Expression Regulation Of Genes Related To Calcium Metabolism By PTHrP Gene In Goat Mammary Gland Epithelial Cell

Parathyroid hormone related protein (PTHrP) has important biological functions incalcium metabolism. It regulated blood calcium and bone calcium dynamic balance underregulation during lactating period, and it has direct effect on dynamic balance of Calcium.Many studies suggested that ΔFosB have a significant research value in human medicinewhich can promote bone formation and increase bone density. And matrixmetalloproteinase-9(MMP-9) could promote degradation of bone matrix, Moreover, MMP-9just like Runx2, Smad4, S100A4and S100A13also involved in the pathway of calciummetabolism and play a great role in it. Therefore it's very important to known therelationships between PTHrP and ΔFosB, PTHrP and MMP-9, PTHrP and the other fourgenes.The aim of this study was to silence the expression of PTHrP by RNA interference andrecombinant adenovirus, and to provide a material to investigate the relative functions ofPTHrP in goat mammary gland epithelial cell (GMEC). The first generation recombinantadenovirus particles (AD-PTHrP-322) were produced and further amplified by transfectingHEK-293cells. The titers of the recombinant adenovirus were determined by TCID50assays.The result of RT-PCR indicated that Ad-PTHrP-572had higher interference efficiency thanthat by infecting GMEC with the recombinant adenovirus, in which PTHrP, ΔFosB, MMP-9,Runx2, Smad4,S100A4and S100A13mRNA expression levels were tested by RT-PCR andWestern blotting. From this experiment, the results that we've got can lay the foundation forfurther clarified the function of PTHrP gene, and conducive to further develop the nutritionalvalue of goat's milk, it also can provide the basis for the study of human metabolic disease.The results were as follows:1Linear products of pAD/PL-DEST/CMV-GFP/U6-322and pAD/PL-DEST/CMV-GFP/U6-NC, digested by Pac I restriction enzyme, was used for transfecting HEK-293cellsby Lipofectamine2000. Then Ad-PTHrP-322and Ad-PTHrP-NC adenoviruses were obtainedby repeated the processing4times. The titer of adenovirus reached2.0×10~9and1.3×10~9 PFU/mL determined by TCID50assays respectively.2We proved the optimum MOI of Ad-PTHrP-322and Ad-PTHrP-NC was200byinfecting GMEC.3The RT-PCR and Western Blotting results indicated that Ad-PTHrP-322has betterinterference efficiency in GMEC,the mRNA expression of PTHrP were decreased16%,38%å'Œ67%respectively.4We infected GMEC by the adenovirus AD-PTHrP-322, which can decrease the PTHrPexpression. We found that ΔFosB gene mRNA expression level can be decrease by37%,56%(P<0.05) after tested by RT-PCR, the protein level was also deregulated by Western Blot. AndMMP-9gene mRNA expression level can be increase by1.1folds,2.0folds (P<0.05).5. The Runx2, Smad4, S100A4and S100A13mRNA expression level can be decrease by50%and62%,42%and60%,31%and42%,40%and52%(P<0.05) respectively.6We infected GMEC by the adenovirus AD-ΔFosB-572for48h and72h, we found thatthe Runx2, Smad4, S100A4and S100A13mRNA expression level can be decrease by47%,30%,28%,32%(P＜0.05) and52%,61%,59%,50%(P＜0.05) respectively.7PTHrP gene could regulate genes' expression of Runx2,Smad4,S100A4and S100A13through ΔFosB.