MHC五聚体方法检测基因疫苗体外诱导的抗原特异性细胞毒T淋巴细胞  

Determination of antigen-specific CTL induced by the gene vaccine in vitro with MHC pentamer

作　　者：李绵洋[1] 朱平[3] 王成彬[1] 王红霞[1] 潘玉玲[1] 丛玉隆[1] 达万明[2] 

机构地区：[1]解放军总医院临床检验科,北京100853 [2]解放军总医院血液科,北京100853 [3]北京大学第一医院血液研究室

出　　处：《中华检验医学杂志》2010年第7期686-690,共5页Chinese Journal of Laboratory Medicine

基　　金：科技部国际科技合作重大项目资助课题（2006DFB31430）

摘　　要：目的 探讨MHC五聚体方法检测在体外用抗B细胞淋巴瘤基因疫苗（简称基因疫苗）刺激PBMC生成抗原特异性CTL的效率.方法 用于细胞培养的4份血液标本来自2例B淋巴细胞肿瘤患者（1例为滤泡性淋巴瘤患者,另1例为毛细胞白血病患者）和2名健康对照者.分别检测上述标本中PBMC受基因疫苗刺激后CTL生成情况.PBMC贴壁培养获得DC前体,在重组细胞因子GM-CSF和IL4的支持下诱导培养DC,采用基因枪方法将基因疫苗质粒导入DC,RT-PCB方法检测转染后细胞IgHV1-GM-CSF mRNA的转录,ELISA方法检测细胞因子GM-CSF的表达,并验证转染是否获得有效表达.转染后的DC再与从PBMC中获得的淋巴细胞共培养,诱导抗原特异性的CTL;共进行2次DC刺激,共计培养24 d,分别在培养前、培养第7天、第17天及第24天收获培养细胞,培养分为转染基因疫苗组和转染对照质粒组,流式细胞术分析培养细胞中CD3＋ CD8＋细胞亚群的水平;用针对基因疫苗抗原肽抗原表位的MHC五聚体荧光抗体检测CTL产生水平.结果 基因枪颗粒轰击方法转染DC后,RT-PCR结果显示转染得到了表达;转染基因疫苗组的DC中GM-CSF含量为（28±6）ng/106个细胞,而转染对照质粒组的DC中GM-CSF含量为（10±3）ng/106个细胞,差异有统计学意义（t=5.191,P〈0.01）.分析培养后的T淋巴细胞亚群,显示随着培养时间的延长,CD3＋CD8＋细胞亚群比例明显增加,转染对照质粒组在培养前、培养第7天、第17天和第24天的比例分别为（34.24±2.72）%,（46.06±3.08）%,（65.34±4.26）%,（73.86±4.85）%,而在转染基因疫苗组,其比例分别为（32.28±2.08）%,（45.32±3.81）%,（63.37±4.21）%,（75.01±3.20）%.两因素方差分析显示培养不同时间点的CD3＋CD8＋细胞亚群比例差异有统计学意义（F培养时间=176.966,P〈0.01）,但转染因素本身对其产生的影响差异无统计学意义（F转染因素=0.657,P〉0.05）.MHC五聚体检测结�Objective To determine the antigen-specific CTL in PBMC induced by a fusional family-gene vaccine of the immunoglobulin heavy chain variable gene framework region combined with the sequence of cytokine CM-CSF in vitro with MHC pentamer. Methods Peripheral blood samples were collected from two healthy donors and two patients. One was follicular lymphoma and another was hair cell leukemia. PBMC were isolated by density gradient centrifugalization with Ficoll and then subsequently differentiated into immature DCs （imDCs） induced by recombinant human GM-CSF and recombinant human IL-4. Gene gun was used to deliver the plasmids of the gene vaccine or the control plasmids into the imDCs. RT-PCR and ELISA assay were used to detect IgHVl-GM-CSF mRNA and GM-CSF in order to validate the transfection of the vaccine. After adding the cytokine cocktail, the imDCs became mature DCs. Then the mature DCs were co-cultured with lymphocytes from the blood samples for the induction of the antigen-specific CTL. The cultured cells were classified into vaccine group and control group and harvested at different time points of 0 d,7d, 17 d and 24 d after transfection. The subset of CD3＋CD8＋ T cells was analyzed by FCM assay. Finally, the CTL levels were detected with fluorescently labeled MHC pentamer antibody targeting vaccine epitopes. Results With the induction of cytokines, the imDCs with typical morphology were generated in PBMC. After delivering, the efficient expressions of the vaccine in the imDCs were determined by RT-PCR. And ELISA results also confirmed that GM-CSF was produced at a level of （28 ±6） ng/106 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group （10 ± 3） ng/106 cells （t = 5. 191, P 〈0.01）. FCM assay result showed that the CD3＋ CD8＋ T cells increased in a stepwise pattern during the culture. For control group, the levels at 0 d,7d, 17d and 24 d were （ 34. 24 ± 2. 72 ）% , （46.06 ± 3.08）%, （ 65. 34 ± 4. 26 ）% and （73.86 ±

关 键 词：疫苗 DNA T淋巴细胞 细胞毒性 组织相容性抗原I类 

分 类 号：R392[医药卫生—免疫学]