Gene expression profiles in dendritic cells from Peyer's patches and epidermal of skin

Many studies have shown that the mucous membranes and skin are at the interface with different external environments and face the disparity of pathogenic effects, such as biological agents, chemical or physical environment. This difference may demand distinct immune responses. However, the mechanism to induce the distinct immune responses in mucous and skin is largely unknown. Dendritic cells of mucosa and skin are crucial in the initiation of immune responses, maintenance of self-tolerance and antigens presentation T cells. The different functions between mucosal and epidermal dendritic cells may play an important role in different immune responses. To compare the different gene expression of the mucosal DC and Langerhans cells (LC), we utilized microarrays to investigate different gene expression profiles in mucosal DC isolated from PPs (PDC) and epidermal LC from skin (ELC). 3548 genes were shown to be differentially expressed between PDC and ELC. According to genes annotations, 105 genes may be involved in immunity process. The genes involved in immune process were categorized to five groups related to DC function, including antigen presentation, antigen uptake, cytokines chemokines, and receptors, cell surface molecules and signal transduction. 11 of the highest expressed genes were selected as the candidate genes and reformed by real-time PCR. These 11 selected genes might be suitable candidates to further study the difference of gene expression between mucosal DC and epidermal LC and would be used for design for new vaccine. Beads and FACS sort were used to isolate dendritic cells from Peyer's patches and epidermal of skin from 80 four-week-old female BALB/c mice. Dendritic cells from Peyer's patches were pooled as one sample, and dendritic cells from epidermal of skin were pooled as another sample. RNA isolation, amplification, cDNA labelling, microarray hybridization and analyses were performed according to the Affymetrix manual book.

多项研究证实，黏膜与皮肤均处于不同外部环境的界面位置，且面临生物制剂、化学或物理环境等不同致病因素的作用差异。此类差异可能促使机体产生差异化的免疫应答。然而，黏膜与皮肤中诱导此类差异化免疫应答的具体机制在很大程度上仍未阐明。黏膜与皮肤中的树突状细胞（dendritic cell, DC）在启动免疫应答、维持自身免疫耐受以及向T细胞呈递抗原的过程中发挥关键作用。黏膜与表皮树突状细胞的功能差异，可能在不同免疫应答的调控中扮演重要角色。为比较黏膜树突状细胞与朗格汉斯细胞（Langerhans cell, LC）的基因表达差异，本研究采用微阵列（microarray）技术，对从佩尔斑（Peyer's patches, PPs）分离的黏膜树突状细胞（PDC）以及皮肤表皮朗格汉斯细胞（ELC）的基因表达谱进行了分析。结果显示，PDC与ELC间共有3548个基因存在差异表达。根据基因注释信息，其中105个基因可能参与免疫相关过程。参与免疫过程的基因可根据DC功能划分为5大类，分别为抗原呈递、抗原摄取、细胞因子与趋化因子、受体及细胞表面分子、信号转导相关基因。本研究选取11个高表达基因作为候选基因，并通过实时荧光定量PCR（real-time PCR）进行验证。这11个筛选得到的候选基因既可作为进一步研究黏膜DC与表皮LC基因表达差异的理想靶点，也可用于新型疫苗的设计开发。本研究从80只4周龄雌性BALB/c小鼠的佩尔斑与皮肤表皮中，通过磁珠分选与荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到树突状细胞；将所有小鼠佩尔斑来源的DC合并为一个样本，皮肤表皮来源的DC则合并为另一个样本。RNA提取、扩增、cDNA标记、微阵列杂交及数据分析均严格按照Affymetrix官方操作手册完成。