Ectopic Intramuscular Expression Of Ghrelin And GRF Gene Leads To Mouse Body Weight Gain

Growth Hormone(GH) releasingfrom pituitary is a polyfunctional hormone , which plays an important role in body metabolism regulation.Recently,a bioactive peptide named"Ghrelin",which was identified during the research of growth hormone secretagogues( GHSs),was found to have great effect on the regulation of GH releasing,gastric acid secretion and food intake etc.GRF(Growth Hormone Releasing Factor)are synthesized and secreted by hypothalamus,which can stimulate pituitary to synthesize and secrete growth hormone,thus,increasing GH concentration in animal and human bodies.In this research,Ghrelin gene was synthesized chemically for four single-chain gene.After amplification for three times by PCR,174bp in length,Including both ends of BamHⅠ,EcoRⅠrestriction site sequence,the products was purified in 2.5% agarose gel electrophoresis,then digested with BamHⅠ,EcoRⅠ.Digested fragments were ligated to pcDNA3.1(-) with the same digestion,and transformed to competent DH5α,positive recombinants were selected and identified by PCR,restriction enzyme digestion and sequencing.GRF gene was amplified by PCR taking pcDNA3-GRF(1-32) plasmid as templet.The products were purified in 2.5% agarose gel electrophoresis. Double digestion fragments of PCR products (EcoRⅠ,BamHⅠ)and pcDNA3.1(-)were prepared and ligated,then transformed to competent DH5α, positive recombinants were selected and identified by PCR,restriction enzyme digestion and sequencing.The three expression vectors were transfected by Lipofectin to prepared CHO cells in 60% confluent according to provided protocols,then total RNA was extracted from transfected CHO cells,the positive result was obtained by RT-PCR.Three expression plasmids were extracted and poly lactic-co-glycolic acid (PLGA) microspheres encapsulating plasmids were prepared by double emulsion-in liquid evaporation process. Entrapment efficiency was achieved to 91.52%,mean particle size 2.61μm and the yield 67.85%;Mice were muscularly injected with plasmid microspheres(1mg plasmid/ kg of BW),bare plasmid(1mg / kg of BW) and saline,the accumulative weight gain of mice were measured and the concentrations of serum IGF-I and GH were measured by radioimmunoassay(RIA)in every 7 days. The results showed that injection of pcDNA3.1(-)-GRF and pcDNA3.1(-)-Ghrelin lead to accumulative weight gain was 40.53%(P<0.05),13.05%(P<0.05) and 13.95%(P<0.05) higher than the groups of the saline group,pcDNA3.1(-) -GRF and pcDNA3.1(-)-Ghrelin at 21 days after injection. The serum IGF-I levels were 95.33%(P<0.01),1.61%(P<0.01) and 73.71%(P<0.01) higher than the groups of the saline group,pcDNA3.1(-)-GRF and pcDNA3.1(-)-Ghrelin.These have a correlation with the accumulative weight gain of mice as well(P<0.05).The results showed that plasmids were expressed in-vivo could promote the secretion of GH and improve the serum IGF-I levels.But the serum GH levels of each group did not have significantly difference,it is possible that the secrete cycle of GH is fairly short,so it is hard to analysis the secretion trend of the whole cycle by a point of data.We draw a conclusion that Ghrelin and GRF produce a marked effect together in animal body do increase IGF-1 concentration and improve animal growth,whose biological activity might be more powerful than GRF and Ghrelin single gene.All above indicated synergy between Ghrelin gene and GRF gene .it may be a fresh approach to improve animal productivity by gene transfection technique.