An Experimental Study Of Signal Transduction Pathway Of Activated Schwann Cell

IntroductionIt is a difficult problem in peripheral surgery when nerve deficiency especially long distant nerve deficiency meets. The artificial nerve is now one of prospective areas with the development of biomaterials and deeper study of tissue engineer nerve. Collagen-Chitosan might be an ideal material for fabrication artificial nerve and activated Schwann cell might be the ideal seed cell for the bio-active nerve because of its high bio-active and quick proliferation. In our three part of this research we will study the reason why activated Schwann cell perior nomal Schwann cell to boosting the regeneration of injuied nerve.Part 1: To explore molecular composition of activated Schwann cells' signal transduction pathwayObjective To explore molecular composition of activated Schwann cells' signal transduction pathway. Methods Activated Schwann cells and normal Schwann cells were abtained by the way of modified Schwann cell from adult SD rats. 7 signal transduction pathway inhibitors of RTK and GPCR were added into activated Schwann cells and normal Schwann cells for an hour before these cells were collected respectively. Western Blot method was used to check the change of p-ERK1/2 to compare the difference between the activated Schwann cell and normal Schwann cell. Results Both of them were positive stained by S-100．ERK1/2 and p-ERK were also compared in activated Schwann cells and normal Schwann cells. There was no statistic difference of ERK1/2 between the two cells. However p-ERK was remarkable different (p<0．01) . The rising of p-ERK1/2 might be the key reason of Schwann cells activated. In activated Schwann cells, changes of p-ERK1/2 by CTX,D609 and B?PTA-AM were prominent but there was no distinct change using other inhibitors (p<0．01) . However in normal Schwann cells, changes of p-ERK1/2 inhibitor U73122 and BAPTA-AM were notably but there was no distinct change using other inhibitors (p<0．01) . Conclusions Activated Schwann cell and normal Schwann cells is the same cell in two different conditions. The rising of p-ERK1/2 might be the key reason of Schwann cells activated. GPCR was the common signal transduction pathway of Activated Schwann cell and normal Schwann cells. But Activated Schwann cells exert their biologic effect through PC-PLC decompounding choline and boosting level of DAG. However normal Schwann cells exert their biologic effect through PI-PLC signal transduction pathway.Part 2: To explore the activated transcription factors of activated Schwann cell using TranSignal^TM Protein/DNA Array techniquesObjective To explore the activated transcription factors of activated Schwann cell. Methods TranSignal^TM Protein/DNA Array techniques were adopted to test the activated transcription factors. Results Lattice of activated Schwann cells showed that there were 5 different transcription factors doubly raised their expression than that of normal Schwann cells. The rising of AP-1 might be the dominant reason of activated Schwann cells' quick proliferation. NF-Atx takes a cooperation role with AP-1．NRF1, however, may supply the activated Schwann cells with energy with its enzyme mtTFA for its' quick proliferation and vigorous secretion. PO-G exerts its action in DNA repair and replication of activated Schwann cells. Finnally PRDI-BF ties closely up with the special condition of activated Schwann cells. Conclusion Total 5 different transcription factors correlated their level with activated Schwann cells' quick proliferation and vigorous secretion condition .Part 3: To examine the signal transduction passway of activated Schwann cell using the method of dual color fluorescence reporter gene system.Objective To examine the signal transduction of activated Schwann cell using the method of dual color fluorescence reporter gene system. Methods Dual color fluorescence reporter gene system made of pGL3 Luciferase Reporter and phRL-SV40 was adopted and vector contained NGF-promotor.Activated Schwann cells were infected by these vectors. After that Schwann cells were divided into 7 teams. They were: 1．Activated Schwann Cell(ASC), 2．ASC+ 0．8μg pGL3 + 40 ng phRL-SV40, 3．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 4．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 0．5h, 5．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 1h,6．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 1．5h, 7．Cell+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 2h. Results Fluorescence of 3th team was the most intensive in the 7 teams. Its comparative value RLU1/RLU2 was also the biggest. However 0．5h after the usage of signal transduction inhibitor D609 the value of ASC decreased suddenly due to the activating of NGF-promotor was restrained. And pGL3 luciferase could not call into play. But alone with the attenuation of D609, the activated level NGF-promotor increase gradually. And pGL3 luciferase resumed their role playing on their substrate and the fluorescence intensity was also increased gradually. Concision The effect of signal transduction of activated Schwann cells on their target were observed directly through Dual color fluorescence reporter gene system. And our molecule composition of signal transduction pathway of activated Schwann cell were proved right directly.