巴细胞处理24 h[(82.66±1.60)%、(73.01±1.51)%、(47.73±4.31)%](t=4.007,P=0.011;t=10.240,P<0.001;t=4.648,P=0.001)。(3)联合组HEC-1A、Ishikawa细胞存活率[(24.32±5.30)%、(23.13±2.83)%]均低于γδT淋巴细胞组[(50.14±3.02)%、(36.48±1.89)%]、卡铂组[(83.18±2.19)%、(84.14±2.62)%](P<0.05)。结论 青年及围绝经期女性外周血来源γδT淋巴细胞对子宫内膜癌细胞均有杀伤作用,呈剂量依赖性增强,青年女性外周血来源γδT淋巴细胞的杀伤效果优于围绝经期女性外周血来源γδT淋巴细胞,联合卡铂可进一步抑制子宫内膜癌细胞存活。

Objective To observe the amplifying ability of γδT lymphocytes derived from peripheral blood in ex-vivo,and to investigate their efficacy on killing endometrial cancer cells.Methods Three young women and 4 perimenopausal women were collected 4 mL of elbow venous blood,and γδT lymphocytes derived from peripheral blood were amplified with Solid-phase antibody coating method (anti-TCR γδ antibody)and interleukin-2 (IL-2).After 14 days,the total number and amplification fold of the γδ T lymphocytes were counted,and flow cytometry was used to detect the proportion of γδT lymphocytes and its subtype Vδ1 and Vδ2 T lymphocytes.Endometrial cancer cell lines HEC-1 Acells and Ishikawa cells were co-cultured with γδT lymphocytes for 24h at an effector to target ratio(E:T)of 1:1,5:1,10:1,20:1 and 40:1,respectively,and the survival rates were detected with CCK-8 method.HEC-1 Acells and Ishikawa cells were treated with carboplatin for 72h(carboplatin group),with γδT lymphocytes from young women at an E:T of 20:1 for 24h (γδT lymphocytes group),and with γδT lymphocytes from young women at an E:T of 20:1 for 24h after carboplatin treatment for 72h(combination group).CCK-8 method was used to detect the survival rates.Results(1)After 14days of amplification,the number and the amplification fold of γδT lymphocytes were larger in young women[(5.76±0.43)×10^(6)/L,(213.50±15.10)times]than those in perimenopausal women[(3.19±0.48)×10^(6)/L,(95.87±11.98)times](t=3.819,P=0.012;t=5.288,P=0.003).The proportions of γδT lymphocytes and Vδ2 T lymphocytes were higher in young women[(92.77±0.69)%,(94.23±0.77)%]than those in perimenopausal women[(70.40±3.26)%,(67.43±2.30)%](t=5.743,P=0.002;t=9.589,P=0.002),and the proportion of Vδ1 T lymphocytes showed no significant difference between young women[(0.72±0.49)%]and perimenopausal women[(1.94±1.32)%](t=0.867,P=0.435).(2)After γδT lymphocytes treatment at an E:T of 5:1,10:1,20:1 and 40:1 for 24h,respectively,the survival rates of HEC-1 Acells and Ishikawa cells were significant different in young women and perimenopausal women(P<0.05),and decreased with the increase of E:T of γδT lymphocytes(P<0.05).The survival rates of HEC-1 Acells at an E:T of 10:1,20:1 and 40:1 after γδT lymphocytes treatment for 24h were lower in young women[(73.71±3.07)%,(50.14±3.02)%,(37.36±0.86)%]than those in perimenopausal women[(82.66±1.60)%,(73.01±1.51)%,(47.73±4.31)%](t=4.007,P=0.011;t=10.240,P<0.001;t=4.648,P=0.001).(3)The survival rates of HEC-1 Acells and Ishikawa cells were lower in combination group[(24.32±5.30)%,(23.13±2.83)%]than those in γδT lymphocytes group[(50.14±3.02)%,(36.48±1.89)%]and carboplatin group[(83.18±2.19)%,(84.14±2.62)%](P<0.05).Conclusion γδT lymphocytes derived from peripheral blood in young women and perimenopausal women can kill endometrial cancer cells in a dose-dependent manner,which is better in young women than in perimenopausal women,while carboplatin can further inhibit the survival of endometrial cancer cells.