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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 investigate the expression of programmed cell death-1 (PD-1) in peripheral blood derived from CIK in cord blood and breast cancer patients and its killing effect on breast cancer MCF-7 cells. Methods: We collected 5 cases of peripheral venous blood from healthy puerpera and breast cancer patients in the No. 105 Hospital of People’s Liberation Army from June 2015 to December 2015. PBMCs were isolated, and CIK cells were cultured and expanded in vitro. Flow cytometry was used to detect the expression of PD-1 in two different sources of CIK cells at different time points. CIK cells from the 7th, 14th, 21st, and 28th days of culture were co-cultured with MCF-7 cells, and cell counts (CCK-8) were measured. The killing rate of CIK cells against MCF-7 cells was determined by the method. The apoptotic changes of CIK cells were observed after co-culture with acridine orange-ethidium bromide staining (AOEB) method. The apoptosis rate of CIK cells was detected by flow cytometry. RESULTS: With the prolongation of culture time in vitro, the expression rate of PD-1 in venous blood-derived CIK cells was gradually increased in umbilical cord blood and breast cancer patients, and the PD-1 expression rate of CIK cells in umbilical cord blood group was lower than that in breast cancer group on the 14th day [ (38.42±4.76)% vs (50.54±3.50)%, P<0.05]. After the 21st day, the expression rates of PD-1 in both groups increased, but the difference was not statistically significant (P>0.05). The killing rates of MCF-7 cells by the two groups of CIK cells on the 7th, 14th, 21st, and 28th days were (18.54±3.54)% and (21.74±4.27)%, (71.86±16.86)%, and (58.78±24.25), respectively. %, (44.32±26.87)%, and (43.96±26.04)%, (43.24±24.27)%, and (40.28±23.69)% of the CIK cells derived from umbilical cord blood on the 14th day were the strongest (P< 0.05). The analysis found that the expression levels of PD-1 in two sources of CIK cells were positively correlated with the apoptosis rate of CIK cells (r=0.971, r=0.900, all P<0.01), and negatively correlated with the killing rate (r=-0.865, r=-0.885, all P<0.01). CONCLUSION: PD-1 is highly expressed on the surface of activated CIK cells, and umbilical cord blood is lower than the PD-1 level on the surface of CIK cells derived from venous blood of breast cancer patients; the apoptosis rate of CIK cells on the 14th day of culture is lower, and its killing ability is even higher. Strong.