肌钙蛋白T迷你基因的构建及其心衰模型表达谱系研究

Construction of cardiac troponin T mini-gene and investigation of its expression level in heart failure model

目的：研究肌钙蛋白T（cardiac troponin T, cTnT）基因外显子5（exon 5, E5）的可变剪接及其在真核细胞和小鼠心力衰竭（心衰）模型的表达情况。方法：通过分段PCR构建包含cTnT外显子2-6及相应的内含子的基因片段，连接至真核表达载体中，转染真核细胞HEK293FT，观察其在细胞的表达和在体心肌中的表达是否有异同；建立小鼠心衰模型，用免疫组织化学法和PCR研究cTnT E5的表达情况。结果：cTnT迷你基因在HEK293FT细胞内表达出3个剪接异构体，分别是1个cTnT E5＋和2个cTnT E5-；小鼠心脏内主要表达3个剪接异构体，组成情况和迷你基因表达产物相似；小鼠心衰模型组的cTnT E5＋比例增高。结论：成功构建cTnT迷你基因，可用于进一步研究cTnT E5的可变剪接表达调控；小鼠心衰模型显示cTnT E5的表达和心肌功能有一定联系。

Objective： To study alternative splicing of cTnT exon 5（E5） in mammalian cells and murine heart failure model.Methods： By segmental PCR strategy, we constructed cTnT mini-gene which consists of its exon 2-6 and the interspersed introns. Then this mini-gene was transfected into HEK293FT cells and then dectected the exon 5 inclusion expression by PCR. Results： We found the cTnT mini-gene expressed three isoforms with one cTnT E5 inclusion band and two cTnT E5 exclusion bands. This was consistent with the expression pattern in murine heart. Moreover, the cTnT E5 inclusion was increased in murine heart failure model compared with that of control group. Conclusion： We successfully constructed a cTnT mini-gene, which is useful for our further study. In addition, dysregulation of cTnT E5 splicing may be responsible for the normal cardiomycyte function.