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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。
Addition of different cytokines is required for adoptive immunity cell culture. The purpose of this study was to compare the lymphocyte differentiation and related gene expression changes after different cytokines and EBV antigen peptides combined with cytokines stimulate lymphocytes. Lymphocytes were stimulated with different cytokines and EBV antigen peptides in combination with cytokines. On the day of culture and days 1, 3, 7 and 10, flow cytometry was used to detect the expression of CD3 + (total T cells), CD3 + CD4 + T helper cells CD3 + CD8 + cytotoxic T cells CD3 + CD8 + CD45RO + memory T cells CD3 + CD8 + CD45RA + naive T cells CD3 + CD30 + CD19 + (B cells), CD56 + (NK cells), CD4 + CD25 + (naïve regulatory T cells), CD4 + CD25 + FOXP3 + (accurately regulate T cells) in total cells before and after culture; The expression of gene transcription factors t-bet (Th1), gata3 (Th2), cytokines ifn-γ (Th1) and il-4 (Th2) The results showed that EBV peptide group CTL cells become the dominant cells, the exact effect of clinical treatment; compared with the EBV polypeptide group and different cytokines cultured group results show that EBV antigen peptide can more effectively stimulate CTL generation, ifn-γ gene expression was significantly increased ; The gene t-bet and gata3 related to auxiliary lymphocyte differentiation were significantly increased; the housekeeping gene mad1 and pten genes did not change much; adding antigen peptides under different cytokine culture environment could stimulate CTL generation more effectively. CONCLUSION: Specific CTL cells can be obtained by co-culturing lymphocytes with EBV polypeptide and cytokines. Different cytokines play different roles in cell differentiation.