| 摘要 | 树突状细胞（dendritic cells, DC）作为机体天然免疫和获得性免疫反应的桥梁和枢纽，发挥着重要的启动和调控作用。随着体外诱导方法的建立和生物学技术的进步，有关DC 的基础生物学研究得到了快速的发展，在诱导方法、个体发生及基因表达和调控等方面，涌现出很多新的、未解的关键问题。同时，随着对粘膜免疫机理研究的深入，DC 在粘膜生态环境中的功能和影响，渐已成为免疫学研究前沿领域中的热点和要点。在本研究中，为了确定DC 体外分化成熟的最短时程，同时为了研究DC 分化成熟相关的基因表达调控，我们建立了快速的DC 体外诱导方法，分析了体外快速诱导 DC 的mi/mRNA 表达谱。此外，在原始分离的女性生殖道共生乳酸杆菌的基础上，以THP-1作为DC 前体细胞的细胞系模型，开展了女性生殖道共生乳酸杆菌刺激活化 THP-1 的研究，希望能够为乳酸杆菌作为生殖道粘膜免疫疫苗的应用提供理论基础。首先，采用外周血单个核细胞（peripheral blood mononuclear cells, PBMC）来源的CD14+细胞为DC 前体，经过GM-CSF 和IL-4 的刺激，1-6 天后得到未成熟DC （immature dendritic cells, iDC），并经成熟因子（TNF-α, IL-1β, IL-6 与PGE2）诱导 1-2 天后，获得成熟DC（mature dendritic cells, mDC）。经过比较和分析，明确了完全分化和成熟各2 天，即“2+2”，为DC 诱导分化的最佳和最短时程，从而证实和建立了DC 体外快速诱导的体系和方法。该方法获得的iDC 与mDC，具有与传统的“6+2” 方法获得的DC 相同的形态与表型，而且，利用该方法获得的DC 总数高于“1+1”， “1+2”与“6+2”的方法，为DC 的生物学研究提供了基础数据。我们进而采用芯片技术，对体外快速分化成熟的DC 进行了mi/mRNA 表达谱分析，确定了DC 不同分化发育阶段特征性的mi/mRNA 表达差异。结果发现，与CD14+ 单核细胞即DC 前体相比，iDC 与mDC 之间具有更加相近的mi/mRNA 表达方式。 miRNA 表达谱分析则表明，不同的miRNA 表达与DC 的不同分化和发育阶段相关。而且，位于同一基因簇内的miRNA，呈现协同表达的情况。特别值得注意的是，本研究发现了在DC 的某些发育阶段特异表达的miRNA，它们在DC 发育过程中的功能，还未得到诠释，它们在DC 某些分化阶段的特异表达，提示了DC 各分化阶段的相关性与特异性。结合mRNA 表达谱分析，我们发现miRNA 的表达与其目的基因的表达在mRNA 水平呈现负相关的特性。同时，免疫相关mRNA 与miRNA 在DC 体外不同发育阶段的表达亦呈现差异，其中，miRNA（如hsa-miR-181a, hsa-miR-223, hsa-miR-155, hsa-miR-146, hsa-miR-106a 与hsa-miR-20a 等）与mRNA（如ALM1 等）参与了特定的与免疫相关的GO（Gene Ontology）与通路（Pathway），提示这些miRNA 与mRNA 可能通过不同的方式调节控制着DC 的体外诱导过程。在有关粘膜生态环境中DC 的分化、成熟及其功能影响的研究中，我们首先通过各种乳酸杆菌鉴定方法的综合应用，确定了6 种原始分离的女性生殖道主要共生乳酸杆菌：发酵乳酸杆菌（L.Fermentum）、约氏乳酸杆菌（L.Johnsonni）、卷曲乳酸杆菌（L.Crispatus）、革氏乳酸杆菌（L.Gasseri）、詹氏乳酸杆菌（L.Jensenii）与德氏乳酸杆菌（L.Delbrueckii ）。其中，德氏乳酸杆菌（L.Delbrueckii）和发酵乳酸杆菌（L.Fermentum）具有较高的产H2O2 的能力。在此基础上，我们在与THP-1 的共同培养体系中，将乳酸杆菌对DC 前体的作用和影响进行了比较和研究。结果发现，L.Crispatus 在分离的各原始菌株中，具有最强的刺激THP-1 活化的能力，而且，在相同刺激比例下，L.Crispatus 活菌具有比死菌更强的免疫刺激能力，表现为明显上调THP-1 细胞表面标志CD40、CD80、CD86、 CD1a、CCR6 与CD324 的表达水平，同时可诱导活化THP-1 上调表达Th1 型细胞因子。通过FITC-Dextran 吞噬实验，我们发现，经过L.Crispatus 刺激的THP-1 细胞，其吞噬外来抗原的能力明显下降，但尚未检测到经过活化的THP-1 细胞刺激T 细胞增殖的能力。通过流式细胞术分析的方法，我们检测了TLR1、TLR2、TLR4 与TLR6 在不同的刺激分化阶段的表达水平，结果表明，THP-1 主要通过TLR2 与TLR6 识别女性生殖道L.Crispatus。综上所述，本研究首先通过对DC 体外分化成熟的最短时程的分析，确立了快速诱导DC 的最佳方法，进而利用芯片技术，研究了快速诱导DC 的mi/mRNA 表达谱，揭示了DC 体外分化发育过程中可能的调控途径，为进一步研究DC 的基础生物学提供了恰当的模型和具有指向性的线索。同时，通过与DC 前体THP-1 的共同培养体系，证实了生殖道共生乳酸杆菌的免疫调节作用，为以乳酸杆菌为载体的生殖道粘膜免疫疫苗的研究和应用提供了实验依据 |
| 其他摘要 | Dendritic cells (DC) represent a central element in the efficient functioning of both innate and adaptive immunity. With the development of generation strategies of DC and the progression of biotechnology, significant studies have been set towards understanding the DC basic biology. Novel questions have emerged with respect of DC generation, ontogeny and gene expression modulation. The key role of DC in mucosal immunity system has also been recently highlighted and has become a key point in the field of immunology. In the present study, in order to decide the minimal and optional time coruse of DC generation, and to investigate the gene expression characteristics of DC, we established a rapid method for DC generation and characterized the mi/mRNA expression signature of the in vitro generated DC. Different strains of lactobacilli from female vagina were isolated and cocultured with DC precursor THP-1. Stimulation of THP-1 by vaginal lactobacillus was studied in order to investigate the capacity of lactobacilli as mucosal vaccine delivery vector. Monocytes from peripheral blood mononuclear cells (PBMC) were used as DC precursors and subsequently cultured with GM-CSF and IL4 for 1-6 days then maturated for another 