干旱区生态环境知识资源中心(Arid): 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

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	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
其他题名	Determination of antigen-specific CTL induced by the gene vaccine in vitro with MHC pentamer
	李绵洋 1; 朱平 2; 王成彬 1; 王红霞 1; 潘玉玲 1; 丛玉隆 1; 达万明 3
来源期刊	中华检验医学杂志
ISSN	1009-9158
出版年	2010
卷号	33 期号:7 页码:686-690
中文摘要	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人DC, RT-PCR方法检测转染后细胞IgHVl-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CM-CSF含量为(286) ng/10~6个细胞, 而转染对照质粒组的DC中GM-CSF含量为(103) rig/10~6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, CD_3+ CD_8+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(34.242.72)%, (46.063.08)%, (65.34t4.26)%, (73.864.85)%, 而在转染基因疫苗组, 其比例分别为(32.282.08)%, (45.323.81)%, (63.374.21)%, (75.013.20)%. 两因素方差分析显示培养不同时间点的 CD_3CD_8+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五聚体检测结果显示转染基因疫苗组的DC能够诱导针对该表位的抗原特异性CTL产生, CTL水平随着DC的刺激逐渐增高; 在培养第 24天, 健康对照者最高获得2. 03%的CTL, 滤泡性淋巴瘤患者的CTL达4.36%, 而毛细胞白血病患者为3. 89qc. 结论MHC五聚体方法能够有效检测基因疫苗诱导的抗原特异性CTL; 该方法对于抗肿瘤细胞免疫的检测、肿瘤患者的细胞免疫状态及早期预后判定可能是一项有前景的临床检验方法.
英文摘要	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 GM-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 CM-CSF and recombinant human IL-4. Cene 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 arid 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 o d, 7 d, 17 d and 24 d after transfection. The subset of CD_3+CD_8+ 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 EUSA results also confirmed that GM-CSF was produced at a level of (286) ng/10~6 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group (103)ng/10~6 cells (t=5.191, P<0.01). FCM assay result showed that the CD_3+CD_8+ T cells increased in a stepwise pattem during the culture. For control group, the levels at o d, 7 d, 17 d and 24 d were (34.242.72)%, (46.063.08)%, (65.344.26)% and(73.864.85)%, respectively. For vaccine group, the results were (32.282.08) %, (45.323.81)%, (63.37 4.21)% and (75.013.20) %. The differences between each time point had statistical significance (F= 176.966, P < 0. 01), but there was no statistical differences between vaccine group and control group(F= 0.657, P>0.05).The MHC pentamer analysis showed that the DCs loaded with IgHVl-GM-CSF fusional vaccine could efficiently induce the antigen-specific CTL response and the CTL levels increased gradually with the culture time, with the highest level of 4. 360/o in the lymphoma blood and 3. 89% in the hair cell leukemia blood. Conclusions MHC pentamer assay could efficiently determine the antigen-specific CTLs response induced by the gene vaccine of family IgHV frame region in vitro. It could be a useful method for monitoring of anti-tumor cell immunity and evaluating of diagnosis and prognosis of the tumors in clinical application.
中文关键词	疫苗 ; T淋巴细胞 ; 细胞毒性 ; 组织相容性抗原I类
英文关键词	DNA Vaccines DNA T-lymphocytes cytotoxic Histocompatibility antigens class I
语种	中文
国家	中国
收录类别	CSCD
WOS类目	MEDICINE GENERAL INTERNAL
WOS研究方向	General & Internal Medicine
CSCD记录号	CSCD:3916971
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/226092
作者单位	1.解放军总医院临床检验科, 北京 100853, 中国; 2.北京大学第一医院血液研究室; 3.解放军总医院临床血液科, 北京 100853, 中国
推荐引用方式 GB/T 7714	李绵洋,朱平,王成彬,等. 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[J],2010,33(7):686-690.
APA	李绵洋.,朱平.,王成彬.,王红霞.,潘玉玲.,...&达万明.(2010).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.中华检验医学杂志,33(7),686-690.
MLA	李绵洋,et al."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".中华检验医学杂志 33.7(2010):686-690.