Effect Of Elastic Stress On Expression Of Core-binding Factorα1 In Human Periodontal Ligament Cells In Vitro

Core-binding factor a l(cbfal) is a transcription factor that closely relates tobone development. Cbfal plays an essential role in bone formation and osteoblast differentiation. It serves as a master gene in regulating osteoblast-specific gene expression. Cbfal gene only expresses in the cells of osteoblast lineage. When it expressed in non- osteoblast cells, these cells could express many osteoblast-like characteristics. In the gene knockout experiments, no evidence of bone formation could be observed in animals that were homozygous for the deletion. In the heterozygote studies, the results indicate that osteoblast function was highly compromised, including a severe reduction in the number of osteoblasts, in the synthesis of bone matrix protein and in the activity of alkaline phosphatase. All studies show that cbfal plays a very important role in bone formation.Current studies about cbfal focused on osteoblast differentiation and bone development, but the study about the relationship between cbfal and orthodontic bone formation was not mentioned. Orthodontic tooth movement depends on the remodeling of periodontal tissues especially surrounding alveolar bone. Periodontal ligament cells play an essential role in alveolar bone remodeling during orthodontic tooth movement. It is reasonable to assume that cbfal isinvolved in these processes. The purpose of this study was to investigate the expression of cbfal in human periodontal ligament cells affectted by mechanical stress in vitro and to enhance our understanding of the role of cbfal in orthodontic bone formation.This study mainly contains the following works:1. Aim of this study is to culture osteoblasts of new-bom SD rats in vitro and to prepare enough cells for the RT-PCR. The SD rat osteoblasts were cultured from the cranial bones by using explant method. The purification of the cells was obtained by plating efficiency difference. The identification of the cells was conducted with ALP activity assay and Von Kossa staining methods. The results showed that ALP activity was positive. Von-Kossa staining showed there was calcium deposition in the nodules. Both results demonstrated that the osteoblasts were successfully cultured in vitro.2. Cloning and sequencing of cbfal cDNA fragment from cultured SD rat osteobasts. The cbfal cDNA fragment about 700bp was obtained by RT-PCR method and was identical to the sequence in the GenBank.3. The cloned cbfal cDNA fragment was subcloned into prokaryotic gene expression vector pRSET A in a certain direction, and the resulted plasmid was used to transform competent E.coli BL21; After selection and identification, the positive clone was induced by FPTG to express fusion protein. SDS-PAGE was employed to detect the expression products and the target protein was purified by Ni-NTA affinity chromatography.4. Rabbits were routinely immunized with the purified protein to produce antisera to Cbfal. The antisera were identified by double immunodifiusion, Western Blot and immunostaining for new-born SD rat cranial bone and MG-63 osteosarcoma cell lines. And also in present study the cbfal cDNA probe was generated byDIG-labelling method.5. Aim of this study is to investigate the osteoblast-like characteristics of human periodontal ligament cells affected by elastic stress in vitro and the role of cbfal in orthodontic bone formation. Human periodontal ligament cells were cultured on the elastic bottom plate and stimulated by elastic stress using mechanical loading system for cultured cells in vitro. The expression of cbfal was observed by immunohistochemistry and in situ hybridization methods. The results showed that cbfal was positive immunoreaction in human periodontal ligament cells stimulated by elastic stress and negative immunoreaction in control' s. Cbfal mKN A showed positive expression in human periodontal ligament cells stimulated by elastic stress and negative expression in control' s. It is suggested that elastic stress play a role in the differentiation process from hum...