Experimental Study On The Effects Of Regulatory-T-cells On The Apoptosis Of Effect-T-cells In Sepsis Mice

Objective:This study was Performed cecal ligation and Puncture(CLP) on mice to induce sepsis,regulatory T cells(Treg) and effect T cells(Teff) were isolated by irnrnunomagnetic bead isolate system,then they were co-cultured together to be observed the capacity of the Tregs to suppress the proliferation of Teff,and the capacity of the Tregs to promot the Teffs apoptosis in sepsis miceMethods:(1) mode operation:30 male BALB/C mice,weight ranged from 18 to 22 grams,8 to 10 weeks old,were utilized and assigned randomly to two groups:Sham operated group(Sham) 15,cecal ligation and puncture group(CLP)15.All mice had nothing 12 hours before the operation.For induction of polymicrobial sepsis,mice were subjected to a CLP.In brief,a laparotomy was performed,and the cecum was isolated, ligated below the ileocecal valve,and punctured through and through with a 12-gauge needle.Sham operation was performed by isolating the cecum without ligation or puncture.Closed the abdominal and lml of sterile saline solution was used for fluid resuseitation.Animals were observed for up to 72 hours to determine survival.(2) Cells isolated and co-culture:Spleens were harvested 24 h after CLP or Sham-treatment,regulatory T cells(Treg) and effect T cells(Teff) were isolated by irnrnunomagnetic bead isolate system,then they were co-cultured together,Treg:Teff were placed into 24-well plates or Transwell plate with the ratio that was 1:1.(3) Flow cytometry test:the proliferative capacity of Teff was analyzed confirmed by following the dilution of carboxyfluoroscein succinimidyl ester(CFSE) at 60^th hour by flow cytometry and the apoptotic rates of Teff were analyzed by FCM used CFSE,allophycocyanin(APC)-Annexin and 7-amino-aetinomycin-D(7-AAD) at 60^th hour.Results:(1) Affirmation of the succeeded CLP model:After operation,But Sham group had the same appearance before or after operation.No mice were dead in Sham group.In CLP group,the mice in CLP group appearanced pressed,drowsiness,clumsy,loss of appetite and so on,the survival ratio was 67%in 72 hours.(2) The result showed that in the cultured cells,the percentage of the Teff cells marked by CFSE in the CLP group was(54.89±2.97)%,in Sham group was (73.60±2.10)%;the cells Transwell cultured cells the percentage of the Teff cells marked by CFSE,in the CLP group was(92.01±1.74)%,in Sham group was (93.25±1.27)%.Compared to the cells in the Sham group,Teff cells in the CLP group has a higher percentage(p＜0.05),showed that Treg cells in the CLP group has a stronger inhibitory activity.Compared to the cells co-cultured,the cells cultured in Transwell plate had a higher percentage(p＜0.05),and there was no significant difference between the two group that the cells separated culture in Transwell plate(p＞0.05),this showed that Treg surprised the proliferation of Teff cell mainly through the cells-cells in direct contact means.(3) The result showed that the apoptosis rate of the co-cultured cells in the CLP group was(38.13±2.55)%,in the Sham group was(15.36±2.42)%;apoptosis rate of the cells separated culture cells in the CLP group was(0.36±0.12)%,in the Sham group was(0.35±0.13)%.The result showed that the apoptosis rate of the co-cultured was higher than Treg in the CLP(P＜0.05),Treg in the CLP has a stronger role in promoting Teff apoptosis.But the cells separated culture in Transwell plate with the lower apoptosis rate compared to the cells co-cultured(p＜0.05),and there was no significant difference between the two group that the cells separated cultured in Transwell plate(p＞0.05),this showed that Treg promoted the Teff apoptosis mainly through the cells-cells in direct contact means.Conclusion:(1) The mouse model by sepsis can be established successfully by the method CLP for experiental observation.The CD4⁺CD25⁺Tregs isolated by magnetic beads were pure and suitable for the subsequent experiments.(2) The Tregs had a stronger inhibitory activity to suppress the proliferation of Teff in sepsis mice mainly through the cells-cells direct contact means. (3) The Tregs had a stronger capacity to promot the Teffs apoptosis in sepsis mice with the cells-cells direct contact means.