| The main function of matrix metalloproteinase MMP-9is the degradation of extracellular matrix and the mediation of cells migration. Previous studies have shown that calcium can regulate expression of the MMP-9gene in renal epithelial cells, while the demand for mammary gland in dairy goat production process in the epithelial cells of the huge amount of calcium ion. Thus we speculate the expression in goat mammary epithelial cells in calcium could be regulated by MMP-9gene. The functions of PTHrP gene and Δ fosB gene have been studied clearly in the experiments of our laboratory, which are calcium metabolism related genes.Therefore, in order to explore the regulation of MMP-9gene, PTHrP gene and Δ fosB gene by calcium in the milk goat mammary epithelial cells (GMECs), we first used the calcium to treat the GMECs in vitro. Secondly, by using the recombinant adenovirus targeting dairy goat PTHrP gene Ad-PTHrP and Ad-PTHrP-322and the recombinant adenovirus targeting dairy goat ΔfosB gene Ad-HA-ΔfosB and Ad-ΔfosB-572, which have been prepared successfully in our laboratory, to treat the GMECs, we detected the expression of MMP-9at mRNA level regulated by the PTHrP gene and AfosB gene. Finally, the recombinant adenovirus targeting dairy goat PTHrP gene Ad-PTHrP and Ad-PTHrP-322and the recombinant adenovirus targeting dairy goat AfosB gene Ad-HA-ΔfosB and Ad-ΔfosB-572were used to treat the GMECs, and the intracellular calcium was detected by specific fluorescent probe calcium Rhod-2and laser scanning confocal microscope.The main results of this research are as follows:(1) With the increase of calcium treated the GMECs, cell apoptosis increased gradually, and at the same time, the expressions of MMP-9gene at mRNA level compared with the blank control group were increased by2times,3times,5times,13times and15times; the expressions of PTHrP gene at mRNA level increased by9times,16times,38times,158times and188times; the expression of AfosB gene in mRNA level were increased by3times,11times,38times,80times and95times. At the same time, the protein expressions of the three genes increased by different degrees. (2) At the same time, regardless of when the overexpression of fosB gene or interference expression, the expression level of MMP-9gene mRNA levels were increased to varying degrees, compared with the control group,24h,48h and72h, expression increased by1.327±0.097times,1.522±0.260times,5.115±0.838times,7.689±0.078times,8.937±0.724and12.227±1.543times.(3) PTHrP gene can regulate the intracellular calcium of GEMCs, and the two is positive correlation. AfosB gene can also regulate the Ca+concentration, and the two is the negative correlation.(4) In GMECs the PTHrP gene can alter the intracellular calcium concentration to regulat the expression of MMP-9gene, and the two is the positive correlation between them. And as the upstream gene, ΔfosB gene can also regulate calcium dairy goat mammary gland epithelial cell concentration, but the mechanism is more complex the gene expression of MMP-9, influence of variation is not reasonable conclusion. |