1-2 days by addition in the culture of TNFα, IL1β, IL6 and PGE2. After analysis, we validated that the minimal and optional time for DC generation was 2 days for immature DC (iDC) and 2 days for mature DC (mDC). Morphology and phenotype of the DC derived from this method (“2+2”) developed typical DC characteristics as traditional “6+2” DC. Compared with different differentiation methods(“1+1”, “1+2” and “6+2”), monocytes differentiated for 2 days and then matured for 2 days showed highest yield efficiency of DC. After mi/mRNA expression analysis of in vitro generated DC, we found that, mi/mRNA expression signature of iDC was similar to that of mDC. Mentioned to the miRNA expression pattern, the miRNA expression of precursors, iDCs and mDCs displaying a stage dependent manner, and miRNAs localized in the same genomic clusters have coordinated expression features. Notably, we found some specific miRNAs in different DC stages, indicating the important function of microRNAs during the generation of DC in vitro. Combined with the mRNA expression analysis, we found that target genes of immune related miRNAs showed reversed corresponding expression at the mRNA level during the DC differentiation and maturation. Some miRNAs, which play critical roles in immune cells development and function, such as hsa-miR-181a, hsa-miR-223, hsa-miR-155, hsa-miR-146, hsa-miR-106a and hsa-miR-20a, and some mRNAs, such as ALM1, participate some immune related GO (Gene Ontology) and Pathway, indicated that their downregulation or upregulation may also contribute to the different developmental stage of DC. In order to investigate the DC’s differentiation, maturation and function modulation, different identification methods were used firstly to isolate 6 main strains of vaginal lactobacilli: L.Fermentum, L.Johnsonni, L.Crispatus, L.Gasseri, L.Jensenii and L.Delbrueckii, among which L.Delbrueckii and L.Fermentum have the strongest ability to secret H2O2. THP-1 and lactobacilli coculture system was established to detect the different strains’ stimulation ability toward DC precursor. After analysis, we found the L.Crispatus induced the strongest activation of THP-1 and live bacteria was better than dead bacteria when they were used to active THP-1 in the same dose and time course. L.Crispatus strongly upregulated the surface marker CD40, CD80, CD86, CD1a, CCR6 and CD324 of THP-1, and it could also induce THP-1 to secret Th1 cytokines. After FITC-dextran uptake analysis, we found that, after stimulation with L.crispatus, the antigen uptake ability of THP-1 was reduced although the T cells stimulation ability was not influenced. Flow cytometry analysis was used to detect the TLR1, TLR2, TLR4 and TLR6 expression level of THP-1 in different stimulation stage, and the results showed that the recognition of L.Crispatus by THP-1 was TLR2 and TLR6 dependent. In conclusion, we validated the minimal and optional time coruse for in vitro generated DC, and by means of mi/mRNA expression profiling, we also demonstrated for the first time that the modulating pathways of DC differentiation in vitro. Results obtained from this study elucidate the important function of this appreciated model and contribute to directe the basic biology research of DC. Coculture system of vaginal lactobacillus and DC precursor THP-1 was established, through which we demonstrated the immune regulated ability of lactobacilli and illustrated the capacities of lactobacilli as mucosal vaccine delivery vector. |
修改评论