| Objective:(1)To explore the activation state of the mTOR signaling pathway in T cells during theprogress of aplastic anemia (AA), evaluate the effect of rapamycin（RAPA） or CTLA4-Igon the expression of the phosphorylated molecules and upstream and downstream negativeregulate factor of T cells in AA.(2) To further clarify the actived mechanisms in T cells ofAA and to guide the search for new treatments and drugs.Methods:(1) To detect the expression of p-Akt, p-Mtor, p-TSC, p-P70S6K, p-S6, p-4EBP1andSirt1in CD3+T cells of37AA patients peripheral blood samples.17peripheral bloodsamples collected from volunteers were used as negative control, leukemia cell line CEMwas used as positive control.(2) In addition, samples of AA patients and CEM above were treated with RAPA for24hours and CTLA4-Ig for72hours and then FCM was used to detect the expression ofp-Akt, p-Mtor, p-TSC, p-P70S6K, p-S6, p-4EBP1and Sirt1in CD3+T cells.(3) To detect the expression of GAS5in CD3+T cells of bone marrow samples of30aplastic anemia patients,20volunteers (as normal controls group) and CEM (as positivecontrol group).(4) In addition, after exposure to RAPA and CTLA-4Ig24h and72h respectively，samples were detected by PCR for the expression of GAS5in CD3+T cells.(5) To compare the difference of p-Akt, p-mTOR, p-TSC, p-P70S6K, p-S6, p-4EBP1, Sirt1between the AA group and the negative control group. Analyse the expression of thephosphorylated molecules, upstream and downstream negative regulate factors before andafter treated with RAPA or CTLA4-Ig to estimate the effect of RAPA and CTLA-4Ig on thepathway of mTOR in AA. Results:(1) The espression of p-Akt, p-TSC, p-mTOR, p-P70S6K, p-S6, p-4EBP1in CD3+T cells ofAA were (1.86±1.51),(2.21±0.21),(1.64±1.28),(1.44±0.31),(1.63±1.04),(8.11±3.89),respectively. The espression of p-Akt, p-TSC, p-mTOR, p-P70S6K, p-S6, p-4EBP1in CD3+Tcells of the control group were (1.11±0.35)ã€(1.60±0.86)ã€(1.11±0.35)ã€(1.12±0.59)ã€(1.01±0.23)ã€(1.32±0.87), respectively. So, the expression of the phosphorylated moleculesin aplastic anemia patients were obviously higher than the control, P<0.05. The espressionof Sirt1and GAS5in CD3+T cells of AA was (0.98±0.07)ã€(2.83±1.52) much lower than thenegative control group(1.68±0.32)ã€(12.43±4.50)，P<0.05.(2) Exposed to RAPA, the espression of p-Akt, p-TSC, p-mTOR, p-P70S6K, p-S6, p-4EBP1in CD3+T cells decreased markedly (P＜0.05) in AA. And the espression of GAS5inCD3+T cells of AA was much higher than before, P<0.05. Through, the espression of Sirt1was no significant different after treated with RAPA, the ratios of Sirt1/p-Akt, Sirt1/p-TSC,Sirt1/p-mTor, Sirt1/p-4EBP1, Sirt1/p-P70S6K were much higher after exposed to RAPA,P<0.05.(3) Treatment with CTLA-4Ig could also cause a significant reduction of theespression of p-Akt, p-TSC, p-mTOR, p-P70S6K, p-S6, p-4EBP1in CD3+T cells in AA. And theespression of Sirt1and GAS5in CD3+T cells of AA was much higher than before, P<0.05.Conclusions:(1) The phosphorylated molecules in the AA group were significantly higher than thenegative control group, but lower than the positive control group. which suggested that themTOR signal pathway in T cells of aplastic anemia was in relative activation, and likely tobe involved in the activation of T lymphocytes in AA group. And the downstream regulatorSirt1and GAS5in AA group was obviously lower than the negative control group, whichsuggested the lack of GAS5and Sirt1further promote the activation of T lymphocytes inAA group.(2) The expression of p-Akt, p-TSC, p-mTOR, p-P70S6K, p-S6, p-4EBP1in CD3+T cells ofthis signal pathway in AA could be suppressed by RAPA or CTLA-4Ig. And the upstreamand downstream negative regulate factors, Sirt1and GAS5were upregulated by RAPA orCTLA-4Ig,which may open a new avenue for immunosuppression treatment of AA. |
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