为过继性免疫治疗培养淋巴细胞时的细胞因子作用和基因表达特征

Role of Cytokines and Gene Expression Characteristics in Cultured Lymphocytes Ex Vivo for Adoptive Immunotherapy

在过继免疫性细胞培养时需要加入不同的细胞因子。本研究旨在比较不同细胞因子及EBV抗原肽联合细胞因子刺激淋巴细胞后淋巴细胞分化方向和相关的基因表达变化特征。分别用不同细胞因子及EBV抗原肽联合细胞因子刺激淋巴细胞,在培养的当天和第1、3、7、10天,用流式细胞术检测各组细胞中CD3+(总T细胞)、CD3+CD4+(辅助T细胞)、CD3+CD8+(细胞毒性T细胞)、CD3+CD8+CD45RO+(记忆型T细胞)、CD3+CD8+CD45RA+(初始型T细胞)、CD3+CD30+(Th2辅助细胞)、CD19+(B细胞)、CD56+(NK细胞)、CD4+CD25+(初始调节T细胞)、CD4+CD25+FOXP3+(精确调节T细胞)在培养前后总细胞中的百分比变化;用RT-PCR技术检测管家基因mad1、pten和辅助T细胞转录调控基因t-bet(Th1)、gata3(Th2)、细胞因子ifn-γ(Th1)、il-4(Th2)基因表达量。结果表明:EBV多肽组CTL细胞成为优势细胞,临床治疗有确切疗效;比较加入EBV多肽组和不同细胞因子培养组结果显示,EBV抗原肽可以更有效刺激CTL生成,ifn-γ基因表达量明显增加;辅助淋巴细胞分化相关的基因t-bet、gata3都有明显的升高;管家基因mad1、pten基因变化不大;在不同细胞因子培养环境下加入抗原肽可以更有效刺激CTL生成。结论:用EBV多肽联合细胞因子共同培养淋巴细胞可以获得特异性CTL细胞,不同细胞因子对细胞分化发挥不同作用。

Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy.This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stmulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines.The experiment was divided into 4 groups.The levels of total T lymphocytes（CD3＋）,T helper lymphocytes（CD3＋CD4＋）,cytotoxic T-lymphocyte（CD3＋CD8＋）,memory T cells（CD3＋CD8＋CD45RO＋）,naive T cells（CD3＋CD8＋CD45RA＋）,Th2 cells（CD3＋CD30＋）,B cells（CD19＋）,NK cells（CD56＋）,naive T regulatory cells（CD4＋CD25＋）,precise T regulatory cells（CD4＋CD25＋FOXP3＋） were detected by flow cytometry.The expression levels of house-keeping gene（mad1,pten）,T helper cells transcriptional regulatory gene t-bet（Th1）,gata3（Th2）,cytokine IFN-γ（Th1）,IL-4（Th2） were detected by using RT-PCR.The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects.Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation.The expression of IFN-γ gene was significantly increased;the T helper differentiation-related gene t-bet,gata3 also increased evidently,while expression change of house-keeping gene mad1 and pten were not evident.Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation.It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines,and the different cytokines play different roles in cell differentiation.