بررسی سازوکار مولکولی فعال‌سازی سامانه ایمنی گاو با استفاده از داده‌های بیان دیجیتالی لوکوسیت‌ها

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی مقطع دکتری، گروه علوم دامی، دانشگده علوم دامی و صنایع غذایی، دانشگاه کشاورزی و منابع طبیعی رامین-خوزستان

2 استاد، گروه علوم دامی، دانشگده علوم دامی و صنایع غذایی، دانشگاه کشاورزی و منابع طبیعی رامین-خوزستان

3 استادیار، گروه علوم دامی، دانشکده کشاورزی و منابع طبیعی، دانشگاه یاسوج

4 استادیار، گروه علوم دامی، دانشگده علوم دامی و صنایع غذایی، دانشگاه کشاورزی و منابع طبیعی رامین-خوزستان

5 استادیار، گروه دام و طیور، پردیس ابوریحان، دانشگاه تهران

چکیده

کاربرد مسیرهای ژنی مؤثر در بروز صفات مختلف در اصلاح نژاد به راحتی امکان‌پذیر نمی‌باشد، اما شناسایی مسیرهای ژنی بدست آمده از برهم کنش داده‌های ترانسکریپتومی تأثیرگذار بر یک فرآیند زیستی، می‌تواند گام مهمی در فهم بهتر سازوکارهای تنظیمی آن فرآیند باشد. در این پژوهش، چگونگی سازوکار مولکولی تقویت پاسخ ایمنی با در نظر گرفتن مسیرهای ژنی فعال شده در لوکوسیت‌ها، مورد بررسی قرار گرفت. برای رسیدن به این هدف، پس از پیاده‌سازی داده‌های حاصل از تکنیک توالی‌یابی رونوشت‌ها (RNA-Seq) با شماره دسترسی GSE37447 از پایگاه داده NCBI، کنترل کیفیت داده‌ها با نرم‌افزار FastQC بررسی گردید. مکان‌یابی ژن‌ها و ایزوفرم‌ها با استفاده از نرم افزار TopHat2 انجام شد و تعیین میزان بیان هر ژن و ایزوفرم با نرم‌افراز HT-Seq انجام گرفت و نهایتاً بررسی ژن‌های متفاوت بیان شده با edgeR انجام گرفت. آنالیز مسیرهای ژنی بر اساس ژن‌هایی که تفاوت بیان داشتند، با پایگاه اطلاعاتی DAVID صورت گرفت. آنالیز مسیرهای ژنی نشان داد، مسیرهای اسپلایسوزم و ترمیم اتصالات نادرست ژنوم در لوکوسیت‌ها فعال می‌شود. بنابراین، مسیرهای ژنی یاد شده می‌توانند چگونگی تقویت پاسخ ایمنی را تفسیر نمایند و به روشن شدن بهتر مسیرهای تنظیمی در این فرآیند بیولوژیکی کمک کنند. به نظر می‌رسد جهت بهبود سامانه ایمنی به کمک برنامه‌های اصلاح نژادی می‌توان از ژن‌ها، فاکتورهای رونویسی، میکرو ریبونوکلئیک اسیدها و یا کوفاکتورهای دخیل در این دو مسیر ژنی (مسیر ژنی اتصال جایگزین و مسیر ترمیم اتصالات نادرست) کمک گرفت، زیرا این دو مسیر ژنی به طور مستقیم در فعال‌سازی و تنظیم فعالیت سامانه ایمنی مؤثر هستند.

