The Effect Of PDK1 Protein Kinase On The Proliferation Of Osteoblasts

Objective: To provide a theoretical basis for understanding the specific mechanisms of proliferation and differentiation of osteoblasts by exploring the effects of PDK1 on the proliferation and differentiation of osteoblasts and its possible mechanisms at the cellular level.Method: The MC3T3-E1 osteoblast precursor cell line was selected as the research object to establish a culture model in vitro.The cells were divided into three groups: group C,the culture medium was complete medium;Group P+,the medium was complete medium + PDK1 activator(10nM);group P-,the medium was complete medium + PDK1 inhibitor(10nM).Related tests were performed in the following order:(1)Identification of osteoblast culture system: MC3T3-E1 cell line was subjected to ordinary culture for 72 hours,followed by alkaline phosphatase staining,and after 14 days of culture,mineralized nodule staining was perfoermed.(2)Determination of logarithmic growth period: cell proliferation detection was performed at 24 h,48h,72 h,96h,and 120 h in group C,P + and P-,respectively.(3)Immunofluorescence staining was performed in group C,P + and P-after 72 h of culture.(4)Propidium Iodide staining was performed in group C,P + and P-after 72 h of culture,and then cell cycle and apoptosis were detected by flow cytometry.(5)mRNA extraction was performed was performed in group C,P + and P-after 72 h of culture,and then the expression of Runx2 and c-fos was detected by RT-PCR.Results:(1)The MC3T3-E1 cell line can successfully differentiate into osteoblasts.(2)The cells at 72 h in group C,P+ and P-were all in the logarithmic growth period.(3)Group C had dense cells and the largest proportion of nuclei in a single cell.(4)The SPF% value of group P-was significantly lower than that of group C and group P+,the apoptosis rate of group P-was significantly higher than that of group C and group P+.(5)The expression of mRNA of Runx2 in cells of group P+ and P-were significantly higher than that of group C.(6)The expression of mRNA of c-fos in cells of group P-were significantly higher than that of group C.Conclusion:(1)In the process of osteoblast differentiation,normal level of PDK1 promotes the proliferation of osteoblasts.(2)At the stage of osteoblast proliferation,normal level of PDK1 maintains the cytoskeletal morphology of osteoblasts,promotes DNA synthesis of osteoblasts,inhibits apoptosis of osteoblasts and the expression of mRNA of Runx2 and c-fos.