脐带血和乳腺癌患者外周血来源CIK细胞PD-1表达及对MCF-7细胞杀伤

Expression of PD-1 in CIK cells derived from umbilical cord blood or peripheral blood from breast cancer patients and the cytotoxicity of CIKs on MCF-7 cells

目的:探讨脐带血和乳腺癌患者外周静脉血来源的CIK细胞程序性死亡分子-l(programmed cell death-1,PD-1)的表达及其对乳腺癌MCF-7细胞的杀伤作用。方法:采集2015年6月至2015年12月在解放军第105医院住院的健康产妇脐带血和乳腺癌患者外周静脉血各5例,分离PBMC,体外培养、扩增CIK细胞。流式细胞术检测不同时间节点两种来源的CIK细胞PD-1表达情况,分别取培养第7、14、21、28天的CIK细胞与MCF-7细胞共培养,细胞计数(CCK-8)法测定CIK细胞对MCF-7细胞的杀伤率,吖啶橙-溴化乙啶双染(AOEB)法观察共培养后CIK细胞凋亡变化,流式细胞术检测CIK细胞凋亡率。结果:随着体外培养时间的延长,脐带血和乳腺癌患者静脉血来源的CIK细胞PD-1表达率均逐渐上升,第14天时脐带血组CIK细胞PD-1表达率低于乳腺癌组[(38.42±4.76)%vs(50.54±3.50)%,P<0.05],至第21天后两组PD-1表达率均升高,但差异无统计学意义(P>0.05)。培养第7、14、21、28天两组CIK细胞对MCF-7细胞的杀伤率分别为(18.54±3.54)%和(21.74±4.27)%、(71.86±16.86)%和(58.78±24.25)%、(44.32±26.87)%和(43.96±26.04)%、(43.24±24.27)%和(40.28±23.69)%,以培养第14天的脐带血来源的CIK细胞的杀伤活性最强(P<0.05)。分析发现,两种来源的CIK细胞PD-1表达水平与CIK细胞的凋亡率呈正相关(r=0.971,r=0.900,均P<0.01),与杀伤率呈负相关(r=-0.865,r=-0.885,均P<0.01)。结论:活化的CIK细胞表面高表达PD-1,脐带血较乳腺癌患者静脉血来源的CIK细胞表面PD-1水平低;培养第14天的CIK细胞凋亡率均较低,其杀伤能力更强。

Objective：To detect the expressions of programmed cell death-1 （PD-1） on cytokine-induced killer （CIK） cells derived from umbilical cord blood or peripheral blood from breast cancer patients, as well as to investigate the cytotoxicity of CIK cells on MCF-7 cells. Methods： Umbilical cord blood from healthy pregnant women （n=5） and venous blood from breast cancer patients （n=5） were collected during June 2015 to December 2015 at the 105th Hospital of PLA. The PBMC was isolated, and CIK cells were differentiated and amplified in vitro. The PD-1 expressions on CIK cells derived from two origins at different time points were detected by FCM; CIK cells at 7th, 14th , 21st and 28th days were used for the co-culture with MCF-7 cells, and the cytotoxicity of CIK cells on MCF-7 cells was determined by CCK-8 assay; CIK cell apoptosis after co-culture was observed by AOEB, and the apoptosis rate of CIK cells was determined by FCM. Results：Along with the extending of incubation time, the expressions of PD-1 on CIK cells of both groups increased gradually; PD-1 expression in CIK cells devived from umbilical cord blood at 14th day was lower than that devived from peripheral blood of breast cancer patients （／[38.42±4.76／]% vs ／[50.54±3.50／]%,P〉0.05）, however, the expressions increased in both groups at 21st day and the difference between two groups was not statistically significant （P〉005）. Cytotoxicity rates of the CIK cells on the MCF-7 cell in the two groups at 7th ,14th ,21st and 28th days after co-culture were （18.54±3.54）% and （21.74±4.27）%,（71.86±16.86）% and（58.78±24.25）%,（44.32±2687）% and （43.96±26.04）% as well as （43.24±24.27）% and （40.28±23.69）% respectively, and among them the cytotoxicity of the CIK cells from umbilical cord blood at 14th day of the culturing was the highest （P〈0.05）. According to the analysis, there was a positive correlation between PD-1 expression and CIK cell apoptosis（r=0.971,r=0.900, all P〈0.01）, and a negative correlation between PD-1 expression and CIK cytotoxicity rate （r=-0.865,r=-0.885, all P〈0.01）. Conclusion：The activated CIK cells had high PD-1 expression,and CIK cells from patients with breast cancer had higher PD-1 expression than CIK cells from umbilical cord blood. The apoptosis rates of CIK cells at 14th day of culture were in both groups lower, and possessed higher cytotoxicities.