Study On The Regulation Of Osteogenic Differentiation And Proliferation Of MSCs By CCN3 And DLL1

Notch signaling pathway plays an important role in biological activities.In recent years,more and more attention has been paid to its role in bone development and regeneration.Current studies have shown that the presence of a secretory ligand called nephroblastoma overexpressed factor(NOV/CCN3)in the ligands of Notch signaling pathway except Jagged1,Jagged2,DLL1,DLL3,DLL4,the regular membrane protein ligands.Previous studies of our research group have showned that DLL1(regular membrane protein ligand)activated Notch signaling can promote osteogenic differentiation of bone morphogenetic proteins(BMPs)induced mesenchymal stem cells(MSCs)through the BMP signaling pathway,while CCN3 inhibits osteogenic differentiation of MSCs.Then,how does CCN3,as a secretory ligand of Notch signal,play different roles with the membrane protein ligand DLL1 respectively,affecting the osteogenic differentiation of MSCs? What are their effects on the proliferation of MSCs? Aiming at the above problems,our study intends to explore the mechanism of CCN3 and DLL1 in regulating the osteogenic differentiation and proliferation of MSCs,and to explore the clinical application potential of CCN3 in bone tissue engineering.Part ?: Objective: To investigate the mechanism of CCN3 and DLL1 in regulating osteogenic differentiation of MSCs.Method: The expression of alkaline phosphatase(ALP),an indicator of early bone formation,was determined by ALP staining;The expressions of osteopontin(OPN),osteocalcin(OCN),runt-related transcription factor 2(Runx2)were detected by immunohistochemistry;The expression of CCN3 was detected by qRT-PCR and ELISA assay;The expression of highly homologous adenovirus E1A-related 300 kDa protein(p300)was detected by qRT-PCR;qRT-PCR and Western Blot were used to detect the expression of Runx2;Acetylation levels of Histone 3(H3)and Histone 3 lysine 9(H3K9)were detected by Western Blot.Result: DLL1 inhibited the expression of CCN3 in extracellular matrix;BMP9 promoted the expression of CCN3;High concentration of CCN3 inhibited osteogenic differentiation,while low concentration of CCN3 promoted osteogenic differentiation of MSCs;CCN3 inhibited the expression of p300,decreased the level of histone acetylation,and inhibited the expression of Runx2,while DLL1 up-regulated the expression of p300,increased the level of histone acetylation,and promoted the expression of Runx2.Conclusion: Notch signaling pathway's two different ligands CCN3(secretory ligand)and DLL1(membrane ligand)interact with BMP9 to influence the level of histone acetylation and synergistically regulate the osteogenic differentiation of MSCs.Different concentrations of CCN3 play different roles.Part ?: Objective: To study the effect and mechanism of CCN3 on MSCs proliferation.Method: The effect of CCN3 on MSCs proliferation was detected by Cell Counting Kit-8(CCK-8)and flow cytometry(FCM);The expressions of Notch pathway classical ligand and receptor,DLL1 and Notch1 were detected by qRT-PCR;The effect of cell density,Notch and MAPK signaling pathways on proliferation promoting effect of CCN3 were detected by CCK-8.Result: High concentration of CCN3 significantly promoted the proliferation of MSCs,while DLL1 had no significant effect on the proliferation of MSCs;The proliferation promoting effect of CCN3 did not decrease with the differentiation of MSCs into osteoblasts;With the increase of cell density,the effect of CCN3 on proliferation was significantly enhanced.Inhibition of Notch and MAPK signaling pathway attenuates the proliferation promoting effect of CCN3.Conclusion: High concentration of CCN3 can significantly promote the cell proliferation of MSCs,which is related to the Notch signaling pathway,which is closely related to cell density,possibly through the MAPK signaling pathway.