为了分析丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinases, MAPK)信号通路基因在肝再生中的表达图谱, 以及探讨MAPK信号通路在肝再生中的作用, 文章利用四氯化碳(Carbon Tetrachloride, CCl4)诱导的小鼠肝损伤再生模型对MAPK信号通路基因的表达进行检测。首先, 采用CCl4腹腔注射的方法建立小鼠肝损伤再生模型, 通过肝脏切片HE染色和测定血清中谷丙转氨酶活性确认模型的质量, 然后, 在注射CCl4后的第0、0.5、1.5、4.5、7 d分别采集小鼠肝脏样本, 应用Affymetrix公司的小鼠基因表达芯片, 检测MAPK信号通路中93个基因的差异表达图谱, 并用荧光实时定量PCR法验证芯片检测的结果。结果表明, 在芯片检测到的93个MAPK信号通路基因中, 有31个在肝再生中有不同程度差异表达, 且经荧光实时定量RT-PCR检测的结果与基因芯片的结果相符合。基因表达谱芯片技术可以筛选出肝再生中差异表达的基因, 在小鼠肝再生中的第0.5和1.5 d, MAPK信号通路中表达水平上调的基因增多, 而在第4.5和7 d, 则表达水平下调的基因明显增多。这一结果表明MAPK信号通路对肝再生不同阶段的双重调控作用。
To investigate the different expression profiles of MAPK pathway genes and their corresponding functions during liver regeneration, we used a CCl4 induced mouse liver regeneration model in this study. Mouse was injected with CCl4 in the abdominal cavity to cause damage in the liver and followed by liver histology examination and measurement of serum ALT levels in blood sample collected at 0, 0.5, 1.5, 4.5, and 7 d after CCl4 injection. Differentially expressed genes in the MAPK pathway during liver regeneration were analyzed using mouse cDNA microarray method (Affymetrix). The results obtained were further subjected to hierarchical clustering study and were validated with real-time PCR. Microarray hybridization identified 31 out of the 93 MAPK pathway component genes, which have significantly altered their expression levels during liver regeneration. Among them, both up- and down-regulated genes were classified into various groups according to clustering studies and functional analysis. At the initial stage of liver regeneration, the number of up-regulated genes was greater than the down-regulated genes, while at the late stage the situation was reversed. Our results suggest that MAPK pathway might play different regulatory roles in responding to different stages of liver regeneration.
[1] Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Bio, 2004, 5(10): 836–847.
[2] Fausto N, Campbell JS, Riehle KJ. Liver regeneration. HepatologY, 2006, 43(2): S45–53.
[3] Michalopoulos GK, DeFrances M. Liver regeneration. Science, 1997, 276(5309): 60–66.
[4] Yuan YS, Wu XY, Ou QX, Gao J, Han W, Tennant, BC, and Yu Y. Differential expression of the genes involved in amino acids and nitrogen metabolisms during liver regeneration of mice. Hepatol Res, 39(3): 301–312.
[5] Ozaki M. Role of Jak/STAT 3 and PI3-K/Akt pathways in liver injury and regeneration. Seikagaku, 2008, 80(5): 399–408.
[6] Diehl AM, Rai R. Review: Regulation of liver regenera-tion by proinflammatory cytoklnes. J Gastroenterol Hepaml, 1996, 11(5): 466–470.
[7] Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver growth by tumor necrosis factor. Proc Natl Acad Sci USA, 1997, 94(4): 144–146.
[8] Wang B, Gao C, Ponder KP. C/EBPbeta contributes to hepatocyte growth factor-induced replication of rodent heaptocytes. Jhepatol, 2005, 43(2): 294–302.
[9] Bruccoleri A, Galhcci R, Germolec DR, Blackshear P. In-duction of early-immediate genes by tumor necrosis factor alpha contribute to liver repair following chemical-induced hepatotoxicity. Hepatolngy, 1997, 25(1): 133–141.
[10] Castro GD, Díaz Gómez MI, Castro JA. DNA bases attack by reactive metabolites produced during carbon tetrachlo-ride biotransformation and promotion of liver microsomal lipid peroxidation. Res Commun Mol Pathol Pharmacol, 1997, 95: 253–258.
[11] Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbontetrachloride as a toxicological model. Crit Rev Toxicol, 2003, 33(2): 105–136.
[12] Fecher LA, Amaravadi RK, Flaherty KT. The MAPK pathway in melanoma. Curr Opin Oncol, 2008, 20(2): 183–189.
[13] Cuschieri J, Maier RV. Mitogen-activated protein kinase (MAPK). Crit Care Med, 2005, 33(12): 417–441.
[14] Punn A, Mockridge JW, Farooqui S, Marber MS, Heads RJ. Sustained activation of p42/p44 mitogen-activated protein kinase during recovery from simulated ischaemia mediates adaptive cytoprotection in cardiomyocytes. Biochem J, 2000, 350(Pt 3): 891–899.
[15] Lee JT Jr, McCubrey JA. The Raf/MEK/ERK signal transduction cascade as a target for chemotherapeutic in-tervention in leukemia. Leukemia, 2002, 16(4): 486–507.
[16] Kyriakis J M, Avruch J. Mammalian mitogen-activated pro-tein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev, 2001, 81(2): 807–869.
[17] Simon JA, Schreiber SI. Grb2 SH3 binding to peptides from Sos: evaluation of a general model for SH3-ligand interactions. Chem Biol, 1995, 2(1): 53–60.
[18] Gao J, Li WX, Feng SQ, Wan DF, Han W, Yu Y. A protein- protein interaction network of transcription factors acting during liver cell proliferation. Genomics, 91(4): 347–355.
[19] Feller SM, Tuchscherer G, Voss J. High affinity molecules disrupting GRB2 protein complexes as a therapeutic strategy for chronicmyelogenous leukaemia. Leuk Lymphoma, 2003, 44(3): 411–427.
[20] Kardinal C, Konkol B, Lin H, Eulitz M, Schmidt EK, Es-trov Z, Talpaz M, Arlinghaus RB, Feller SM. Chronic myelogenous leukemia blast cell proliferation is inhibited by peptides that disrupt Grb2-SOS complexes. Blood, 2001, 98(6): 1773–1781.
[21] Jun N, Kazuya K1, Atsushi H, Jun-ichiro I, Zhaodan C, Kunihiro M. The kinase TAK1 can activate the NIK-IB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature, 1999, 398(6724): 252–256.
[22] Takaesu G, Kishida S, Hiyama A, Yamaguchi K, Shibuya H, Irie K, Ninomiya-Tsuji J, Matsumoto K. TAB2, a novel adaptor protein,mediates activa
