【作者】 王辉；

【导师】 王顺启；

【作者基本信息】 南昌大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 黑灵芝在中国被用做传统医药和功能食品的历史已达几千年。多糖被认为是黑灵芝发挥其功能的主要成分之一。本研究以黑灵芝多糖组分PSG-1为原料,通过体外诱导树突状细胞(Dendritic cells,DCs),研究黑灵芝多糖PSG-1对DCs的免疫调节作用,并深入探讨其免疫调节活性作用机制。主要结论总结如下:(1)PSG-1可以直接调节DCs的免疫功能。PSG-1促进DCs分泌大量细胞因子(TNF-α、IL-12 p70、IL-10和IL-6)和趋化因子(RANTES和MIP-1α),增强DCs表面MHC II分子和共刺激分子CD80/CD86表达水平,同时提高DCs促进T细胞增殖的能力,且MAPK通路是PSG-1调节DCs免疫功能的一条关键通路。(2)基于RNA-Seq技术,获得PSG-1刺激DCs的转录本序列信息。处理组与对照组样本的测序总量分别达6350423700 bp、6749478634 bp、6922562700 bp、6501046800 bp、6608047500 bp和7174258200 bp。其中,对照组Clean Data分别为6174227174 bp、6552993304 bp和6687389756 bp,多糖组Clean Data分别为6260597776 bp、6272268250 bp和6830301050 bp。测序数据质控分析和表达量质量分析结果表明,RNA-seq的测序结果质量良好。(3)以Ensembl的小鼠基因组数据库为参考,测序结果匹配至基因组比率达98.37%。两组样本共比对到2231个差异表达基因,其中上调的有1014个,下调的有1217个。经KEGG pathway分析,PSG-1刺激DCs差异基因涉及的信号通路达240个。在最显著的20个信号通路中,涉及的免疫相关通路不仅包括MAPK通路,也包括PI3K/Akt通路和JAK-STAT通路等。此外,涉及的细胞因子受体相互作用、ECM受体相互作用、补体、粘着和粘附分子等通路,表明PSG-1改变了DCs分泌功能、粘附功能和迁移能力。(4)挑出部分RNA-Seq数据中涉及NF-κB与PI3K/Akt信号通路的差异表达基因做进一步验证,RNA-Seq结果和RT-q PCR结果一致。NF-κB以及PI3K/Akt通路抑制剂可显著降低PSG-1诱导DCs分泌的细胞因子(TNF-α和IL-6)和表面分子(MHC II分子、CD80和CD86),表明NF-κB与PI3K/Akt通路也是PSG-1作用的关键通路,且RNA-Seq结果可靠、可重现。(5)PSG-1可以在肠道上皮细胞阻隔的情况下,间接调节DCs免疫。体外建立Caco-2-DCs共培养模型,发现高剂量PSG-1可以间接的促进DCs表面MHC II分子、CD80和CD86的表达,增强DCs TNF-α和IL-6的表达,且不会影响Caco-2单层膜的通透性。更多还原

【Abstract】 The history of Ganoderma atrum being used in traditional medicine and functional food has been for thousands of years in China.Polysaccharides were found to be the main active ingredient of Ganoderma atrum.PSG-1 was a polysaccharide component extracted from Ganoderma atrum.The present study was designed to investigate the immunomodulatary effects and mechanisms of PSG-1 on dendritic cells(DCs)induced in vitro.The main research results obtained in this dissertation were concluded as follows:1.PSG-1 has direct immunomodulatory effects on DCs.PSG-1 increased cell surface molecule expression of MHC-II,CD80 and CD86,and enhanced the production of TNF-α,IL-12 p70,IL-6,IL-10,RANTES and MIP-1a in DCs.PSG-1-treated DCs promoted the proliferation of splenic T lymphocyte of mouse in mixed lymphocyte reaction.MAPK pathway is a key pathway for PSG-1 to regulate the immune function of DCs.2.Based on RNA-Seq technology,transcriptional sequence information of PSG-1 stimulated DCs was obtained.The total quantity of sequencing data of treatment group and control group were 6350423700 bp,6749478634 bp,6922562700 bp,6501046800 bp,6608047500 bp and 7174258200 bp respectively.The quantity of clean data in control group was 6174227174 bp,6552993304 bp and 6687389756 bp respectively.The quantity of clean data in PSG-1 group was 6260597776 bp,6272268250 bp and 6830301050 bp respectively.The quality control analysis of the sequencing data and the quality analysis of the expression quantity showed that the results of RNA-seq sequencing were of good quality.3.Genome database of Ensembl was used as a reference,and the percentage of sequencing results matching to mouse genome was up to 98.37%.There were 2231 differentially expressed genes between the two groups,in which 1014 genes were Up-regulated and 1217 genes were down-regulated.In KEGG pathway analysis results,240 signal pathways were involved in PSG-1 stimulated DCs.In the most significantly changed 20 signaling pathways,the immune related pathways not only involved the MAPK pathway,but also inclued PI3K/Akt pathway and JAK-STAT pathway.In addition,transformations in cytokine receptor interaction,ECM receptor interaction,complement,adhesion and adhesion molecules and other pathways indicated that PSG-1 changed secretory function,adhesion function and migration ability of DCs.4.In the RNA-Seq data,a part of differentially expressed genes which involved in the NF-κB and PI3K/Akt signaling pathway were selected for further verification,and the RNA-Seq results were consistent with the RT-q PCR results.Secretions of cytokines(TNF-α and IL-6)and surface molecules(MHC II molecules,CD80 and CD86)of DCs via PSG-1 inducing were rapidly reduced by NF-κB and PI3K/Akt pathway inhibitors,which showed that the results of RNA-Seq were reliable and reproducible.5.PSG-1 has indirect immunomodulatory effects on DCs in the case of intestinal epithelial cell barrier.Based on a Caco-2-DCs co-culture model,it was found that expression of MHC II molecules,CD80 and CD86 on DCs and production of TNF-α and IL-6 of DCs were indirectly promoted by high dose of PSG-1.But PSG-1 would not affect the permeability of Caco-2 monolayer.更多还原