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目的利用Cx43基因剔除(KO)小鼠模型,观察TGFβ2在心脏流出道隔的表达,探讨Cx43基因剔除小鼠心脏流出道隔心肌化异常与TGFβ2的关系;采用胚心脏体外培养技术,研究外源性TGFβ2是否促进流出道隔心肌化过程。方法选用胚E12.5~E15.5 d的Cx43基因剔除纯合型(Cx43-/-)、杂合型(Cx43+/-)及野生型(Cx43+/+)C57/BL6小鼠作为研究对象,采用PCR方法鉴定基因型,免疫组织化学法测定TGFβ2;选用野生型E12.5进行胚心脏体外培养至E15.5,同时加用TGFβ2不同剂量干预处理,免疫组织化学法测定肌节肌动蛋白-αSCA的表达。结果Cx43 KO小鼠胚期心脏近端流出道隔心肌细胞明显减少,尤其以E13.5和E14.5的Cx43-/-小鼠为著。与Cx43+/+小鼠对照,Cx43-/-小鼠心脏近端流出道隔TGFβ2的表达明显降低,以E14.5较为显著;E14.5的Cx43+/-小鼠表达也有降低。然而TGFβ2不同剂量干预组均没有见到明显的促进离体心脏心肌化的作用。结论Cx43 KO小鼠心脏近端流出道隔存在明显的心肌化延迟。TGFβ2表达减少可能参与了Cx43-/-小鼠心肌化异常的发病机制。体外应用TGFβ2可能难以模拟在体的时空表达模式,或者心肌化的进行需要精确的TGFβ2的剂量和严格的培养条件。
Objective To detect the expression of TGFβ2 in the cardiac outflow tract by using the Cx43 knockout (KO) mouse model and to explore the relationship between TGFβ2 and myocardial abnormalities in cardiac outflow tract of Cx43 knockout mice. Using embryo heart in vitro culture technique, Whether or not TGFβ2 promotes efflux septal muscularization. Methods The Cx43 gene knockout homozygous (Cx43 - / -), heterozygous (Cx43 +/-) and wild type (Cx43 + / +) C57 / BL6 mice from embryo E12.5 to E15.5 d were selected as research objects, Genotypes were identified by PCR and TGFβ2 was detected by immunohistochemical method. The embryos were cultured in vitro to E15.5 with wild-type E12.5 and treated with different doses of TGFβ2. Immunohistochemistry was used to determine the expression of sarcomeric actin- αSCA expression. Results The Cx43 KO mice showed a marked decrease of cardiac myocytes in the proximal cardiac outflow tract, especially in Cx43 - / - mice of E13.5 and E14.5. Compared with Cx43 + / + mice, the expression of TGFβ2 in the proximal cardiac outflow tract of Cx43 - / - mice was significantly decreased to E14.5, while the expression of Cx43 +/- mice in E14.5 was also decreased. However TGFβ2 intervention group did not see any significant promotion of cardiac myocytes in vitro. Conclusion The cardiac outflow tract septum in Cx43 KO mice has obvious delayed myocardialization. The reduced TGFβ2 expression may be involved in the pathogenesis of myocardial abnormalities in Cx43 - / - mice. In vitro application of TGF [beta] 2 may be difficult to mimic the spatial and temporal patterns of in vivo expression, or the progression of cardiacization requires precise dosage of TGF [beta] 2 and stringent culture conditions.