The Effects On Fibroblasts Activity By Co-cultured With Notch Signaling Manipulated Keratinocytes

Objective：To investigate the effects on fibroblasts proliferation, expression of Collagen-1,Fibronectin, KGF, and cytokine, which are co-cultured with keratinocytes occurredNotch signaling.Methods：Use Jagged1-Fc peptide or DAPT pretreat keratinocytes to regulate Notch signalingbefore the process of inducing terminal differentiation. Raising up to the air-liquidinterface and stimulated with serum for3days, keratinocytes are induced intodifferentiation and multiple layer growth. Then, it can be used for co-culturing withfibroblasts. First of all, we use real-time PCR test Notch receptor, paired Notch ligand,downstream genes to find out Notch signaling activated or not after the inducing differentiation. Then, observing he effects on fibroblasts which are co-cultured withkeratinocytes occurred Notch signaling, including cell counting test cell proliferation,real-time PCR test mRNA levels and ELISA test protein levels of the expression infibroblasts of Collagen-1, Fibronectin, KGF, and some cytokine.Results：Use Fc fragment or DMSO pretreat the keratinocytes, the expression of p21and p63,who are the downstream genes of Notch signaling, has no significant differencecompared to Blank control(p>0.05).But using Jagged1-Fc peptide pretreat thekeratinocytes, the expression of Notch1, Jagged1, p21and p63are up-regulation inkeratinocytes, which means Notch signaling is activation. Using DAPT pretreat thekeratinocytes, there have an opposite results: Notch signaling are inhibited compared tothe Blank control. The cell proliferation is enhanced, and the expression of Collagen-1,Fibronectin, KGF and TGF-β1are up-regulated in fibroblasts when co-cultured withkeratinocytes had activation of Notch signaling(p>0.05). Contrast to the fibroblastsWhich co-cultured with keratinocytes had inhibition of Notch signaling, fibroblastsproliferation has no significant decline(p>0.05), but the expression of Collagen-1,Fibronectin, KGF and TGF-β1are down-regulated(p>0.05).Conclusion：Notch signaling may involve into the hyperplasia progress of scarring. Theexpression of Collagen-1, Fibronectin, KGF and TGF-β1can be inhibited in fibroblastswhen co-cultured with keratinocytes blocked Notch signaling. KGF and TGF-β1produced by fibroblasts may involve into this regulation.