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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (1) : 61-66     DOI: 10.1007/s11684-009-0007-8
RESEARCH ARTCILE |
Influence of retinoic acid on TBX1 expression in myocardial cells induced by Shh and Fgf8
Miao LIU, Xiaoyan WU, Jiawei XU, Runming JIN()
Department of Paediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
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Abstract  

The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3×10-7 mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 (P<0.05) and 1.10 times (P<0.05), respectively. In comparison with the control group, under the concentration of 5×10-7 mol/L RA, they were up-regulated by 2.21 (P<0.05) and 2.38 times (P<0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times (P<0.05) and 80% (P<0.05) separately compared with the control group after stimulation of 3×10-7 mol/L RA, and they were up-regulated by 3.48 (P<0.05) and 2.04 times (P<0.05) individually after stimulation of 5×10-7 mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.

Keywords retinoic acid      Tbx1 protein      Shh protein      Fgf8 protein     
Corresponding Authors: JIN Runming,Email:jinrunm@public.wh.hb.cn   
Issue Date: 05 March 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0007-8     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I1/61
geneprimer sequencetemperaturecycleslength/bp
all-actinS: GTG GGG CGC CCC AGG CAC CA55°C35548
A: CTC CTT AAT GTC ACG CAC GAT TTC
ShhS: CCA AAG CTC ACA TCC ACT GTT C55°C35500
A: CCG GTT GAT GAG AAT GGT G
Fgf8S: GCC TCC AAG CCC AGG TAA CT55°C35333
A: TGA AGA CGC AGT CCT TGC CTT
Tab.1  The primer sequence and amplification conditions of genes
Fig.1  The cardiomyocytes in culture (× 200)
Fig.2  Purification of cardiomyocytes detected by FACS. M1 represents negative cells; M2 represents positive cells.
Fig.3  Expression of Shh and Fgf8 mRNA in cultured cardiomyocytes by RT-PCR. The relative level of Shh and Fgf8 were determined by quantification of RT-PCR bands and were normalized against the level of all-actin. M: marker; 1: Shh mRNA in cultured cells; 2: Shh mRNA in cultured cells added with 0.1% DMSO; 3: Shh mRNA in cultured cells added with 0.1% DMSO and 1 × 10mol/L RA; 4: Shh mRNA in cultured cells added with 0.1% DMSO and 3 × 10 mol/L RA; 5: Shh mRNA in cultured cells added with 0.1% DMSO and 5×10 mol/L RA; 6: Fgf8 mRNA in cultured cells; 7: Fgf8 mRNA in cultured cells added with 0.1% DMSO; 8: Fgf8 mRNA in cultured cells added with 0.1% DMSO and 1 × 10 mol/L RA; 9: Fgf8 mRNA in cultured cells added with 0.1% DMSO and 3 × 10 mol/L RA; 10: Fgf8 mRNA in cultured cells added with 0.1%DMSO and 5×10 mol/L RA. RA: retinoic acid; DMSO: dimethyl sulphoxide.
Fig.4  Expression of Shh and Fgf8 protein in cardiomyocytes by Western blot. The relative level of Shh and Fgf8 were determined by quantification of Western blot bands and were normalized against the level of GAPDH. M: marker; 1: cultured cells; 2: cultured cells added with 0.1% DMSO; 3: cultured cells added with 0.1% DMSO and 1 × 10 mol/L RA; 4: cultured cells added with 0.1% DMSO and 3 × 10 mol/L RA; 5: cultured cells added with 0.1% DMSO and 5 × 10 mol/L RA. RA: retinoic acid; DMSO: dimethyl sulphoxide.
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