Effects Of Wharton's Jelly-derived Mesenchymal Stem Cells From Human Umbilical Cord On Regulatory T Cellsand Foxp3 Gene Of Severe Aplastic Anemia Patients

Objetive:To investigate the effects of Wharton's jelly-derived mesenehymal stem cells (WJCs) of human umbilical cord on regulatory T cells and Foxp3 gene and the immunomodulatory mechanisms of servere aplastic anemia (SAA) patients.Methods:WJCs were isolated and expanded from human umbilical cord by the collagenase digestion, which were identified by the morphology of the cells in culture and the flow cytometry of the stem cell markers; Using density gradient centrifugation, T-lymphocytes (TLCs)were isolated from human peripheral blood of patients with SAA; TLCs (1×105) of patients with SAA were cultured in the absence of phytohemagglutinin(PHA). In the experimental group the WJCs (1×103, 1×104, 1×105) were co-cultured with TLCs for 3 days, and then inhibitory ratio of human UC-MSCs at various concentrations on TLCs was measured by MTT assay; The percentage of CD4+CD25+T cells of patients with SAA was detected by flow cytometry, and the expression of Foxp3 gene was detected by reverse transcription polymerase chain reaction(RT-PCR).Results:(1) WJCs were generated successfully by the collagenase digestion. The third generation of MSCs had typical morphology of fiberblast,and were high expression of CD 166, CD29, CD13,CD44,low expression of CD34, CD45, HLA-DR. (2) WJCs showed inhibitory effect on TLCs from patients with SAA in dose-dependent manner,and their inhibitory rate were 56.2±12.1%,62.9±11.9%,44.8±9.6%,2.64±7.2%. (3) In all of the experiments, the increase of percentage of CD4+CD25+T cells in the peripheral CD4+T cell was observed when TLCs were co-cultured with WJCs,and the percentage were 11.88±1.54%,18.35±2.78%,22.46±3.05%. (4)The level of Foxp3 mRNA in the experimental groups was significantly higher than that in the controls, and was negatively associated with the value of representing TLCs proliferation. The level of Foxp3 mRNA were 1.75±0.21,1.07±0.14,0.63±0.11.Conclusion:WJCs specifically inhibits the proliferation of TLCs of patients with SAA in dose-dependent manner and one of mechanism could be that up-regulated expression of Foxp3 promote the CD4+CD25+regulatory T cells to perform its immunoregulation function.