抑制乳腺癌患者自体CD3AK细胞PD-1的表达对杀伤MCF-7/adr细胞的影响  

Improvement of Cytotoxicity of Autologous CD3AK Cells in Breast Cancer Patients to MCF-7/adr Cells by Suppressing PD-1 Expression

作　　者：刘力伟 刘波 周敏 孙宏浩 潘跃银[4] LIU Liwei LIU Bo ZHOU Min SUN Honghao PAN Yueyin(Bioengineering and Food College of Hubei University of Technology, Key Laboratory of Fermentation Engineering, Ministry of Education, Wuhan 430068, China Department of Biological Treatment Center, 105th Hospital of PLA, Hefei 230031, China Anhui Kedgene Biotechnology Co. Ltd., Hefei 230036, China Department of Oncology, Anhui Provincial Hospital, Hefei 230035, China)

机构地区：[1]湖北工业大学生物工程与食品学院发酵工程教育部重点实验室,武汉430068 [2]解放军第一0五医院生物治疗中心,合肥230031 [3]安徽柯顿生物科技有限公司,合肥230036 [4]安徽省立医院肿瘤科,合肥230035

出　　处：《肿瘤防治研究》2017年第9期580-584,共5页Cancer Research on Prevention and Treatment

摘　　要：目的探讨抑制PD-1表达后的乳腺癌患者自体CD3AK细胞对乳腺癌MCF-7/adr细胞的杀伤作用。方法采用慢病毒介导反转录技术将已构建的PD-1重组质粒转染入CD3AK细胞抑制PD-1的表达,流式细胞法检测PD-L1的表达情况;CD3AK细胞与MCF-7/adr细胞共培养,CCK-8法测定各组的杀伤率,分别由荷瘤BALB/c裸鼠尾静脉注入空白质粒组和PD-1质粒组细胞和0.9%氯化钠溶液,观察各组的抑瘤作用。结果反转录技术可有效抑制CD3AK细胞PD-1的表达(P<0.01);RT-PCR结果显示lentiviral vector/PD-1使CD3AK细胞PD-1基因表达水平降低;CCK-8结果显示培养第14、21天的空白质粒组和PD-1质粒组对MCF-7/adr细胞的杀伤率分别为42.98%和62.68%,47.22%和66.95%(P<0.01);裸鼠体内实验表明,PD-1质粒组CD3AK细胞能够显著抑制肿瘤的生长,其抑瘤率可达74.8%。结论 MCF-7/adr细胞表面高表达PD-L1,抑制PD1的表达可有效提高CD3AK细胞的杀伤能力。Objective To investigate the cytotoxicity of autologous anti-CD3 antibody-induced activated killer cells（CD3AKs） from patients with breast cancer towards MCF-7/adr cells after suppressing PD-1 expression.Methods Lentivirus-mediated reverse transcription technology was applied to transfer PD-1 recombinant plasmid into CD3 AK cells to inhibit PD-1 expression.PD-1 expression on CD3 AK and PD-L1 on MCF-7/adr cells surface were detected by flow cytometry（FCM）.After co-cultivation of CD3 AKs and MCF-7/adr cells,the killing rates of each group towards MCF-7/adr cells were measured by CCK-8 assay.Blank plasmids,PD-1-transfected cells and normal saline（NS） were intravenously injected into BALB/c tumor-bearing nude mice through tail vein.The antitumor effects on tumor-bearing nude mice engrafted with MCF-7/adr cells were observed and evaluated among the three groups through tumor size,weight and tumor inhibition rate.Results Reverse transcription technology exhibited promising effect on inhibiting PD-1 expression in CD3 AKs.On the 14 th and 21 th days of cultivation,PD-1 expression rates on CD3 AKs obtained from breast cancer patients were 44.13% and 60.18%,respectively,however,PD-1 expression rates of PD-1 recombinant plasmid transfection groups were 15.35% and 13.98%（P〈0.01）,respectively.RT-PCR results presented that lentiviral vector/PD-1 decreased PD-1 gene expression in CD3 AKs.CCK-8 assay demonstrated that on the 14 th,21 th days of cultivation,the killing rates of CD3 AKs cultivated with blank plasmids and PD-1 recombinant plasmids in MCF-7/adr cells were 42.98% and 62.68%,47.22% and 66.95%（P〈0.01）,respectively.In vivo experiment of nude mice showed that recombinant PD-1 transfected CD3 AKs exhibited remarkable inhibition on tumor growth.The anti-tumor rate reached 74.8%.Conclusion High PD-L1 expression on MCF-7/adr cell surface is demonstrated and the inhibition of PD-1 expression can improve the cytotoxicity of CD3 AK cells.

关 键 词：PD-1 自体CD3AK细胞 MCF-7/ADR PD-L1 杀伤作用 

分 类 号：R737.9[医药卫生—肿瘤]