ABSTRACT
Objective To identify the rale of NKX2-5 gene in cardiomyocyte differentiation and its mechanism.Methods P19 cells were divided into transfected and non-transfected groups.In the transfected group,P19 cells were with stable expression of NKX2-5 gene.The P19 cells were cultured in suspension for 4 days,and the formed aggregates were transferred to Petri dish for adherent culture.On days 4,8,12,and 16 of the adherent culture,the expressions of ct-saicomeric actin(α-SA)and cardiac troponin T(cTnT)were detected with double-labeling immunofluorescence and Western blot.The ultrastruetural changes were observed on day 16.Results In the transfected group,no expression of α-SA and cTnT was found on day 4,and the expression of these 2 proteins or co-expression existed on days 8,12,and 16.There were early cell junction and myofilament-like structure in the cytoplasm of some cells in the transfected group.In the non-transfected group,these 2 proteins were negative,and no differentiated cell was found.Conclusion Stable expression of NKX2-5 gene can induce cardiomyocyte differentiation from P19 cells,but the P19 cells with stable expression of JVKX2-5 gene is not suitable to be an in vitro model of cardiac development.
ABSTRACT
The Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and cardiac malformations. Mutations in the TBX5 gene cause HOS and have also been associated with isolated heart and arm defects. Interactions between the TBX5, GATA4 and NKX2.5 proteins have been reported in humans. We screened the TBX5, GATA4, and NKX2.5 genes for mutations, by direct sequencing, in 32 unrelated patients presenting classical (8) or atypical HOS (1), isolated congenital heart defects (16) or isolated upper-limb malformations (7). Pathogenic mutations in the TBX5 gene were found in four HOS patients, including two new mutations (c.374delG; c.678G > T) in typical patients, and the hotspot mutation c.835C > T in two patients, one of them with an atypical HOS phenotype involving lower-limb malformations. Two new mutations in the GATA4 gene were found in association with isolated upper-limb malformations, but their clinical significance remains to be established. A previously described possibly pathogenic mutation in the NKX2.5 gene (c.73C > 7) was detected in a patient with isolated heart malformations and also in his clinically normal father.
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Objective:To investigate mRNA expression of cardiac related genes of NKX2.5,TBX5 and GATA4 in patients with tetralogy of fallot(TOF). Methods:A total of 10 TOF patients(TOF group)from 4 months to 8 years with the mean age of 3.5 years were recruited in our hospital from June to December 2005.The patients were diagnosed by typical clinical manifestation and cardiac color echocardiogram, the diagnosis was confirmed by cardiac surgery.6 non-congenital heart disease children were selected as Control group, and they were from 4 months to 9 years with the mean age of 3.8 years.The myocardial total RNA was extracted,the related cDNA was obtained by RT-PCR.The product cDNA was amplified with fluorescent quantitative PCR in order to compare the differences of NKX2.5,TBX5,GATA4 and GAPDH mRNA expression between TOF group and Control group. Results:NKX2.5 mRNA expression in TOF group was statistically decreased than that in Control group,while there were no statistical changes found in TBX5 and GATA4 mRNA expression between TOF group and Control group. Conclusion:The mRNA expression of NKX2.5,TBX5 and GATA4 were found in myocardium development.TOF was possibly related to decreased NKX2.5 mRNA expression.
ABSTRACT
Objective To study the expression of NKx2.5 on the heart of offspring during the development of embryo,whose mother is deficient of folic acid.Methods 1.Control group involving 18 rats and study group involving 18 rats were chosen from the total 36 adult female SD rats randomly copulate with the male normal rats after feeding different fodder for 2 weeks.The heart of the 13.5 days,17.5 days embryos and the newborns were obtained;2.the expression of NKx2.5mRNA by RT-PCR was observed;3.the expression of NKx2.5 protein by Western-blotting was investigated.Results 1.The expression of NKx2.5 mRNA of study group was weaker than control group in heart of the 13.5 days,17.5 days embryos and the newborns(P