1,Effects Of Pamidronate On Proliferation, Differentiation Of Osteoblast And Fibroblast Derived From Osteogenesis Imperfecta Patients 2,Establishment Of Immortalized Osteogenesis Imperfactacell Lines

ObjectiveOsteogenisis Imperfecta(OI) is a sort of autosomal hereditary disease caused by mesenchymal dysgenesis and collagen disorders with some other name of Fragililis ossium and Idiopathic osteopsathyrosis, Periosteal dysplasia. The surgical therapy of osteogenisis imperfecta is osteotomy fixation with intramedullary pin. Bisphosphonate drugs are widely used in the treatment of OI patient for it can improve bone density and decrease the frequency of fractures.Bisphosphonates can decrease bone turnover and inhibit bone resorption. It is usually used in the group of diseases with bone resorption hyperthyroidism, likepostmenopausal osteoporosis, Puget's and so on. Bisphosphonates can affect the activity and apoptosis of osteoclast (OC), the main mechanism of its effect on OC is that it inhibit the key enzyme of mevalonate pathway , which leads to the G protein can not be isoprenylated. In recent years, some researches have found that bisphosphonates can also inhibit osteoclast by the way of affecting osteoblast. The effects of bisphosphonates on the proliferation and differentiation of bone marrow stormal cells and osteoblast are also reported recently. The mechanism of bisphosphonates'effect on fibroblast is mainly foused on osteonecrosis of the jaw (BRONJ), an in side disease induced by long-time bisphosphonates treatment. Researchers found that zoledronate can induce fibroblast apoptosis when its concentration is higher than 10μM. The apoptosis mechanism of fibroblast is partly caused by the pathway of mevalonate intracellular. Lower concentration of zoledronate could promote the fibroblast proliferation and the expression of IL-6,IL-8. In this research, different concentration of pamidronate was used to treat primary osteoblast and fibroblast derived from OI patients. Its effect on the proliferation and differentiation were studied.Methods1. Culture of primary ostoblasts and fibroblasts derived from clinical OI patients and dislocation of the hip (DHH) patients. Three primary cells was adopted in the experimental group (OI) and control group (DHH). Primary osteoblasts and fibroblasts were tested by ALP staining before drug treatment.2. Pamidronate with the eight different doses ranging from 10-3M to10-10M was added to the experimental and control group cells.MTT assay was performed to test cell proliferation after 48h and 72h drug treatment. Alkaline phosphatase activity was analyzed to observe cell differentiation by ELISA at the same time points as above.Results1. From cell morphology observation and BCIP/NBT ALP staining, primary osteoblsats and fibroblasts derived form OI and DHH patients were successfully cultured and they have the ability to proliferation. High expression of ALP was observed in osteoblasts and lower expression was seen in fibroblasts.2. Proliferation was observed after 48h and 72h's incubation with drugs. While the remarkable increasement comes after 72hours(P<0.01). Proliferation inhibition was appeared both in primary osteoblasts and fibroblasts when the drug's concentration was in the range of 10-3M -10-4M. Increasement in the cell proliferation was seen when drug's concentration falls from10-5M to 10-10M. The optimal proliferation pamidronate concentration for experimental (OI group) osteoblasts and fibroblasts were 10-8M and10-6M ( P<0.01 ) , with their corresponding proliferation rate was 43% and 39% respectively. In the control group, optimal proliferation concentration was 10-7M in ostoblast and 10-5M in fibroblast with the rate of 37% and 28%, respectively. 4. We found the effect of the pamidronate on ALP activity is corresponding with the concentration on the proliferation. When the drug's concentration was 10-8M in osteoblast and and10-6M in fibroblast, the highest ALP activity of OI experimental group was 102U/L and 32U/L.ConclusionsPamidronate has great effect on cell proliferation and differentiation of both osteoblasts and fibroblasts and its effect is dose-dependent. ObjectiveMost researchers mainly use primary cultured cells for their studies,which cost lots of time and money and result in relative complex preparation steps in manipulation and in common variations in cell physiology.Consequently, it is difficult to compare experiments while using different passages of cells.Immortalised cell lines provide more advantages than primary cells,such as uniformity, availability and easier genetic manipulation. The objective of this research was to design and construct eukaryotic expression vector of PCI-neo-hTERT,which is useful for the immortalization of primary cells. Then tranfect primary fibroblast derived from OI patients to built immortalised cellsMethods1.Construction of PCI-neo-SV40T recombinant vector PAAV-SV40T and PCI-neo-hTERT vectors were digested by XhoI and SalI enzymes to obtained PCI-neo and SV40T fragment. After ligation reaction, recombinant fragment was transformed into DH5α. Recombinant plasmid was prepared and identified by both digestion reaction and PCR.2.PCI-neo-SV40T recombinant plasmid was transfected into primary fibroblast cultured from OI patient.Results1. PCI-neo-SV40T recombinant vector are successfully constructed by the test of PCR and double enzyme digestion reaction.2. Primary fibroblasts died gradually after the traqnsfection of PCI-neo-SV40T plasmid.ConclusionsPCI-neo-SV40T recombinant vector was constructed successfully. Further transfection experiment need to be done to built immortalised OI fibroblast cells.