The Influence Of FK506and Rapamycin On The Proliferation, Migration And Expression Of Associated Protein Of Schwann Cells

Objiective: The aim of this study was to evaluate the influence ofRapamycin on the proliferation，migration，and the expression andactivity of related protein (ERK, p-ERK, NCAM; GAP43) and nerve growthfactor (NGF)of Schwann cells in vitro,compared with FK506. Toinvestigate the possibility of application and related mechanism ofrapamycin in promoting peripheral nerve regeneration, to provide thetheory basis and evidence for the large ring lactone classimmunosuppressive treatment of peripheral nerve injury.Method:1. Schwann cells were grown in DMEM culture medium supplementedwith10%fetal calf serum(FCS)and100u/ml penicillin and100u/mstreptomycin at37℃in a5%CO2incubator. Schwann cells were treatedwith different concentrations of FK506or Rapamycin(1.53nM,6.1nM,24.4nM,97.6nM,390nM,1.56microM,6.25microM,25microM,100microM,400microM) for6h、12h、24h、36h、48h、60h and72h.The effectsof FK506or Rapamycin on the proliferation of Schwann cells were studiedby3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-Tetrazoli-um (MTT)assay.2. The influence of Rapamycin or FK506on the cell cycle of Schwanncells was measured by the flow cytometer.3. Schwann cells were grown inDMEM culture medium streptomycin at37℃in a5%CO2incubator. Schwanncells were treated with different concentrations of Tacrolimus (6.25microM,100microM) or Rapamycin (1.53Nm,25microM).The effectsof FK506or Rapamycin on the migration of Schwann cells were measuredby scrath assay respectively at0h,12h,24h,36h.4. Schwann cells were grown in DMEM culture medium supplemented with10%fetal calf serum(FCS)and100u/ml penicillin and100u/m streptomycin at37℃in a5%CO2incubator. Schwann cells were treated with differentconcentrations of Tacrolimus (6.25microM,100microM) or Rapamycin (1.53Nm,25microM) for48h.Western blot and immunoprecipitation-tyrosinekinase assay were used to study the express and activity of ERK,p-ERK,GAP43,NCAM of Schwann cells.5. The effects of FK506or Rapamycinon the secretion of NGF of Schwann cells were studied by enzyme-linkedimmunosorbent assay (ELISA).Results:1. FK506promotes Schwann cells’ proliferation in adose-dependent way,as the dose increase from1.53nM,the proliferationof Schwann cells more obvious,the best beneficial effect was seen whenthe dose get to6.25microM (p<0.05). When the dose get to400microM, FK506begin to inhibit the proliferation of Schwann cells, Schwann cells growthsignificant decreased compared with other FK506treated group (p<0.006).Rapamycin promotes Schwann cells’ proliferation was different fromTacrolimus,there was not in a dose-dependent way.When the dose wassmall(1.53nM), Rapamycin can promotes Schwann cells’ proliferationmore obvious(p<0.05). As the dose add,the proliferation of Schwann cellswas not dose-dependent increase.,and there were no significantdifference between the Rapamycin treated group. When the dose get to100microM, Rapamycin begin to inhibit the proliferation of Schwann cells,Schwann cells growth significant decreased compared with other Rapamycintreated group (p<0.005).2. Both FK506and Rapamycin can increased Schwann cells’ cellproliferation index and S phase, Further moer FK506increased Schwann cells’ cell proliferation index and S phase more obviously compared withthe Rapamycin treated group (P<0.05).3. Both FK506and Rapamycin can significantly promote Schwann cells’migration compared with the control group (p<0.006). Further moerRapamycin increased Schwann cells’ cell migration more obviouslycompared with the FK506treated group (P<0.05).4.There were no significant difference between the treated group and thecontrol group for the ERK expression of Schwann cells(P>0.05),The sameas p-ERK.Both FK506and Rapamycin treated group can significantly promoteSchwann cells’ NCAM expression compared with the control group(p<0.01).And Rapamycin treated group can significantly promote Schwanncells’ NCAM expression compared with the FK506treated group (p<0.05).Both FK506and Rapamycin treated group can significantly promote Schwanncells’ GAP43expression compared with the control group (p<0.01). AndRapamycin treated group can significantly promote Schwann cells’ GAP43expression compared with the FK506treated group (p<0.05).5. Both FK506-100microM and Rapamycin-1.53nM treated group cansignificantly promote Schwann cells’ NGF secretion compared with othergroup (p<0.05). But there were no significant difference betweenFK506-100microM and Rapamycin-1.53nM treated group.(p>0.05).Conclusion：1. Both FK506and Rapamycin can increase the proliferationof Schwann cells in vitro, and FK506increased Schwann cells’ cellproliferation more obviously compared with the Rapamycin,the optimalconcentration of FK506is6.25microM; the optimal concentration ofRapamycin is1.53nM.2. FK506or Rapamycin can promote the migration of Schwann cells in vitro,And rapamycin’s promoting effect is more obvious.3. FK506and Rapamycin have no effect to the ERK and p-ERK expressionof Schwann cells,but FK506and Rapamycin could increase the expression of NCAM,GAP43and NGF of Schwann cells in vitro, And rapamycin’spromoting effect is more obvious.