Functional phenotypes alteration on dendritic cells between tolerance and immnunity

Dendritic cells (DC) are key regulators of immunological responses, immunity and tolerance. Due to their differentiation capacities, they have emerged as a promising tool for therapeutic applications. However, there are poorly understood functional DC-specific markers and cellular mechanisms for in-vitro research and clinical usage. This study’s aim is to evaluate the gene expression profiling of up-regulated or down-regulated by DC maturation and explore their function. The dendritic cells were prepared using GM-CSF and IL-4 (imDC) and subsequently maturated by adding type II collagen as antigen with LPS (mDC) or TNF-alpha(tDC). Also, surface markers, cytokine profiles, and induction of Treg cell were examined in same DC. Then, transcriptional profile was analyzed by using cDNA microarray and gene clustering programs. The selected genes, marker candidates were validated by using quantitative real-time PCR. By supervised selection, we identified 75 signitures , which 63 genes were consistently upregulated in mDC and 12 genes were expressed only in tDC . In addition, we introduced 10 genes overexpressed or equally expressed in both tDC and mDC. These selected genes were clustered into various functional categories according to GO ontology analysis. Hspa1 and Scin as tDC-specific genes, Orm1, Pdlim4, and Enpp2 as mDC-specific genes were found correlation between microarray data and mRNA expression by qRT-PCR. This work may contribute to identifying specific modulating markers for mDC and tDC, which could in the future serve as therapeutic targets in the treatment of cancer and autoimmune diseases. Total RNA obtained from isolate Bone-marrow derived Dendritic cell subset, immature DC, semi-mature or tolerogenic DC, and fully mature DC were generated in-vitro, used microarray Chip : Illumina MouseRef-8 v2 Expression BeadChip

树突状细胞（Dendritic cells, DC）是免疫应答、免疫防御与免疫耐受的关键调控因子。鉴于其分化潜能，树突状细胞已成为极具前景的治疗应用工具。然而，目前适用于体外研究与临床应用的DC特异性功能标志物及细胞机制仍缺乏充分认知。
本研究旨在分析受DC成熟过程调控的上调与下调基因的表达谱，并探索其功能机制。
本研究采用粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）与白细胞介素-4（interleukin-4, IL-4）诱导制备未成熟树突状细胞（immature dendritic cells, imDC）；随后分别以脂多糖（lipopolysaccharide, LPS）联合II型胶原作为抗原，以及肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）诱导其成熟，分别得到成熟树突状细胞（mature dendritic cells, mDC）与耐受型树突状细胞（tolerogenic dendritic cells, tDC）。同时，本研究对上述三类DC的表面标志物、细胞因子谱以及调节性T细胞（regulatory T cell, Treg）的诱导能力进行了检测。随后，通过cDNA微阵列与基因聚类分析软件对转录组表达谱进行分析，并采用实时荧光定量聚合酶链反应（quantitative real-time PCR, qRT-PCR）对筛选得到的候选标志物基因进行验证。
通过监督筛选策略，本研究共鉴定得到75个特征基因：其中63个基因在mDC中持续上调，另有12个基因仅在tDC中特异性表达。此外，本研究还筛选得到10个在tDC与mDC中均高表达或表达水平无显著差异的基因。
经基因本体（Gene Ontology, GO）注释分析，上述筛选得到的基因可被划分为多个功能类别。经qRT-PCR验证，tDC特异性基因Hspa1与Scin，以及mDC特异性基因Orm1、Pdlim4和Enpp2的mRNA表达水平与微阵列检测结果具有显著相关性。
本研究可为mDC与tDC的特异性调控标志物筛选提供参考，上述标志物未来有望成为癌症与自身免疫性疾病治疗的潜在靶点。本研究从体外培养的骨髓来源树突状细胞亚群（包括未成熟DC、半成熟/耐受型DC与成熟DC）中提取总RNA，采用Illumina MouseRef-8 v2表达微珠芯片进行转录组微阵列检测。