Chemerin And GPR1 Gene Clone And Tissue Expression And The Role Of Chemerin In Granulosa Cells

Chemerin(chemotactic factor)belong to the members of the family of chemokines,studies have shown that Chemerin directly or indirectly involved in inflammation,and regulation of fat metabolism process,and through the transcriptome sequencing GPR1 found that the gene and its receptor expressed in chicken eggs period ovarian volume is higher,speculated that Chemerin genes may by combining its receptors are involved in regulating the function of the ovarian follicles.Therefore,this test first obtained the CDS of chemerin gene region sequence,lengthof the gene and its receptor GPR1 quantitative testing their distribution in chicken tissues and expression.Then,using different concentration of chemerin follicular granulosa cells in vitro chicken protein processing,and through the quantitative detection of apoptosis,fatty acid metabolism and steroid synthesis of key gene expression changes.The results showed that:(1)Nest PCR of chemerin CDS zones amplification,and analyze mRNA expression.Results show that using forecast sequence's nest PCR amplification,found that after three rounds of PCR amplification were the band now,did not find the transcription of the two prediction;Quantitative analysis found that Roman laying.hens organizations,chemerin genes are expressed in various tissues have distribution,expression quantity is the most abundant in the liver,the amount of fat,also have higher expression in lung,and is also expressed in the hypothalamus,pituitary,ovary;Quantitative detection ranks before follicle and follicle mRNA expression,chemerin gene with the continuous development of follicular,the expression of general present a rising trend.The expression of the above results indicate that chemerin measuring tool has tissue specificity,and associated with the development of follicles,speculated that chemerin may participate in the regulation of follicular and help laying hens lay eggs.(2)Chemerin must be combined with its receptor to function,through's nest PCR amplification GPR1 gene sequence length area,and analysis of mRNA expression.Results show that through the RACE-PCR technology identified the chicken three new GPR1 gene splicing subtypes,the three subtypes have different CDS region and 3 'UTR,GPR1-vb relative to GPR1 val and GPR1 va2 did less 426 nucleotide sequences,protein spatial structure prediction found these three subtypes have different spatial structure.Quantitative analysis found GPR1-va and GPR1-vb is widely expressed in various organizations and has the very high tissue specificity,expressed in fat tissue volume and the highest;(3)In the different levels of follicle for three subtypes of GPR1 gene expression rule of exploration,found that with the continuous growth and development of follicles,amount of mRNA expression of the three subtypes also grow.The above results show that the different way of splicing GPR1 gene produce different splicing subtypes,and different subtypes have different functional protein,suggesting that the GPR1 gene of different subtypes in the regulation of follicular may have different functions.(4)By adding chemerin chicken protein in vitro culture of follicular granulosa cells,to explore chemerin protein caused by chicken follicular granulosa cell apoptosis,and the influence of steroid synthesis fatty acid metabolism.After adding different concentration of chemerin protein processing,high concentration(100,1000 nm/mL)of chemerin processing chicken follicular granulosa cells after GPR1 and the expression of apoptosis related gene significantly increased(P<0.05),indicating high levels of chemerin may by promoting the expression of GPR1,to raise promoting apoptosis gene expression;(100,1000nM in high density dealing group/mL)fatty acid synthesis of key genes(ACLY,ACC1 and FASN)and fatty acid beta oxidation speed limit key enzyme gene(CPT1 and CTP2)amount of express were significantly higher than that of low concentration treatment group(1,10 nM)and the control group(0 nM)(P<0.05),TG and the critical speed of enzyme synthesis DGAT2 gene expression of the quantity in each group were not significant difference(P>0.05);Responsible for steroids transshipment STAR gene expression quantity presents the fluctuation,,there is no significant difference between the groups(P>0.05),and the catalytic steroidogenesis pregnenolone CYP11A and catalytic pregnenolone generation progesterone 3 beta HSD gene expression of the trend of decline,and 3 beta HSD gene expression in high density dealing group volume is much lower than low concentration treatment group and control group(P<0.05).The above results show that high levels of Chemerin may by promoting the expression of GPRI,promote the expression of apoptosis gene,to raise and to maintain a relatively stable within the cell,the Bcl-2 gene expression quantity also rise;Chemerin fatty acids can promote beta oxidation,and no large effects on the synthesis of TG;Chemerin to chickens follicular granulosa cells of steroid hormone synthesis related genes with dose regulation effect,high levels of chemerin in chickens of follicular granulosa cells may be inhibit the synthesis of steroid hormones.Above all,regardless of Chemerin and its receptor on the tissue or cell,all show that can promote the synthesis of fat and follicular development ability,the apoptosis of cells and the synthesis of steroid has different degrees of influence.Therefore,this study concluded that chemerin mainly through GPR1 to control the effect of follicle and granulosa cells.This study can be further explore Chemerin in chickens regulation function in the process of follicular and revealing the molecular mechanism of chicken of follicular differences provide a reference basis.