The Effect Of β-TCP On Gene Expression Of Osteoblast And The Purification Of Osteonectin

Calcium phosphate materials have been widely used in the biomedical fields, especially in bone repairing and reconstruction. More and more researchers are concentrating on the mechanism that how the calcium phosphate materials are involved in the life in recent years. In this paper, works were carried out to investigate this effect and disscuss the biomineralization of the calcium phosphate materials by cell biology and biomolecular methods.The osteoblsts were isolated from the new born Wister rats by enzymatic cell isolation and explanted culture methods. In this experiment the cells were acquired from the calvarial bones of rats successfully, and the osteoblasts were purified by Nash's method. After cultured the osteoblasts for some days, the cells were identified by cell morphology, ALP activity, and calium nodule. The result showed that the osteoblasts acquired by the two methods displayed high alkalin phosphatase activity and calcified nodule-forming activity, which can testify if the osteoblasts have good biological characteristic. It was turned out that the cells can satisfied the need of our research, and the cells can be used for the further experiment. As well, in our experiment the same tissue were cultured for several times, and the osteoblasts were acquired the osteoblasts successfully. After the osteoblasts were stained with alkalin phosphataste, the osteoblasts acquired from the same tissue also exhibited a good biological characteristic. The results testified that the osteoblast acquired by continuing tissue culturing method have good biological characteristics, and can apply into the further experiment.Through the co-cultured experiment of osteoblast andβ-TCP in vitro, the endocytosis phenomina was observed, theβ-TCP can be endocytosised by osteoblasts. The MTT assay showed that theβ-TCP also can promote growth and proliferation of the osteoblasts. After theβ-TCP and qstoblasts co-cultured for a period of time, the total RNA were extracted from the osteoblasts, and the mRNA expression level of the Alkalin phosphatase(ALP), Osteonectin (ONN), osteopontin (OPN) were tested through the method of the RT-PCR and PCR test assay. The experiment showed that after the osteoblasts were co-cultured withβ-TCP, the osteoblasts' mRNA expression level of ALP , ONN are higher than the control group, andβ-TCP can affect the gene expression level of osteoblasts. The experiment implied that maybeβ-TCP play an important role in the process of bone bio-mineration.The above experiments have turned out thatβ-TCP can affecte the ONN mRNA expression. In order to investigate the protein's function in the process of biomineration, an expression vector pHX201/QNN which can express the ONN protein in vitro was constructed in the experiment. And the pHX201/ONN expression vector as well as another vector pGEX-KG₂/ON which have been construced in previous experiment were inserted in the E.coli BL21(DE3)pLysS and R1L, and the pGEX-KG₂/ON expression vector displayed a more expression efficiency than the pHX201/ONN expression vector. The expression condition of pGEX-KG₂/ON was modified, and the interested proteins were acquired through bacteria culture, ammonium sulphate precipitation, ultrfiltration membrane separation, affinity chromatograph purification with GST affinity coloum. At last, the purified ONN protein was acquired successfully. The protein can be applied into the further experiment.