| 1.The more effective method of isolating the osteoblastic cells from calvariae of embryonic chicken was built in order to supply the further way to study the osteoporosis of cage laying hens. The osteoblastic cells was harvested by sequential enzyme digestion(0.2% cllogenase II and 0.25% trypsin) from bone chips isolated from calvariae of about 15-day embryonic chicken .The good second passage culture is used for identify. The cells were a classic characteristic of osteoblast: be fibroblastic, shape of shuttle, triangle and stellate; Black deposits of AKP, red deposits of promatrix were showed by cytochemical staining and form mineralized node for long culture. Osteoblastic cells could be harvested from the mixed sequential digestion method.2. To study the effect of different concentration of 1P98 on the osteoblastic cells about proliferation and liveness of AKP based on the method of isolating the osteoblastic cells from calvariae of embryonic chicken. The osteoblastic cells was harvested by sequential enzyme digestion (0.2% cllogenase II and 0.25% trypsin) from bone chips isolated from calvariae of embryonic chicken, Through identified, cells were of characteristic by osteoblast. The good second or third passage culture is used for research , which were seeded in 96-well plates at the density of 1.5 x104, which are cultured with 10-4-10-10M IP98 culture medium for 72h. Then MTT and PNPP method were made to assay the proliferation of and activity of AKP of osteoblastic cells. 10-4M IP98 distinctly inhibited the osteoblastic function, but the 10-6M IP98 obviously improved their function, the other concentratin of IP98 has no effect on the osteoblastic cells. The sequential digestion method could obtain pure chicken osteoblastic cells. The appropriate concentration of IP98 may promote the chicken osteoblastic cell's function, namely, accelerating proliferation and differentiation.3. 500 ISA cage layers were divided into 5 groups. A group is control, IP98 was addto B, C, D, E groups at the dosage of 15ppm, 25ppm,50ppm, 100ppm respectively. The test was carried out for 70 days. Once each month, hen blood was collected to check the Ca, P, BGP, AKP, E2 and the hens were weighted, bone mineral content was tested. Each day, the number of eggs and damaged eggs and the weight of eggs were noted. The egg laying rate of C group was significantly higher than that of control and the other groups, the laying rate of D, E group were apparently lower than that of A, B, C groups. The mean egg weight and shell quality of C group were best. The body weight of C group is lightest, but these were not significant; the serous level of phosphor of C group and calcium of B group was significantly lower. The serous level of AKP and BGP of C group were significantly higher. There were no changes observed in serous level of E2 of all experiment groups. The study demonstrated that the appropriate dosage of IP98 may improve and the production of cage layer during the late cycle and bone metabolism.4. In order to analysis the restrictive fragment length polymorphism of insulin-like growth factor- I of ISA cage layer hens, the sequence of IGF- I gene is amplified exactly by polymerase chain reaction (PCR). Red cells were collected from the hens and the genetic DNA were extracted, as the template, the 621bp fragment located about 7Kb upstream of IGF- I promoter sequence was amplified by the improved Nagaraja's method, its specificity was checked firstly by gel electrophoresis, and was cloned in pMD18-T vector, then sequenced. The sequencing data was compared with the previously published with the DNAstar software. It showed the 621bp gene fragment by 1% gel electrophoresis, there was 99% homologous between amplified gene and standard one. It is successfully amplify the objective gene, and supply the basis of the study of PCR-RFLP of IGF- I .5. To study the association between restrictive fragment length polymorphism (RFLP) and osteoporosis of ISA cage layer hens. Choos... |
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