Studies Of Effects Of Chicken IGF-Ⅰ On Osteoblastic Functions And Its Correlation Of Gene Polymorphism To Bone Metabolism In Cage Layers

Osteoporosis in laying hens is difined as a progressive loss of structural bone, leading to increased fragility and susceptibility to fracture during the laying period. Cage feeding, nutrition, hormones and genetic factors may play some role in forming of osteoporosis, but the exact mechanism is unknown. Thinking about that insulin-like growth factor- I is the most abundant growth factor in bone and play an important role in remolding of skeleton, we carried out our studies from two aspects as osteoblastic biofunctions of IGF-1 and relationship between the polymorphism of IGF- I and bone metabolism. The mainly objective was to elucidate the mechanism of osteoporosis partly and provide the theoretic standard for preventing this disease.1 Molecular Cloning of Chicken Insulin-Like Growth Factor I and Its Expression in Escherichia coliBased on the published nucleotide sequence of chicken insulin-like growth factor I , a pair of RT-PCR primers was designed and synthesized. Total RNA, isolated from young chicken liver, was used as template to generate complementary DNA by reverse transcription. The IGF- I DNA fragment was amplified by polymerase chain reaction, and cloned into PMD18-T vector. DNA sequencing, restriction enzyme digestion and PCR amplification all confirmed the inserted fragment was a complete chicken IGF- I with the code for signal peptide, which had the identities of 100% with the chicken IGF- I gene published in the GenBank. Another pair of primers was designed to sub-clone the gene coding chicken IGF- I mature protein. Then the sub-cloned IGF- I gene has been successfully inserted to expression vector pRLC and expressed in E.coli through the changes of temperature, The Tricine-SDS-PAGE and Thin line chromatography of recombinant protein showed that the cloned recombinant protein which with molecular weight of 7.5KD was amount to 23 % of the whole protein in the E.coli cell.2 The Study of Refolding, Renaturarion and Bioactivity of Chicken Insulin-Like Growth Factor I Recombinant ProteinBase on the high expression of pRLC- IGF-1 , the E.coli cell induced by rising temperature for 5 hours were collected. The Tricine-SDS-PAGE result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 7.5KD, which was subsequently dissolved in 7M guanidine chloride and renatured with dilution in refolding buffer containing 0.5M arginine. In order to obtain pure protein, the renatured chicken IGF- I was desalting and purified by Hiprep Sephacryl S-200 chromatography. The biological activities of IGF-1 product was assayed in NIH 3T3 cells and chick embryo fibroblasts by using MTT method. The results show that the expressed IGF- I can obviously stimulate NIH3T3 cells and chick embryo fibroblasts cells to proliferate at the concentration ranging from 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, suggesting that the protein has its biological activities.3 Study on Isolation and Culture of Osteoblastic Cells from Calvaria of Embryonic Chicken and Effect of rcIGF- I on ItBased on the more effective method of isolating osteoblastic cells from calvariae of embryonic chicken, the effect of recombinant chicken IGF- I on the osteoblastic cells about proliferation and activity of AKP was studied. The osteoblastic cells were harvested by enzyme digestion(0.1%cllogenase II) from bone chips isolated from calvariae of about 15-day embryonic chicken. The good second passage culture was used for identify by AKP staining and seeded in 96-well plates cultured with different concentration of rcIGF- I for 48h.Then MTT and PNPP method were made to assay the proliferation and activity of AKP of osteoblastic cells. Phase contrast inverted microscopy showed that cultured cells were characteristic of osteoblast: with the shape of shuttle, triangle and stellate; 80%-90% of osteoblasts showed positive alkaline phosphatase; The appropriate concentration of rcIGF- I may promote the chicken osteoblastic cells' proliferation and differentiation.4 Effect of rcIGF- I on...