Effects Of Rapamycin On Mouse Memory T Cells In Vitro

Effects of rapamycin on mouse memory T cells in vitroPART ONE Proliferation of memory T cells in mice by rejection after skin transplant modelObjective To harvest a stable proliferation of memory T cells by T cell proliferation in C57BL/6 mice with skin transplant model.To prepare for the drug effect on the cell in vitro.Methods We transplanted DBA/2 donors'skin to C57BL/6 recipients. At the same time,transplanted C57BL/6 donors'skin to C57BL/6 hosts as control.The proportion of memory T cells in SPM(spleen mononuclearcell)from recipient C57BL/6 mice was examined by FCM (flow cytometer)from 1 to 8 weeks after skin transplant(n=6).Results(1)Heterologous series of skin transplant group:Compared with the number of memory T cells from 1-3th week,there was a significant increase from 4th week after operation(P＜0.01);as well as in 5-8th week compared with the 4th week after operation(P＜0.01);There was no significant increase in the number of memory T cells in 1-3th(P＞0.05)week after operation as well as in 5-8th week(P＞0.05).(2) Homologous series of skin transplant group:There was no significant increase in the number of memory T cells in 1-8 th week after operation(P＞0.05).Conclusion Memory T cells of mice have its homeostasis proliferation during the exposure to skin allogenic antigen in 4-5 weeks.This model has a stable increased state of memory T cells in mice which could be useful for transplant study.PART TWO Effects ofrapamycin on mouse memory T cells in vitroObjective To investigate the effects of rapamycin on proliferation and levels of interleukin-2(IL-2)of mouse memory T cells in vitro.The sensitive difference to rapamycin was compared in naive T cells and memory T cells of mice mouse naive T cells,and investigate a mechanism of rapamycin on mouse memory T cells.Methods Harvest mononuclearcell from C57BL/6 mouse's spleen,and then separate memory T cells by Magnetic Cell Sorting System.Harvest mononuclearcell from C57BL/6 mouse's spleen,which memory T cells have been induced by frontal experiment.Separate memory T cells by fluorescence-activated cell sorting(FACS).Various concentrations of repamycin(0 ng/ml,10 ng/ml,20 ng/ml,30 ng/ml)were used to culture the memory T cells for 72 hours,the activity of cells and levels of IL-2 were measured by MTT(methylthiazlyltetrazolium)assay and Elisa.At the same time,we detected memory T cells' cell cycle by Flow cytometer cultured with 10ng/ml repamycin stimulation.Finally,the expression of IL-2 receptor(α,β,γsubset)mRNAwas detected by RT-PCR. Results Proliferation of naive and memory T cells and expression of IL-2 were suppressed by rapamycin medium when cultured with rapamycin(P＜0.01).This suppression effect is in a dose-dependent manner(P＜0.01).And at the same concentrations of repamycin, memory T cells are less suppressed compared with naive T cells. (P＜0.01).The cell population increseased in the G₀G₁ phase (P＜0.01)and decreased in the S phase(P＜0.01).The expression of the IL-2R(α,β,γsubset)mRNA were significantly reduced in experimental group.Conclusion In vitro,rapamycin is a potential inhibitor that blocks the proliferation and the expression of IL-2 of both naive and memory T cells,memory T cells are less suppressed compared with naive T cells.Rapamycin inhibit proliferation of memory T cells by blocks the expression of IL-2,IL-2R(α,β,γsubset)mRNA and cell cycle.