Genetic Characteristics Of Yak PDGF-A,PDGFR-A And Their Relation With Adaptation To Hypoxia In Different Organs

Platelet derived growth factors(PDGFs)and Platelet derived growth factor receptor(PDGFR)are crucial growth factors in mammalian physiological regulation.They are synthesised and screened by diffrent types of cell through paracrine and autocrine systems.Foue(4)gene types of PDGFs(PDGFA,PDGFB,PDGFC and PDGFD)in different chromosome,play relevant biological functions by combining with different specific PDGFR.PDGFA is a kind of important platelet-derived growth factor screening by several kinds of cell.In modulating process of cell proliferation,migration and cell apoptosis,the PDGFA/PDGFR-A signaling pathway are activated by PDGFA combines with cellular surface PDGFR-A,to start their unique physiological or pathological biological effects.In the absent of oxygen,thrombin,and relevant cytokines,PDGFA makes the body adapt the stress and modulate the growth factors.Yak lives in high altitude and low oxygen environment,and some of their physiological structures differ from related mammals in oxygen-rich regions.This thesis aimed to at exploring the regulation mechanism of PDGFA in Yak.Below are parameters that were considered and the findings.(1)Bioinformatics analysis and functional prediction for PDGFA and PDGFR-A.The gene PDGFA and PDGFR-A were cloned by Reverse transcription-polymerase chain reaction(RT-PCR).Characteristics of These two genes characters and their evolutionary relationship between in Yak and other mammals were analyzed by bioinformation method to predict their biological functions and uniqueness.The results showed that the PDGFA sequence could be amplified in Yak by RT-PCR(GenBank NO.: KU851801).Its coding region included 636 bases,encoding 212 amino acid,which was highly conserved sequence in the evolution process and 98.1% homology compared to cattle and bubalus with 7 nucleotides and 4 amino acid changed.The PDGFR-A gene sequence(GenBank NO.: MH152569)included coding region 3270 bases,encoding 1090 amino acid with highly conserved sequence,which 99.6% homology compared cattle with 15 nucleotides and 10 amino acid mutations.According to the gene prediction analysis,the subcellular localizations of PDGFA and PDGFR-A in yak subcellular were different,providing theoretical foundation for revealing the physiologic modulating mechanism of PDGFA and PDGFR-A.(2)Gene expression differential display for PDGFA and PDGFRA by qRT-PCR in yak various tissues and organs.The first proper antigens against PDGFA and PDGFR-A were selected based on analysis of gene characteristic in chapter one.To explore the organic specificity of PDGFA and PDGFR-A,their protein expression and location were evaluated in various organs of yak by Western Blot and immunohistochemistry.The results showed that PDGFA and PDGFR-A expressed were lowest in yak spleen,liver and kidney at both gene and protein levels without significant difference,while highly expressed in heart,lung and testis with significant difference.The PDGFA and PDGFR-A expressed were highest in the testis,than the lung and heart with expression in the heart being the lowest among the 3 organs.The protein PDGFA and PDGFR-A were mainly located in the myocyte,vascular endothelial cell in all kinds of organs,and in sustentacular cells in the testis.These results showed that PDGFA and PDGFR-A were highly expressed in organs that functioned to modulate oxygen concentration and reproduction than other organs,which had similar physical function using VEGF in hypoxia state.(3)The expression of PDGFA and PDGFR-A in normal yak central nervous system at both gene and protein levels.By testing the expression specificity of PDGFA and PDGFR-A in yak brain tissues,their expression were highly observed than in in cerebrum and cerebellum,than in the hippocampus and spinal cord.PDGFA and PDGFR-A were in spongiocyte of cerebrum,cerebellum and hippocampus,pyramidal cells of cerebrum,and Purkinje cells of cerebellum.These variations could be relative with long term living under the hypoxia stress,which could provide information for the functional studies on the PDGFA and PDGFR-A involved in hypoxia regulating and neuroprotection.(4)Mechanism study on sertoli cells in yak testis.The primary sertoli cells of yak testis was cultured as target cell model.To evaluate the effects of HIF-1A in the hypoxia microenvironment,the PDGFA,PDGFR-A and apoptosis related genes and proteins were tested,(by comparing to sertoli cells with antagonist PDGFA and PDGFR-A),to ascertain the effects of PDGFA and PDGFR-A in apoptosis and hypoxia adaptability.The results showed that HIF-A significantly increased in low-oxygen stress.The expression of PDGFA and PDGFR-A were modulated in relation to apoptosis in the low-oxygen environment.The expression of HIF-1A,Bax and Bcl-2 could be regulated by exogenous PDGFA,which reduced cell injury.Our results would be helpful for in furthering studies on PDGFs involved in physiological low-oxygen state and pathological tumor conditions.