The Effect Of KLF4 On Osteoblast Osteogenetic Differentiation And Matrix Secretion In Mice

Periodontal disease is one of the common diseases that threaten human oral health.It is a chronic infectious disease that occurs in periodontal tissues and is induced by bacteria and metabolites.The main clinical symptoms of periodontitis are alveolar bone absorption,where the pathological change is the lack of bone,which is caused by the imbalance of bone formation and bone resorption.Periodontitis expresses a high incidence rate and is closely related to the body health.Currently the clinical treatments include symptomatic treatment and inhibiting the progress of periodontitis,which shows no effective repair for the bone defects.Thus it is necessary to study the basic molecular mechanism of bone homeostasis.Bone homeostasis is a dynamic process.The balance between bone formation caused by osteoblasts and bone resorption caused by osteoclasts is the premise to maintain steady bone metabolism.A large number of studies have shown that,the imbalance of less bone formation than bone resorption will lead to osteoporosis,periodontal disease,osteoarthritis,rheumatoid arthritis and other bone diseases,otherwise lead to bone sclerosis.Among them,osteoblasts are one of the key links of bone metabolism balance.Osteoblasts ar e main functional cells of bone formation,of which the activity can be regulated by a variety of factors.Therefore,the study of the molecular mechanism of bone formation is of great clinical significance to improve the effect of bone repair.More and more studies have focused on the generation,proliferation,and differentiation of osteoblasts,suggesting the importance of osteoblasts in bone regeneration in bone-related diseases.Based on the above analysis,this study aims to establish the isolation and culture of osteoblasts firstly,preparing basis for the completion of the later experiments.Secondly,the effect of overexpressed KLF4 on osteoblast proliferation and differentiation is detected,followed by the studying of the effect of KLF4 on the early osteoblast differentiation of osteoblasts.Finally,this study aims to the matrix secretion that involving in the KLF4-regulated bone homeostasis in osteoblast differentiation,to further provide a theoretical basis for the treatment of periodontitis.Methods:1.In this study,the primary cells were obtained from the newborn mice skull fragments by stepwise enzymatic digestion.The morphology of the cells was observed under the microscope.Cells were cultured in osteogenic medium for 9 d and 21 d,then the osteogenic induction was identified by alkaline phosphatase and alizarin red staining.2.Using immunofluorescence technique to detect the expression of KLF4 in mice osteoblast..The transfection efficiency was detected by measuring the diffe rent multiplicity of infection(MOI)with flow cytometry.Osteoblasts were randomly distributed into con trol group(no-transfection),GFP group(Ad-GFP transfection)and KLF4 group(Ad-KLF4 transfection).KLF4 protein was detected by Western blot,growth curve was analyzed by MTT,and a real-time PCR was used to illuminate the expression variations of osteogenic genes alkaline phosphatase(ALP),bone sialoprotein(BSP)and runt-related transcription factor2(RUNX2).3.The expressions of KLF4,MMP2 and COL1 were detected by Western blot.After the treatment of si RNA KLF4 or Ad-KLF4 for 0,2 and 4 days,the effects of KLF4 on the protein and gene expressions of MMP2 and COL1 were detected by Western blot and Real-time PCR in osteoblasts and in the early osteoblasts differentiation.Results:1.In this study,the primary osteoblasts obtained from the newborn mice skulls by enzymatic digestion were polygonal and cubic.Alkaline phosphatase staining showed positive results of cytoplasmic blue staining.Alizarin red staining showed red mineralized nodules.Through the observation of cell morphology and identification by staining,it was shown that the primary osteoblasts could be isolated via the stepwise enzymatic dige stion,thus the foundation was laid for this experiment.2.By PCR amplification,gene sequencing successful build Ad-KLF4.At the optimal multiplicity of infection(MOI-100 PFU/ml),the protein level of KLF4(P<0.01)and cell proliferation increased obviously,while the mRNA expressions of osteogenic genes ALP,BSP and RUNX2 decreased significantly.3.Along with the osteoblast differentiation,the expression of KLF4 increased,accompanied by decreased expression of MMP2 and increased expression of COL1.Then the expressions of KLF4 gene and protein were significantly decreased after infecting osteoblasts with 40 nmol/L KLF4 si RNA,while the expression of MMP2 was increased and the expression of COL1 was decreased.On the contrary,the expression of KLF4 was significantly increased after infection with Ad-KLF4(MOI = 100 PFU/ml),and the expression of MMP2 was decreased.Oddly,the expressions of COL1 gene and protein still showed a downward trend.Conclusion:1.The primary cells were successfully obtained by stepwise enzymatic digestion.Cells of passage two grew well and had good activity,which could meet the requirements of follow-up experiments.2.Osteoblasts transfected by adenovirus-mediated KLF4 gene can promote the proliferation of osteoblasts and inhibit the expression of ALP,BSP and RUNX2,thereby inhibiting bone formation.3.In bone homeostasis,KLF4 can negatively regulate the expression of MMP2,regulate the secretion of matrix in the process of bone formation,thereby regulating bone formation.