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采用干扰素-γ、抗CD3单克隆抗体和IL-2体外诱导扩增外周血单个核细胞成为CIK细胞,并于诱导培养前及培养第15 d时分别收集细胞样本。在对培养前后细胞的增殖、形态及表面标志变化检测的同时,提取总蛋白进行定量、双向电泳和银染。利用ImageMasterTM软件对培养前后表达相同和不同的蛋白质点进行分析,并选择其中24个蛋白质点进行质谱鉴定。对于部分培养前后具有代表性的蛋白,进一步采用qPCR技术分析其的转录情况。结果表明,培养前后细胞的蛋白质组学特征是完全不同的,相同表达蛋白点主要与基因的转录因子和细胞骨架相关,诱导后特异表达蛋白主要与细胞生长、增殖相关。虽然在转录与蛋白水平上呈现出部分负相关现象,由于蛋白质组才是基因表达的最终形式,结合蛋白差异研究结果提示,经细胞因子诱导后,CIK细胞的大量扩增与细胞内蛋白表达改变相关。
Interferon-γ, anti-CD3 monoclonal antibody and IL-2 were used to induce peripheral blood mononuclear cells (PBMCs) to become CIK cells in vitro. Cell samples were collected before induction and 15 days after culture. Before and after the culture of cells proliferation, morphology and surface marker changes detected at the same time, the total protein extracted quantitative, two-dimensional electrophoresis and silver staining. ImageMasterTM software was used to analyze the same and different protein spots before and after culture, and 24 protein spots were selected for mass spectrometry identification. For some representative proteins before and after culture, the transcription of qPCR was further analyzed. The results showed that the proteomics characteristics of the cells before and after culture were completely different. The same protein spots were mainly associated with the transcription factors and cytoskeleton of the gene. The induced proteins were mainly related to cell growth and proliferation. Although the transcriptional and protein levels showed a partially negative correlation, as the proteome was the final form of gene expression, the results of the binding protein differences suggested that after induction by cytokines, CIK cells were largely expanded and the intracellular protein expression was changed Related.