کلیدواژه‌ها


Anders, S., Pyl, P.T. and Huber, W. (2014). HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics. btu638.
Andrews, S. (2010). FastQC: A quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc.
Arce, L., Yokoyama, N. and Waterman, M. (2006). Diversity of LEF/TCF action in development and disease. Oncogene. 25(57): 7492-7504.
Atamas, S.P. (1997). Alternative splice variants of cytokines: making a list. Life of Sciences. 61(12): 1105-1112.
Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L. et al. (1999). Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science. 285(5428): 727-729.
Blanco, M., Casasús, I. and Palacio, J. (2009). Effect of age at weaning on the physiological stress response and temperament of two beef cattle breeds. Journal of Animal Science. 3(01): 108-117.
Boehm, U., Klamp, T., Groot, M. and Howard, J. (1997). Cellular responses to interferon-γ. Annual Review of Immunology. 15(1): 749-795.
Bolger, A.M., Lohse, M. and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. btu170.
Brierley, D.J. and Martin, S.A. (2013). Oxidative stress and the DNA mismatch repair pathway. Antioxidants and Redox Signaling. 18(18): 2420-2428.
Burke, N.C., Scaglia, G., Boland, H.T. and Swecker, W.S. (2009). Influence of two-stage weaning with subsequent transport on body weight, plasma lipid peroxidation, plasma selenium, and on leukocyte glutathione peroxidase and glutathione reductase activity in beef calves. Veterinary Immunology and Immunopathology. 127(3): 365-370.
Cole, S.W. (2008). Social regulation of leukocyte homeostasis: The role of glucocorticoid sensitivity. Brain, Behavior and Immunity. 22(7): 1049-1055.
Cooke, M. and Perlmutter, R. (1989). Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. The New Biologist. 7(1): 66-74.
Cosman, D., Müllberg, J., Sutherland, C.L., Chin, W., Armitage, R., Fanslow, W. et al. (2001). ULBPs, novel MHC class I–related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity. 14(2): 123-133.
Davidson, D., Viallet, J. and Veillette, A. (1994). Unique catalytic properties dictate the enhanced function of p59fynT, the hemopoietic cell-specific isoform of the Fyn tyrosine protein kinase, in T cells. Molecular and Cellular Biology. 14(7): 4554-4564.
Dhabhar, F.S. (2009). A hassle a day may keep the pathogens away: the fight-or-flight stress response and the augmentation of immune function. Integrative and Comparative Biology. 49(3): 215-236.
Diefenbach, A., Jamieson, A.M., Liu, S.D., Shastri, N. and Raulet, D.H. (2000). Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nature Immunology. 3(2): 119-126.
Dizdaroglu, M. (2012). Oxidatively induced DNA damage: mechanisms, repair and disease. Cancer Letters. 327(1): 26-47.
Dornan, S., Sebestyen, Z., Gamble, J., Nagy, P., Bodnar, A., Alldridge, L. et al. (2002). Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction. Journal of Biological Chemistry. 277(3): 1912-1918.
Elenkov, I.J. and Chrousos, G.P. (1999). Stress hormones, Th1/Th2 patterns, pro/anti-inflammatory cytokines and susceptibility to disease. Trends in Endocrinology and Metabolism. 10(9): 359-368.
Ergun, A., Doran, G., Costello, J.C., Paik, H.H., Collins, J.J., Mathis, D. et al. (2013). Differential splicing across immune system lineages. In: Proceedings of the National Academy of Sciences, Ayla, Ergun, p. 14324.
Felix, T.M., Hughes, K.A., Stone, E.A., Drnevich, J.M. and Leips, J. (2012). Age-specific variation in immune response in Drosophila melanogaster has a genetic basis. Genetics. 191(3): 989-1002.
Gajewski, T.F. and Fitch, F.W. (1988). Anti-proliferative effect of IFN-gamma in immune regulation. I. IFN-gamma inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. The Journal of Immunology. 140(12): 4245-4252.
Galyean, M., Perino, L. and Duff, G. (1999). Interaction of cattle health/immunity and nutrition. Journal of Animal Science. 77(5): 1120-1134.
Grigoryev, Y.A., Kurian, S.M., Nakorchevskiy, A.A., Burke, J.P., Campbell, D., Head, S.R. et al.(2009). Genome-wide analysis of immune activation in human T and B cells reveals distinct classes of alternatively spliced genes. Plos One. 4(11): e7906.
Halliwell, B. and Gutteridge, J.M. (2015). Free radicals in biology and medicine. Oxford University Press, USA. pp: 670-675.
Henn, V., Slupsky, J.R., Gräfe, M., Anagnostopoulos, I., Förster, R., Müller-Berghaus, G. et al. (1998). CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature. 391(6667): 591-594.
Hermiston, M.L., Xu, Z. and Weiss, A. (2003). CD45: a critical regulator of signaling thresholds in immune cells. Annual Review of Immunology. 21(1): 107-137.
Hickey, M.-C., Drennan, M. and Earley, B. (2003). The effect of abrupt weaning of suckler calves on the plasma concentrations of cortisol, catecholamines, leukocytes, acute-phase proteins and in vitro interferon-gamma production. Journal of Animal Science. 81(11): 2847-2855.
Huang, T.T. and D'Andrea, A.D. (2006). Regulation of DNA repair by ubiquitylation. Nature Reviews Molecular Cell Biology. 7(5): 323-334.
