ABSTRACT
BACKGROUND:Mesenchymal stem cells(MSCs) have immunoregulation,can reduce the occurrence rate of graft versus host disease following transplantation,and treat autoimmune disease. OBJECTIVE:To observe the immunoregulation of human umbilical cord(UC) -MSCs on bone marrow T-lymphocyte of patients with aplastic anemia. DESIGN,TIME AND SETTING:A randomized grouping design,in vitro cytological controlled study. Cases were obtained from the Department of Hematology,First Hospital and Second Hospital Affiliated to Nanchang University. Experiments were conducted at the Institute of Hematology,Second Hospital Affiliated to Nanchang University from September 2007 to November 2008. PARTICIPANTS/MATERIALS:A total of 22 patients with aplastic anemia were enrolled at the Department of Hematology,First Hospital and Second Hospital Affiliated to Nanchang University. There were 6 cases of severe aplastic anemia(3 males and 3 females) ,with a median age of 38(16-68) years;16 cases of chronic aplastic anemia(9 males and 7 females) ,with a median age of 41(25-59) years. Umbilical cord was obtained from healthy full-term fetus by normal uterine-incision delivery for isolation,culture and determination of human UC-MSCs. METHODS:MSCs were extracted from human umbilical cord. T-lymphocytes were isolated from bone marrow of patients with aplastic anemia by density gradient centrifugation. MSCs were cocultured with T-lymphocytes. In the control group,T-lymphocytes were incubated at 1?105/well. In the UC-MSCs group,human UC-MSCs were incubated at various densities of 1?105/well,0.5?105/well,0.1?105/well and 0.05?105/well. In the experimental group,T-lymphocytes were cocultured with UC-MSCs at 1:1,1:0.5,1:0.1,1:0.05. MAIN OUTCOME MEASURES:Inhibitory rate of human UC-MSCs at various concentrations on T-lymphocytes of aplastic anemia patients was measured by MTT assay. CD4+/CD8+ changes of the T cells were analyzed by flow cytometry. Expression of the two marrow hematopoietic factors(interleukin-2 and ?-interferon) in the supernatant was evaluated by ELISA. RESULTS:The second passage of MSCs had typical morphology of fibroblast,and highly expressed CD166,CD29,CD54,but lowly expressed CD13,CD34,CD45. MSCs could be induced into adipocytes. UC-MSCs showed inhibitory effect on lymphocyte proliferation of aplastic anemia patients when UC-MSCs and lymphocytes mixed at the ratio of 1:1,0.5:1,0.1:1,and their inhibitory rate were(56.2?12.1) %,(43.7?10.4) %,(28.6?8.9) %. The effect was the strongest at the ratio of 1:1. UC-MSCs adjusted the proportion of CD4+ and CD8+,inhibited the secretion of interleukin-2 and ?-interferon by T cells of aplastic anemia patients. CONCLUSION:Human UC-MSCs can mediate an immunoregulation effect on T lymphocytes of aplastic anemia patients in vitro and the inhibitory effects are dependent on the amount of UC-MSCs.