| In the early research subsidized by the National Natural Science Foundation,the use of porcine growth hormone antibodies as target,random 12-peptide library of phage is used to screened phage that specifically bind to antibody.Then the ssDNA were extracted and the sequences were analyzed,and two groups of high amino acid sequence homology were screened,at the same time combined with the computational biology analysis,determined the core sequence is PCLPHCNSTSD.Based on this sequence,we synthesized porcine growth hormone mimetic peptides.By laser confocal microscopy,flow cytometry analysis and other technical means demonstrated that the porcine growth hormone mimetic peptides specifically bind to growth hormone receptor in liver cell surface and we have proved that porcine growth hormone mimetic peptides activate growth hormone receptor by inducing conformational dimerization.However,whether mimetic peptide of the GH trigger same signal transduction pathway as does GH is not yet clear.Growth hormone plays a physiological role after binding to growth hormone receptor through activating molecules which regulate transcription of target gene into nucleus.Therefore,whether porcine growth hormone mimic peptide can activate intracellular signal transduction molecules is one of the main observation index judging function activity.Therefore,this study used somatic cell model(rat primary liver cell model)and cell line model(3T3-F442A,3T3-L1 cell line)generally acknowledged by research of growth hormone receptor as test material to compare and verify whether porcine growth hormone mimic peptide plays an active role like growth hormone by activating intracellular signal transduction pathway,according to the research of two capital intracellular signal transduction pathway,JAK-STAT and MAPK-ERK.By the following experimental design and a series of experiment,the results are as follows:(1)Wistar rat liver cells are isolated by a modified two-step collagenase perfusion method,hepatocyte morphology is in good condition and survival rate is about 94.23%.the secretion of Albumin and Blood Urea Nitrogen was the highest in 3-5d during cell culture,at the same time,the secretion of Lactate dehydrogenase minimum,determined that the best period of rat hepatocytes is in 3-5d after culture.The best time of rat liver cells viability was 3～5d after culture which determined by MTT colorimetric assay.Less influence of growth hormone receptors induced by cell separation was identified by specific antibody MAB263 and its surface receptor function is in good condition.According to the above research,we established the liver cell model used to study membrane receptor in vitro.(2)Receptor competitive binding technology and Confocal laser were used to prove that both the porcine growth hormone mimic peptide and pGH can combine with growth hormone receptor on rat hepatocytes by carrying out binding,co-localization and competitive binding experiment.Similarly,carrying out experimental research on both the porcine growth hormone mimic peptide and pGH can specifically bind to growth hormone receptor in 3T3-F442A,laid experimental foundation of credible research of signal transduction pathway.(3)Western-blot and flow cytometry analyses were used to analyze the signal transduction pathway of porcine growth hormone mimic peptide in liver cells and 3T3-F442A cells,results show that the growth hormone mimic peptide is able to trigger identical signal transduction pathway like pGH and can stimulate phosphorylation of STAT1,STAT3,STAT5 and ERK1/2,and the growth hormone mimetic peptides stimulate the optimal phosphorylation at 20 for 40 min and the best concentration at 45-90nM,indicating that the growth hormone mimetic peptides exert a physiological role which is similar to porcine growth hormone in rat liver cell model(indirect effect cell model)and 3T3-F442A cell model(immediate action cell model).(4)Supplement of pGH during cell culture proved that porcine growth hormone mimic peptide can affect the secretion of IGF-I in hepatocytes,at the same time,found porcine growth hormone mimic peptide also can promote the proliferation and differentiation,proved the physiological action of porcine growth hormone mimic peptide in cell level.Through specific murine cell models,it has been observed taht the porcine growth hormone mimic peptide can activate growth hormone receptor and further activate multiple pho sphorylation reaction of intracellular signal transduction pathway,JAK-STAT and MAPK-E RK,proved the porcine growth hormone mimetic peptides exert a physiological bioactivity and function which is similar to porcine growth hormone.Study on the function of porcine growth hormone mimetic peptides laid the theory and technology foundation of preparing peptides of bioactive micromolecule substance and growth hormone receptor agonist. |
