Watanabe Ritsuo, Hanawa Haruo, Yoshida Tsuyoshi, Ito Masahiro, Isoda Manabu, Chang He, Toba Ken, Yoshida Kaori, Kojima Mayuko, Otaki Keita, Ding Limin, Hao Kazuhisa, Kato Kiminori, Kodama Makoto, Aizawa Yoshifusa
Division of Cardiology, Department of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Transl Res. 2008 Sep;152(3):119-27. doi: 10.1016/j.trsl.2008.07.006. Epub 2008 Aug 20.
Cardiomyocytes with myocarditis compared with the normal state are thought to change the expressions of various genes greatly, some of which may be new biomarkers or new biologic medicinal products. However, until now, little comprehensive analysis has been made of gene-expression changes in cardiomyocytes with myocarditis. In this study, we performed a DNA microarray analysis by using cardiomyocytes from rat experimental autoimmune myocarditis (EAM). On day 0, rats were immunized with porcine cardiac myosin and cardiomyocytes were isolated and purified from EAM hearts and normal hearts by a method that is hardly thought to change gene expressions in cardiomyocytes. RNA from normal cardiomyocytes and cardiomyocytes of EAM on day 18 was analyzed for 7711 gene expressions by DNA microarray. Some gene expressions showed over 10-fold changes. In particular, the regenerated gene (Reg)2/pancreatitis-associated protein (PAP)1 messenger RNA (mRNA) level most markedly increased in the genes, which were clearly expressed in cardiomyocytes rather than in noncardiomyocytes, and it was approximately 2000-fold greater in cardiomyocytes under active myocarditis than normal by real-time reverse transcription polymerase chain reaction analysis. Moreover, we demonstrated that Reg2/PAP1 proteins determined by Western blot analysis and immunohistochemistry and other Reg/PAP family gene expressions were remarkably increased in EAM hearts; in addition, interleukin (IL)-6 expression was significantly related to Reg2/PAP1. It seemed that these data were useful as a reference database of gene-expression changes in cardiomyocytes with myocarditis. The Reg/PAP family, which was found to show dramatically increasing gene expressions by DNA microarray analysis, was suspected to play an important role in myocarditis.
与正常状态相比，患有心肌炎的心肌细胞被认为会极大地改变各种基因的表达，其中一些基因可能是新的生物标志物或新的生物药物。然而，到目前为止，对于患有心肌炎的心肌细胞中的基因表达变化，几乎没有进行过全面的分析。在本研究中，我们使用来自大鼠实验性自身免疫性心肌炎（EAM）的心肌细胞进行了DNA微阵列分析。在第0天，用猪心肌肌凝蛋白免疫大鼠，并通过一种几乎不会改变心肌细胞中基因表达的方法，从EAM心脏和正常心脏中分离并纯化心肌细胞。通过DNA微阵列分析了正常心肌细胞和第18天EAM心肌细胞的RNA中的7711个基因表达。一些基因表达显示出超过10倍的变化。特别是，再生基因（Reg）2/胰腺炎相关蛋白（PAP）1信使核糖核酸（mRNA）水平在基因中增加最为明显，这些基因在心肌细胞中而非非心肌细胞中清晰表达，并且通过实时逆转录聚合酶链反应分析，在活动性心肌炎下的心肌细胞中比正常情况大约高2000倍。此外，我们通过蛋白质印迹分析和免疫组织化学确定的Reg2/PAP1蛋白以及其他Reg/PAP家族基因表达在EAM心脏中显著增加；此外，白细胞介素（IL）-6表达与Reg2/PAP1显著相关。这些数据似乎可作为患有心肌炎的心肌细胞中基因表达变化的参考数据库。通过DNA微阵列分析发现Reg/PAP家族基因表达显著增加，怀疑其在心肌炎中起重要作用。
