CD3/CD28协同刺激逆转HIV-1易感性的细胞机制及m/miRNA表达谱分析和KIR受体的功能研究
文献类型：学位论文
| 作者 | 徐雯雯 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张华堂 ; 毛炳宇 |
| 关键词 | HIV-1 易感性 CD3/CD28协同刺激 杀伤细胞免疫球蛋白样受体 配体 |
| 其他题名 | The Cellular Mechanisms and m/miRNA expression signatures of CD3/CD28 Costimulation Induced Resistance to HIV-1 Infection and The Functions of KIR Receptors |
| 学位专业 | 细胞生物学 |
| 中文摘要 | CD3/CD28协同刺激后的CD4+T细胞，能够逆转对HIV-1的易感性并清除病毒，这一现象发现多年，但其细胞和分子机制至今尚不清楚。本研究利用CD3/CD28磁珠包被的抗体，成功的复建这一独特的逆转模型，并以未刺激和PHA/IL2刺激的细胞为对照，采用mRNA和miRNA芯片技术，联合生物信息学方法对其进行细胞和分子机制的研究。 我们发现协同刺激后的CD4+T细胞高度增殖和活化，呈现均一的高水平表达活化受体CD25、CD69和CD45RO的一个记忆型抗性细胞亚群。HIV-1的主要协同受体CCR5的表达下调，可能是是逆转HIV-1易感性的重要原因。通过mRNA芯片分析发现有7,824个基因在CD3/CD28协同刺激前后差异表达，其中6个具有代表意义的基因谱系包含的1,345个基因能将逆转易感HIV-1和易感HIV-1的两种细胞状态截然分开。通过共表达分析、基因功能分析、信号转导网络分析等，我们发现肌动蛋白细胞骨架、细胞周期监视和蛋白酶体功能在CD3/CD28共刺激后细胞内的上调，可能与逆转HIV-1易感性有关。通过miRNA芯片分析，在CD3/CD28协同刺激后的细胞内，发现了多种表达上调或下调的miRNA，可能与逆转易感性相关。本研究共筛选得到了在CD3/CD28协同刺激前后差异表达的60个靶标基因和17个靶标miRNA，其中大部分尚未报道与HIV-1相关，为进一步的功能验证提供了基础，为HIV-1易感性和抵抗性的研究提供了新的线索。 自然杀伤细胞（NK细胞）作为天然免疫的一个重要组成部分，依靠多个杀伤细胞免疫球蛋白样受体（KIR受体）在肿瘤发生和病毒感染中发挥重要的作用。尽管抑制性的KIR受体能与MHC I类分子相结合，但对激活性的KIR受体的配体却知之甚少。鉴于抑制性和激活性KIR受体结构的相似性，和之前的零星报道，我们推测激活性的KIR受体也能结合MHC I类分子。因此，本研究在体外表达了可溶性的激活性KIR受体KIR2DS1/2/5，并以抑制性的KIR2DL2做对照，来寻找其可能的配体。 我们成功的克隆表达了上述四个KIR受体蛋白，利用其载体上所带的His标签和生物素化标签，进行纯化，分别制成四聚体和磁珠抗体，与5株代表不同HLA-C类型的B细胞系和2例外周血人单个核细胞染色。流式细胞术和磁珠结合实验结果提示，KIR2DS1能够与HLA-C2结合，KIR2DL2能够结合HLA-C1和HLA-C2，而未能发现KIR2DS2和KIR2DS5的MHC I类分子的配体。激活性的KIRSD1/2受体也不结合CMV、EBV、HCV等病毒的蛋白。对KIR2DS1的功能进一步的研究，首次发现糖基化程度和反应温度可直接影响KIR2DS1与HLA-C2的结合。本研究明确了KIR2DS1的主要配体为HLA-C2，并首次证实KIR2DL2的配体包括HLA-C1和HLA-C2，对于进一步研究KIR受体的功能，理解NK细胞和KIR受体在免疫系统中的作用，提供了重要基础。 |
| 英文摘要 | Upon co-stimulation with CD3/CD28 antibodies, activated CD4+T cells were found to lose their susceptibility to HIV-1 infection, exhibiting an induced resistant phenotype. This rather unexpected phenomenon has been repeatedly confirmed but the underlying cell and molecular mechanisms are still unknown. We first replicated the reported system using the specified Dynal beads with PHA/IL-2-stimulated and un-stimulated cells as controls. Genome-wide expression and analysis were then performed by using Agilent whole genome and miRNA microarrays and established bioinformatics tools. We showed that following CD3/CD28 co-stimulation, a homogeneous population emerged with uniform expression of activation markers CD25 and CD69 as well as a memory marker CD45RO at high levels. The downregulated expression of CCR5, the established co-receptor of HIV-1, might contribute to the induced resistance. These cells differentially expressed 7,824 transcripts when compared with the controls on microarrays. Series-Cluster analysis identified 6 distinct expression profiles containing 1,345 genes as the representative signatures in the permissive and resistant cells. Co-expression networks analysis identified 137 “key regulatory” genes holding hub positions in the gene interactions. By mapping these genes on KEGG pathways, the predominance of actin cytoskeleton functions, proteasomes, and cell cycle arrest in induced resistance emerged. Following comparative combined analysis of the mRNA and miRNA array data, 60 marker genes and 17 marker miRNAs were selected as potential regulators of the induced HIV-1, providing new clues on the study of HIV-1 susceptibility. Natural killer (NK) cells play an important role in the control of viral infections, which recognizes virally infected cells through a variety of activating and inhibitory killer-immunoglobulin(Ig)-like receptors (KIRs). Although the ligand identity of inhibitory KIRs is known to comprise MHC class I protein, the ligand specificity activating KIR receptors remains unknown. Here we expressed the soluble extracellular domains of KIR2DS1, KIR2DS2, KIR2DS5 and KIR2DL2 and studied their ligand specificity to HLA-C subtypes. The extracellular domains of the KIR genes were amplified and cloned into a containing a His-tag and a biotin acceptor peptide coding sequence. The KIR expressing constructs were transfected into 293T cells and after 72 hours, supernatants were collected and purified by using nickel chelate affinity columns and size exclusion FPLC. Low and high molecular weight preps comprising differentially glycosylated KIR species were sub-fractioned and tested separately. Biotinylated KIR proteins were tetramerised by the addition of avidin conjugated to the PE fluorchrome, and were used as staining reagents in subsequent flow cytometry based assays. Five B cell lines expressing different HLA-C subtypes were tested to detect the specificity of these KIR-tetramers. FACS results showed that KIR2DS1 tetramers bound cell lines expressing the HLA-C2 subtypes, but not to HLA-C1. This binding could be affected by temperature and glycosylation. KIR2DS2 and KIR2DS5 did not interact with any of the tested B cell lines. Different published data, we found that KIR2DL2 could interact with both HLA-C1 and HLA-C2 subtypes. No binding was found between any above KIRs to CMV or EBV or HCV proteins. In conclusion, we have successfully produced high quality soluble activating KIRs and identified the ligand of KIR2DS1. We also clarified that the ligand of KIR2DL2 includes both HLA-C1 and HLA-C2. Our findings will help to uncover the functions of KIR receptors and NK cells. |
| 语种 | 中文 |
| 公开日期 | 2014-07-02 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7924]  |
| 专题 | 昆明动物研究所_分子免疫生物学 昆明动物研究所_发育生物学 |
| 推荐引用方式 GB/T 7714 | 徐雯雯. CD3/CD28协同刺激逆转HIV-1易感性的细胞机制及m/miRNA表达谱分析和KIR受体的功能研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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