Effects Of Overexpression Of NICD On Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Objective: To study the effect of overexpression of Notch intracellular domain NICD on proliferation and osteogenic differentiation of human periodontal ligament stem cells.Methods: The third generation cells were used for related experiments,and the stable overexpression of NICD cells(hPDLSCs/NICD)was set as the experimental group.Untreated cells(hPDLSCs)as blank control group,and cells transfected with empty vector(hPDLSCs/K)as negative control group.The effect of overexpressing NICD on proliferation ability of hPDLSCs was detected by CCK8(Cell Counting Kit-8,CCK-8),construction of NICD overexpression vector by Real-time quantitative Polymerase chain reaction and Western blottingAlizarin,Red S calcium staining,real-time quantitative polymerase chain reaction and Western Blot detect the effects of NICD on Notch Signal Pathway Gene Notch1 and cementum attachment proteins(CAP),Osteocalcin(OCN),Runt-Related Transcription Factor 2(Runt-Related Transcription Factor 2,RUNX2),Osteogenic Proteins RUNX2 and flag Marker Proteins(Used to mark NICD).Results:The results of CCK8 showed that compared with two control groups,the cell proliferation rate of hPDLSCs/NICD group was significantly faster(P<0.05).Q-PCR and WB results showed that the expression of Notch pathway related genes Notch1 and flag marker protein in hPDLSCs/NICD group was higher than that in the other two groups,indicating that the NICD overexpression vector was successfully constructed.Alizarin red staining showed that compared with two control groups,the mineralized nodules stained by Alizarin red in hPDLSCs/NICD group were small in area,and small in number.Q-PCR results showed that compared with two control groups,the expression level of Notch signaling pathway related molecule Notch1 in hPDLSCs/NICD group was significantlyincreased(P<0.05),but the expression levels of osteogenic marker genes CAP,OCN,RUNX2 in hPDLSCs/NICD group were significantly decreased(P<0.05).Western Blot results showed that compared with two control groups,the protein expression of flag in hPDLSCs/NICD group increased significantly(P<0.05),but the protein expression of RUNX2 decreased significantly(P < 0.05).Conclusion: Overexpression of NICD in Notch's intracellular domain promotes the proliferation of hPDLSCs but inhibits their differentiation and osteogenesis.