Isolation, amplification and identification of human natural CD4⁺CD25⁺ regulatory T cells in vitro / 中南大学学报(医学版)

OBJECTIVE@#To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy.@*METHODS@#Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay.@*RESULTS@#Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner.@*CONCLUSION@#An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.

Serum vitamin D levels in postmenopausal women with type 2 diabetes mellitus / 中南大学学报(医学版)

OBJECTIVE@#To investigate the correlation between serum vitamin D levels and index of glucose and lipid metabolism in postmenopausal women with type 2 diabetes mellitus (T2DM).@*METHODS@#A total of 44 postmenopausal women with T2DM and 41 healthy postmenopausal women were matched with age, body mass index and menopausal duration. The serum vitamin D was detected by enzyme-linked immuno-sorbent assay (ELISA).@*RESULTS@#Compared with the control group, the level of 25(OH)D3 in postmenopausal women with T2DM was lower, with no statistical significance. Multiple regression analysis revealed that only BMI(bj'=-0.372, P<0.05) was independently related to 25(OH)D3 with statistical significance. The percentages of 25(OH)D3 deficiency in all subjects in the control group and in the T2DM group were 84.7%, 80.5%, and 88.6%, respectively. The 25(OH)D3 deficiency in the T2DM group was more prevalent than that in the control group, with no statistical difference (P=0.372). The binary logistic regression analysis showed the serum 25(OH)D3 level was not related to the risk of diabetes.@*CONCLUSION@#Compared with the control group, a lower 25(OH)D3 level and a higher rate of 25(OH)D3 deficiency is found in T2DM subjects. When rectified by BMI, these is no significant difference. In postmenopausal women, hypovitaminosis D is associated with obesity and dyslipidemia, but not with the risk of T2DM.

Role of glucagon-like peptide-1 analogue liraglutide played in the proliferation of CD4+ CD25-T cells in normal people and type 1 diabetic patients in vitro / 中华内分泌代谢杂志

Objective To study the role of glucagon-like peptide-1 (GLP-1) analogue liraglutide played in the proliferation of CD4+CD25 T cells in normal people and newly-onset type 1 diabetic patients,and to evaluate the possible immune regulatory role of liraglutide in the therapy of type 1 diabetes.Methods CD4+ CD25-T cells of 10 normal people and 10 newly-onset type 1 diabetic patients were separated from peripheral blood by MACS immunomagnetic beads and stimulated by Human T-Activator CD3/CD28 Dynabeads to proliferate.CFSE labeling technique was used to evaluate the proliferation of CD4+ CD25-T cells by flow cytometry.Liraglutide of different concentrations(0,25,50,and 100 nmol/ml) was added to the proliferation system,then the proliferation of CD4+CD25-T cell was measured.Results (1) Liraglutide suppressed the proliferation of CD4+ CD25-T cells from either normal people or type 1 diahetic patients with dose-dependent manner (P ＜ 0.05).(2) Under the different concentrationsofliraglutide,the proliferation ofCD4+CD25 T cells from diabetic patients was mueh more robust than that of normal people (P＜0.01).(3) The inhibitory effects of liraglutide on CD4+ CD25-T cells proliferation in normal people and diabetic patients were similar (P＞0.05).Conclusion The proliferation of CD4+ CD25 T cells in type 1 diabetic patients was more robust than normal people,which indicated cellular immune dysfunction in type 1diabetes.Liraglutide inhibits the proliferation of CD4+ CD25-T cells of type 1 diabetic patients in vitro.The immunosuppression effect of liraglutide may have potential value in the treatment of type 1 diabetes.