特异性细胞毒性T淋巴细胞体外培养体系的建立与鉴定及对卵巢癌细胞的影响  

Establishment and identification of specific cytotoxic T lymphocyte in vitro culture system and the effect on ovarian cancer cells

作　　者：司丽慧[1] 刘剑[1] 许志光 许天敏[1] SI Li-Hui;LIU Jian;XU Zhi-Guang;et al.(Department of Gynecology and Obstetrics, the Second Hospital of Jilin University, Changchun, Jilin 130000, China)

机构地区：[1]吉林大学第二医院妇产科,吉林长春130000

出　　处：《中国妇幼保健》2018年第11期2575-2578,共4页Maternal and Child Health Care of China

摘　　要：目的探讨特异性细胞毒性T淋巴细胞(CTL)体外培养体系的建立与鉴定及对卵巢癌细胞的影响,为开发一种高效、安全的抑制杀伤卵巢癌细胞的免疫疗法提供依据。方法提取人外周血单个核细胞(PBMC),加入Cp G模体的寡脱氧核苷酸序列(Cp GODN),人参皂苷Rg1药物浓度分别为3.75μg/ml、7.5μg/ml、15μg/ml、30μg/ml、60μg/ml与不加药物的对照组比较,采用单一变量控制法,验证Cp GODN,人参皂苷Rg1和人类表皮生长因子受体α(HER2/neu)抗原肽的最佳比例。用流式细胞仪检测Cp GODN,人参皂苷Rg1和HER2/neu混合物刺激的PBMC表面标记物CD3、CD4和CD8,并检测细胞核型。用人参皂苷Rg1和HER2/neu混合物刺激的PBMC形成的CTL作用到SKOV-3细胞中,培养一段时间观察CTL的抑制作用。结果随着Cp GODN/人参皂苷Rg1药物浓度的增加,CTL作用显著增强,差异有统计学意义(P<0.05),当药物浓度达到30μg/ml时,抑制效果进入平台,药物浓度再增加,抑制效果不变。因此,以Cp GODN和人参皂苷Rg1有效浓度30μg/ml与HER2/neu抗原肽片段10μg/ml混合,加入到提取的PBMC中,培养34 d,建立CSR-CTL体系。流式细胞仪检测,随着培养时间增加CD3、CD4和CD8阳性细胞数量显著增多,差异有统计学意义(P<0.05)。CSR-CTL对卵巢癌细胞SKOV-3生长有显著的抑制作用,观察组与对照组比较差异有统计学意义(P<0.05)。结论采用Cp GODN,人参皂苷Rg1和HER2/neu抗原肽混合物刺激PBMC可建立安全、特异性高的细胞毒性T淋巴细胞体外培养体系CSR-CTL,并且CTL可抑制人卵巢癌SKOV-3的生长。Objective To explore the establishment and identification of specific cytotoxic T lymphocyte（ CTL） in vitro culture system and the effect on ovarian cancer cells,provide a basis for developing an effective and safe immunization therapy for inhibiting and killing ovarian cancer cells. Methods The mononuclear cells in human peripheral blood were extracted and the concentrations of Cp GODN and ginsenoside Rg1 were 3. 75 μg/ml,7. 5 μg/ml,15 μg/ml,30 μg/ml,and 60 μg/ml,respectively,the results were compared with control group. The optimal ratio of Cp GODN,ginsenoside Rg1 and HER2/neu antigen peptide was verified by single variable control method. The peripheral blood mononuclear cell surface markers CD3,CD4,and CD8 were stimulated by Cp GODN,ginsenoside Rg1,and HER2/neu mixture,then the results were detected by flow cytometry,the karyotype was detected. CTL formed by peripheral blood mononuclear cells stimulated with ginsenoside Rg1 and HER2/neu mixture was used in SKOV-3 cells to observe the inhibitory effect of CTL for a period of time. Results With the increase of Cp GODN/ginsenoside Rg1 concentration,the effect of CTL increased significantly（ P〈0. 05）. When drug concentration reached 30 μg/ml,the inhibitory effect entered the platform,the inhibititory effect maintained stable when the drug concentration increased. Therefore,Cp GODN and ginsenoside Rg1（ effective concentration： 30 μg/ml） and HER2/neu antigen peptide fragment（ 10 μg/ml） were mixed and added into the extraction of PBMC,then the mixture was cultured for 34 days to establish CSR-CTL system. Flow cytometry showed that the numbers of CD3,CD4 and CD8 positive cells increased significantly with the increase of culture time（ P〈0. 05）. CSR-CTL significantly inhibited the growth of ovarian cancer cell line SKOV-3,and there was statistically significant difference between observation group and control group（ P〈0. 05）. Conclusion Safe and specific cytotoxic T cell culture system CSR-CTL can be established by stimulation o

关 键 词：细胞毒性T淋巴细胞 免疫治疗 佐剂 

分 类 号：R737.31[医药卫生—肿瘤]