Differentially Expression Of Reference Genes And Candidate Genes In Uterine Tissues Of Polycystic Pyometra Bitches

Cnaine Pyrometra, which is a common clinical disease of bitch.Accumulation diseases of canine uterus are always induced by abnormal endometrial functions, which are related to several growth factors and the number of the receptors. The current study that do about Pyrometra are into the level of molecular biology, the effects of different growth factors on canine endometrium is insufficient. Thus, we investigated the stabilization of reference genes and select suitable reference gene for canine pyometra study. Then, we detected the gene expressions of IGF2(insulin-like growth factor2,IGF2) and IGF1R(insulin-like growth factor1 receptor) by the suitable reference, at the same time, we analyzed the expressions of IGF2 and IGF1 R proteins in uterine tissue.7In this study, we screed the best reference genes from 7 candidate genes(18SrRNA.ACTB, B2 M, GAPDH, HPRT, RPL13 A,YWHAZ). The software programs, GeNorm, Normfinder, Best Keeper and RefFinder and the method of K.H.Sadek were used to evaluate the stability of gene expression in both pyometra and health(control) uterine tissues samples. RT-qPCR were used to inspect the expression of IGF2 and IGF1 R in Pyrometra and health uterine tissues at the gene level. Immunohistochemistry SP were used to inspect the expression of IGF2 and IGF1 R in Pyrometra and health uterine tissues at the cell level. Western blot were used to detect the expression of IGF2 and IGF1 R in Pyrometra and health uterine tissues at the protein level.Our study identified YWHAZ was the best reference gene which could be used as an accurate internal control gene for canine pyometra study by bioinformatics analysis. Between the two groups, the result showed that the expression of IGF1 R was remarkably altered when different reference gene was used. The most unstable reference gene will lead to inaccurate analysis results. The selection was necessary and effective. The positive expression of IGF2 and IGF1 R protein were mainly located in the cytoplasm. The IGF2 protein level of endometria from pyometra group was extremely significant higher than that from control group(P<0.01). The IGF2 mRNA expressions in pyometra group was extremely significant higher than that from control group(P<0.01). The IGF1 R protein level of endometria from pyometra group was extremely significant higher than that from control group(P<0.01). The IGF1 R mRNA expressions in pyometra group was extremely significant higher than that from control group(P<0.01). The correlation analysis of IGF-2 and IGF-1R in canine pyometra showed that the result of immunohistry was low correlation(r=0.423),the result of Western Blotting was high correlation(r= 0.927). The data suggest that increased expression of IGF-2, IGF-1R plays an important role in occurrence of canine pyometra.In conclusion, we selected the most suitable reference gene for canine pyometra and health. We also detected the effection of IGF2 and IGF1 R expression in uterine tissue in pyometra.The results can not only offer certain theoretical basis in molecular research of pyometra but also clarify the correlation of IGF2 and IGF1 R in development process of pyometra. It may be a new research direction in the prevent and clinical therapy to pyometra...