|
Previous research has shown that PTHrP gene is related to the deposition of milk calcium during lactation, and△FosB, S100A4, S100A13, Smad4, Runx2and Stat5a genes are related to calcium metabolism and expressed in the goat mammary gland. In order to study if PTHrP gene plays any role in metabolism of calcium in goat mammary gland and its relationship with calcium metabolism related genes, we constructed the recombinant adenovirus of PTHrP gene Ad-PTHrP and evaluated its expression level by Real-Time quantitative PCR and western blot from mRNA and protein level. The results indicates that mRNA expression level increased104times,230times and520times than negative control after the goat mammary epithelial cells (GMECs) were infected by Ad-PTHrP24h,48h and72h, respectively, also the protein expression level improved.We infected over-expression recombinant adenovirus Ad-PTHrP and interference recombinant adenovirus carrying anti-goat PTHrP shRNA AD-PTHrP-322(stored by our labratory) with the goat mammary epithelial cells to explore the regulation of PTHrP on some genes related to calcium metabolism (△FosB, S100A4, S100A13, Runx2, Smad4and Stat5a) by Real-Time quantitative PCR. the results indicated that with the increase of PTHrP expression,△FosB, Smad4, Runx2gene mRNA expression levels decreased inordinately, meanwhile, S100A4and S100A13genes mRNA expression level increased; With the reduction of PTHrP expression, the expression level of△FosB, Smad4and Runx2genes mRNA expression level increased, and S100A4, S100A13genes mRNA expression level decreased; In other words, PTHrP genes may regulate the AFosB, Smad4and Runx2genes mRNA expression positively, and regulate S100A4and S100A13gene negatively, but there is no regulation function to stat5a gene. In conclusion, the results showed that PTHrP can effect the mRNA expression of△FosB, S100A4, S100A13, Runx2, Smad4genes, but not the Stat5a gene. It indicates that PTHrP could participate in molecular mechanism of metabolism of calcium in goat mammary epithelial cells.
|