模拟炎性环境下强直性脊柱炎患者MSC的基因表达
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摘要
【目的】研究炎性环境下强直性脊柱炎(AS)患者间充质干细胞(MSC)中的全基因组表达谱基因的表达情况。【方法】首先从AS患者骨髓腔中分离培养MSC并利用流式技术鉴定细胞特定表型;然后利用TNF-α(10 ng/m L)和IFN-γ(10 ng/m L)刺激模拟炎症环境;接着分别提取不同条件刺激下AS患者和正常人MSC中RNA并进行RNA质控;然后行全基因组基因芯片分析,并应用GO分析法和KEGGPathway分析法分析其中的差异表达基因和寻找相关信号通路;最后利用q RT-PCR方法验证上述基因芯片中筛选出的关键差异表达基因真实性。【结果】全基因组基因芯片分析结果显示炎性模拟环境培养下AS-MSC中差异表达基因数量明显增加,GO分析结果显示这些差异表达基因中,56.1%与生物学活动相关,28.99%与分子功能相关,14.91%与细胞构成相关。Kegg-Pathway分析结果显示这些差异表达基因主要与TLR信号通路和MAPK信号通路这两条通路密切相关,其中TLR信号通路包含8个差异表达基因,MAPK信号通路包含14个差异表达基因。q RT-PCR验证结果显示基因表达与基因芯片结果符合。【结论】炎性模拟环境下,AS-MSC中的差异表达基因数量明显增加,并且这些差异表达基因主要以TLR信号通路和MAPK信号通路为主。
【Objective】Toexplore the gene expression and signaling pathway activation of mesenchymal stem cells(MSC) from ankylosing spondylitis(AS) cultured in mimic inflammatory environment. 【Methods】First, flow cytometry was used to identify thespecific immunophenotype of cells cultured from the bone marrow of AS patients. Then, TNF-α(10 ng / m L) and IFN-γ(10 ng /m L) were added to mimic the inflammatory environment. The total RNA was extracted by the Trizol method and qualified by the RNA electrophoresis. Next, Whole genome expression profilinganalysis was performed and differentially expressed genes related to AS were analyzed by GO term analysis and KEGG pathway analysis. Finally, the above results were verified by q RT-PCR to ensure their authenticity and reliability. 【Results】The whole genome expression profiling analysis showed that under mimic inflammatory environment, the number of differentiated expressed genes in AS-MSC was significantly increased. The Go-term analysis suggested that among these genes, 56.10% participated in biological process, 28.99% played a role in molecular function, and 14.91%associated with cellular component. KEGG pathway analysis showed that he two highest scores of indicators in the AS group were TLR signaling pathway(including 8 differentially expressed genes) and MAPK signaling pathway(including 14 differentially expressed genes). The results of q RT-PCR verified the expression variation of the above genes mentioned. 【Conclusion】Under the mimicinflammatory environment, the number of differentiated expressed genes in AS-MSC is significantly increased and these genes are mainly involved in TLR signaling pathway and MAPK signaling pathway.
【Objective】Toexplore the gene expression and signaling pathway activation of mesenchymal stem cells(MSC) from ankylosing spondylitis(AS) cultured in mimic inflammatory environment. 【Methods】First, flow cytometry was used to identify thespecific immunophenotype of cells cultured from the bone marrow of AS patients. Then, TNF-α(10 ng / m L) and IFN-γ(10 ng /m L) were added to mimic the inflammatory environment. The total RNA was extracted by the Trizol method and qualified by the RNA electrophoresis. Next, Whole genome expression profilinganalysis was performed and differentially expressed genes related to AS were analyzed by GO term analysis and KEGG pathway analysis. Finally, the above results were verified by q RT-PCR to ensure their authenticity and reliability. 【Results】The whole genome expression profiling analysis showed that under mimic inflammatory environment, the number of differentiated expressed genes in AS-MSC was significantly increased. The Go-term analysis suggested that among these genes, 56.10% participated in biological process, 28.99% played a role in molecular function, and 14.91%associated with cellular component. KEGG pathway analysis showed that he two highest scores of indicators in the AS group were TLR signaling pathway(including 8 differentially expressed genes) and MAPK signaling pathway(including 14 differentially expressed genes). The results of q RT-PCR verified the expression variation of the above genes mentioned. 【Conclusion】Under the mimicinflammatory environment, the number of differentiated expressed genes in AS-MSC is significantly increased and these genes are mainly involved in TLR signaling pathway and MAPK signaling pathway.
引文
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[2]DOUGADOS M,BAETEN D.Spondyloarthritis[J].The Lancet,2011,377(9783):2127-2137.
[3]AGGARWAL S,PITTENGER MF.Human mesenchymal stem cells modulate allogeneic immune cell responses[J].Blood,2005,105(4):1815-1822.
[4]WONG RS.Role of stem cells in spondyloarthritis:pathogenesis,treatment and complications[J].Human Immunol,2015,76(10):781-788.
[5]WU Y,REN M,YANG R,et al.Reduced immunomodulation potential of bone marrow-derived mesenchymal stem cells induced CCR4+CCR6+Th/Treg cell subset imbalance in ankylosing spondylitis[J].Arthr Res Ther,2011,13(1):R29.
[6]WANG P,LI Y,HUANG L,et al.Effects and safety of allogenic mesenchymal stem cells intravenous infusion in active ankylosing spondylitis patients who failed nsaids:a 20 week clinical trial[J].Cell Transpl,2014,23(10):1293-303.
[7]BEDAIWI M,SARI I,TBAVANESWARAN A,et al.Fatigue in ankylosing spondylitis and nonradiographic axial spondyloarthritis:analysis from a longitudinal observation cohort[J].J Rheumatol,2015,42(12):2354-2360.
[8]VANDERLINDEN S,VALKENBURG H A,CATS A.Evaluation of diagnostic criteria for ankylosing spondylitis.A proposal for modification of the New York criteria[J].Arthr Rheum,1984,27(4):361-368.
[9]LI Y,WANG P,XIE Z,et al.Whole genome expression profiling and signal pathway screening of MSCin ankylosing spondylitis[J].Stem Cells Int,2014,2014(1):11.
[10]DOMINICI M,LEBLANC K,MUELLER I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells:The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[11]SALIM T,SERSHEN CL,MAY EE.Investigating the role of TNF-alpha and IFN-gamma activation on the dynamics of iN OS gene expression in LPS stimulated macrophages[J].Plo S one,2016,11(6):e0153289.
[12]VAN DER MEIDE PH,SCHELLEKENS H.Cytokines and the immune response[J].Biotherapy(Dordrecht,Netherlands),1996,8(3/4):243-249.