环状RNA100783及其靶分子网络在人巨细胞病毒慢性潜伏再激活诱导T细胞免疫衰老的调控机制研究

The persistent herpesviruses human CMV resulted to the extremely high prevalence almost to 100% in the elderly. Acting as a potential chronic antigenic stressor, the chronic molecular regulation between hCMV latency-and-reactivation and the accumulation of CD8+ nonproliferative, apoptosis-resistant memory cells that have lost the expression of the costimulatory molecule CD28, the key part of the global phenomenon termed immune senescence remained unexplored largely due to time-dependent chronicity and complexity. In our previous study, we have found circular RNA 100783, or ciR783 (circRNA, which is highly expressed as a novel type of competing endogenous RNA to miRNA) might participate on hCMV induced T cell immunosenescence. To further explore the role of ciR783 in the chronic hCMV- host immune interaction, an in vitro platform of co-culture TPA-stimulated hCMV-carried K3 and PBMC to mimic the interaction between hCMV latency-and-reactivation and CD8+CD28- T cell progression. The CD8+CD28- T cells were sorted at all time point independently from the chronic platform. ciR783 and its core targeting miRNAs and mRNA/genes were estimated by bioinformatics analysis and detected by corresponding mixed microarray. After ciR783-miRNAs-mRANs interactive molecular lines which has been involved into molecular network has been acquired at different time point, the time-dependent data have been followed. Simultaneously, the time-dependent hCMV quantitation and CD8+CD28- T cells ratio expression were integrated to propose a network of interactive ciR783-miRNAs-mRANs regulating the immunity aging progression. Using overexpress and knocked- down technique, the expression of ciR783 in the CD8+CD28- T cells were altered by which functional targeting genes of have been identified. In conclusion ,by the step-by-step detection and optimization, the network regulation by ciR783-targeted genes have been shown in the hCMV-reactivated T cell immunosenescence.

人巨细胞病毒的老年人携带率极高，其慢性潜伏再激活诱导外周血免疫衰老T细胞CD8+CD28-逐年上调的分子调控机制也一直是免疫衰老研究的核心瓶颈。本研究组发现环状RNA100783（简称ciR783，是一种新确定的小RNA内源竞争分子）可能参与该调控。我们创建了一个上游病毒反复诱导下游T细胞免疫衰老的体外经时共培养体系；体系上分选经时免疫衰老T细胞，用ciR783靶分子网络芯片获取各时间点ciR783表达相关性网络（ciR783-miRNA-gene分子链）；将多时间点分子链数据分析优化获得经时表达相关性ciR783靶分子网络；结合上游病毒和下游T细胞衰老的经时定量数据推测出调控机制相关性ciR783靶基因谱；在衰老细胞上过表达/敲除ciR783，对ciR783靶基因谱进行功能验证，明确在hCMV慢性潜伏再激活过程中，哪些ciR783依赖性靶基因参与诱导了T细胞免疫衰老的慢性分子网络调控机制

人类预期寿命不断增加，全球老龄化是目前全球公共卫生中最重要的问题。衰老的生理、病理和分子生物学机制非常复杂，免疫衰老（Immunosenescence）是老年人群高发病率和高死亡率的重要促进机制，持续低水平炎性细胞因子的表达，诱导了老年人群常见的慢性疾病，例如动脉粥样硬化，代谢综合征，骨关节炎等，免疫衰老的机制非常复杂，其中潜伏在骨髓干细胞的人巨细胞病毒（CMV）慢性潜伏再激活诱导的T细胞免疫衰老，是其中重要机制之一。为了探索潜伏在骨髓干细胞CMV是如何通过一个分化偶联的痕量反复激活诱导T细胞免疫衰老的机制，我们通过比对外周血CMV特异性T细胞克隆的比对,发现了circRNA100783可能参与CMV慢性潜伏再激活诱导免疫衰老。本研究首先体外构建的免疫衰老的研究平台，通过CMV感染的干细胞共培养T细胞细胞，构建免疫衰老的实验模型，验证circRNA100783如何调控免疫衰老。我们初步构建了体外CMV潜伏单次诱导T细胞免疫衰老的模型，反复诱导的T细胞模型稳定性较低，细胞凋亡率高，获取的circ100783相关调控网络数据，重复性差。为了解决这个问题，我们转向在体研究，探索免疫衰老的分子调控机制。一个是通过高敏感度单细胞液滴式PCR/DDPCR进行人骨髓干细胞分化依赖的CMV检测，另一个是通过非人灵长类动物模型大量获取活性骨髓干细胞和分化亚群，进行单细胞测序获取CMV潜伏相关单细胞数据，两个在体研究均取得了进展，且结果互相印证，发表SCI文章一篇，待发一篇。此外，我们从CMV潜伏再激活诱导免疫激活的下游CMV特异性T细胞表型进行逆向比对，结合高通量检测和数据分析，找到另一个非编码靶分子NRON参与免疫衰老的证据，以及NRON可能参与circ100783的调控网络，发表SCI文章一篇。本研究结果从多个角度对非编码RNA参与CMV慢性潜伏再激活诱导T细胞免疫衰老的分子调控机制，进行了探索，并且构建了单细胞研究平台，为从人-微生物共生角度延缓衰老，奠定了坚实的基础。