| Porcine Growth hormone(pGH) is a polypeptide hormone that consists of 191 amino acids and plays a vital role in the regulation of postnatal growth and development. The liver is the most important targets organ to porcine growth hormone, many biological functions of pGH can be achieved on hepatocytes. In traditional theory, by binding to growth hormone receptor(GHR), pGH can activate a series of conformational change to play its physiological role. However, recent mechanism studies of action of human growth hormone(hGH) in rat hepatocytes suggest that, GH and GHR still perform important biological functions after they internalize into cells. The main functional site can be clarified by subcellular localization after its internalization. There is no study about mechanism and intracellular localization of GH on the same kind of somatic cell.In this experiment, we chose primary porcine hepatocytes as the model to give pGH and growth peptides that simulate the action of pGH which is prepared in our laboratory, compare the differences of internalized amount into cells and observe subcellular localization of porcine growth hormone and growth peptides, study whether there are differences on functional site and binding manner between pGH and growth peptides after internalization. The purpose of this test is to study the phenomenon of internalization and subcellular localization of porcine growth hormone, to provide experimental basis for revealing metabolism of GH on same kind of somatic cell.After experimental design, we carried out the following studies:Firstly, we identified cell apoptosis and the expression of GHR on cell surface by flow cytometry and western-blot to verify the activity of isolated hepatocytes. The results showed that viability of piglet hepatocytes reached 80.94 %. Isolated primary hepatocytes in vitro that cultivate in different time prove the expressions of GHR are the same and no significant difference was seen between the groups. The availability and consistency of isolated piglet hepatocytes were proved.Secondly, we have studied internalization of pGH and growth peptide when cells were stimulated after different period of time by confocal microscopy. Results show that pGH in the cytoplasm and nucleus has a parabolic trend within the overall. With begining of internalization, the amount of internalized pGH gradually increased, and decline after reaching the maximum. There are significant differences on internalization between cytoplasm and nucleus. In the cytoplasm, it reached the maximum about 30 minutes. Well in the nucleus, it reached the maximum about 60 minutes. It suggests that there is difference in binding and acting in different site. The change of growth peptide was similar to that of pGH, but there is significantly accelerated in the velocity that it reached the maximum in the cytoplasm and nucleus is about 15 minutes.Thirdly, we used TEM to identify effects of pGH and growth peptide on cell morphology. The results demonstrated that, compared with the control, cell shape had no obvious differences when pGH and growth peptide reached the maximum, to lay the foundation for subcellular localization experiments.Finally, we use immunization electron microscope to do the observation of subcellular localization experiments. Results showed that after stimulating liver cells for 20 minutes, pGH was internalized into cells. It was found that phenomenon of the combination in the mitochondria, nuclear, rough endoplasmic reticulum and cell membrane; and we have konwed that most pGH located at mitochondria, part of pGH located at the nucleus, and a little bit pGH bound to the rough endoplasmic reticulum and cell membrane. In turn reducing the amount of bound, revealed that main site of pGH that has effect. By the observation of subcellular localization test we can know that mitochondria and nucleus are main binding sites of pGH with hepatocytes, and this is prompted pGH play a major role in the site. The result is similar with study of rats.In summary that both of the pGH and growth peptide can have the phenomenon of internalization, and the overall trend is very similar; we can know that the main sites are mitochondria, nuclear, rough endoplasmic reticulum and cell membrane by subcellular localization. |
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