Ishitani, A. and Geraghty, D.E. (1992). Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. In: Proceedings of the National Academy of Sciences, Ishitani, p. 3947.
Karin, M. (2006). Nuclear factor-κB in cancer development and progression. Nature. 44(7): 431-436.
Karin, M. and Ben-Neriah, Y. (2000). Phosphorylation meets ubiquitination: the control of NF-κB activity. Annual Review of Immunology. 18(1): 621-663.
Kim, Y.-J. and M Wilson III, D. (2012). Overview of base excision repair biochemistry. Current Molecular Pharmacology. 5(1): 3-13.
King, P.D., Sandberg, E.T., Selvakumar, A., Fang, P., Beaudet, A.L. and Dupont, B. (1995). Novel isoforms of murine intercellular adhesion molecule-1 generated by alternative RNA splicing. The Journal of Immunology. 154(11): 6080-6093.
Lynch, E.M., McGee, M., Doyle, S. and Earley, B. (2011). Effect of post-weaning management practices on physiological and immunological responses of weaned beef calves. Irish Journal of Agricultural and Food Research. 4: 161-174.
Lynch, K.W. and Weiss, A. (2000). A model system for activation-induced alternative splicing of CD45 pre-mRNA in T cells implicates protein kinase C and Ras. Molecular and cellular biology. 20(1): 70-80.
Lynch, K.W. and Weiss, A. (2001). A CD45 polymorphism associated with multiple sclerosis disrupts an exonic splicing silencer. Journal of Biological Chemistry. 276(26): 24341-24347.
Mabb, A.M., Wuerzberger-Davis, S.M. and Miyamoto, S. (2006). PIASy mediates NEMO sumoylation and NF-κB activation in response to genotoxic stress. Nature Cell Biology. 8(9): 986-993.
Macpherson, P., Barone, F., Maga, G., Mazzei, F., Karran, P. and Bignami, M. (2005). 8-Oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSα. Nucleic Acids Research. 33(16): 5094-5105.
Mallory, M.J., Jackson, J., Weber, B., Chi, A., Heyd, F. and Lynch, K.W. (2011). Signal-and development-dependent alternative splicing of LEF1 in T cells is controlled by CELF2. Molecular and Cellular Biology. 31(11): 2184-2195.
Martinez, N.M., Pan, Q., Cole, B.S., Yarosh, C.A., Babcock, G.A., Heyd, F. et al. (2012). Alternative splicing networks regulated by signaling in human T cells. RNA. 18(5): 1029-1040.
Medzhitov, R. and Janeway, C.A. (1997). Innate immunity: the virtues of a nonclonal system of recognition. Cell. 91(3): 295-298.
Moore, M.J. and Proudfoot, N.J. (2009). Pre-mRNA processing reaches back totranscription and ahead to translation. Cell. 136(4): 688-700.
Moretta, A., Bottino, C., Vitale, M., Pende, D., Cantoni, C., Mingari, M.C. et al. (2001). Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annual Review of Immunology. 19(1): 197-223.
Muller, F.L., Lustgarten, M.S., Jang, Y., Richardson, A. and Van Remmen, H. (2007). Trends in oxidative aging theories. Free Radical Biology and Medicine. 43(4): 477-503.
Nastala, C.L., Edington, H.D., McKinney, T.G., Tahara, H., Nalesnik, M.A., Brunda, M.J. et al.(1994). Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production. The Journal of Immunology. 153(4): 1697-1706.
O’Loughlin, A., Lynn, D.J., McGee, M., Doyle, S., McCabe, M. and Earley, B. (2012). Transcriptomic analysis of the stress response to weaning at housing in bovine leukocytes using RNA-seq technology. BMC Genomics. 13(1): 1.
O’Loughlin, A., McGee, M., Waters, S.M., Doyle, S. and Earley, B. (2011). Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress. BMC Veterinary Research. 7(1): 1.
Radosavljevic, M., Cuillerier, B.t., Wilson, M.J., Clément, O., Wicker, S., Gilfillan, S. et al. (2002). A cluster of ten novel MHC class I related genes on human chromosome 6q24. Genomics. 79(1): 114-123.
Robinson, M.D., McCarthy, D.J. and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 26(1): 139-140.
Rothrock, C., Cannon, B., Hahm, B. and Lynch, K.W. (2003). A conserved signal-responsive sequence mediates activation-induced alternative splicing of CD45. Molecular Cell. 12(5): 1317-1324.
Sancar, A., Lindsey-Boltz, L.A., Ünsal-Kaçmaz, K. and Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry. 73(1): 39-85.
Saul, A.N., Oberyszyn, T.M., Daugherty, C., Kusewitt, D., Jones, S., Jewell, S. et al. (2005). Chronic stress and susceptibility to skin cancer. Journal of the National Cancer Institute. 97(23): 1760-1767.
Sen, R. and Baltimore, D. (1986). Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism. Cell. 47(6): 921-928.
Stamm, S. (2002). Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome. Human Molecular Genetics. 11(20): 2409-2416.
Trapnell, C., Pachter, L. and Salzberg, S.L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 25(9): 1105-1111.
Waterman, M.L., Fischer, W.H. and Jones, K.A. (1991). A thymus-specific member of the HMG protein family regulates the human T cell receptor C alpha enhancer. Genes and Development. 5(4): 656-669.
Wernicki, A., Urban-Chmiel, R., Kankofer, M., Mikucki, P., Puchalski, A. and Tokarzewski, S. (2006). Evaluation of plasma cortisol and TBARS levels in calves after short-term transportation. Revue de Médecine Vétérinaire. 157(1): 30.
Yang, Y., Bazhin, A.V., Werner, J. and Karakhanova, S. (2013). Reactive oxygen species in the immune system. International Reviews of Immunology. 32(3): 249-270.