Effect Of Mechano Growth Factor On Proliferation Of Human Peripheral Blood Regulatory T Cells In Vitro

Part1 Selection and identification of CD4 + CD25 + CD127-regulatory T cells in peripheral blood?Objective?Sorted high purity of CD4 + CD25 + CD127-regulatory T cells in human peripheral blood to provide the guarantee for the experimental accuracy of subsequent cells.?Methods?20 ml of peripheral blood of healthy volunteers was taken and kept on ice for anticoagulation.The mononuclear cells in peripheral blood were isolated by density gradient centrifugation using lymphocyte separation solution.The resulting mononuclear cells were incubated with antibodies and passed.The CD4+CD25+CD127-regulatory T cells were sorted by immunomagnetic bead sorting.The obtained cells were incubated with antibody stained by flow cytometry and tested for purity by flow cytometry.?Results?The purity of CD4 + CD25 + CD127-regulatory T cells obtained by immunomagnetic bead sorting was above 90%.?Conclusion?Immunomagnetic beads sorting of peripheral blood CD4 + CD25 + CD127-regulatory T cells,resulting the high purity cells,and can meet the follow-up experiments.Part2 Effects of mechano growth factor on the proliferation of regulatory T cells in human peripheral blood?Objective? To investigate the effect of mechano Growth Factor(MGF)on Proliferation of CD4 + CD25 + CD127-regulatory T Cells in Human Peripheral Blood.?Methods?(1)CD4 + CD25 + CD127-regulatory T cells obtained from 20 ml of human peripheral blood were sorted by immunomagnetic beads and then stimulated with CD3,CD28,and IL-2 by labeling them with carboxyfluorescein acetoacetate(CFSE)After the cells were treated with MGF(0n M,10 n M,100 n M)for 6 days,the cells were stained by flow cytometry.(2)Peripheral blood mononuclear cells(PBMC)were isolated from human peripheral blood and stimulated with CD3,CD28 and IL-2.After intervention with different concentrations of MGF(0 n M,10 n M and 100 n M).TREG cells accounted for the proportion in PBMC.?Results?(1)Six days after intervention,in Purified TREG cells,the percentage of second passage cells to total cells after MGF intervention was 49.66% ± 9.86% in the 0n M MGF group,58.66% ± 5.95% in the 10 n M MGF group and 62.6% ± 4.66% in the 100 n M MGF group.The proliferation efficiency of 100 n M MGF group was higher than that of 0n M MGF group,the difference was statistically significant(P <0.05).(2)One day after the intervention,the percentage of TREG cells in PBMCs was 17.82% ± 1.23% in the 0 n M MGF group,20.73% ± 1.61% in the 10 n M MGF group,20.29% ±1.46% in the 100 n M MGF group,10 n M MGF group and 100 n M MGF group The proportion was higher than 0n M MGF group(P <0.05).?Conclusion? Mechano Growth factor(MGF)can promote the proliferation of CD4 + CD25 + CD127-regulatory T cells in human peripheral blood in vitro.Part3 Effect of mechano growth factors on Th17 / Treg ratio in human peripheral blood mononuclear cells?Objective? To investigate the changes of Th17 / Treg cells in peripheral blood mononuclear cells after mechano growth factor intervention in peripheral blood mononuclear cells.?Methods? Peripheral blood of 4 ml healthy volunteers was collected and mononuclear cells were isolated by lymphocyte separation.After being stimulated with CD3,CD28 and IL-2,with different concentrations of MGF(0,10,100 n M)for 1 day,Stained and incubated.The ratio of Th17 to Treg in PBMC was detected by flow cytometry and calculate the Th17 / Treg ratio at different drug concentrations in PBMC.?Results? After 1 day of intervention of PBMCs with different concentrations of mechano growth factor,the proportion of Treg cells in each group accounted for PBMC,0n M MGF group: 21.80% ± 1.02%,10 n M MGF group: 23.55% ±1.16%,100 n M MGF group: 23.28% ± 0.70%;TH17 cells in each group accounted for PBMC ratio,0n M MGF group: 10.04% ± 2.08%,10 n M MGF group: 9.29% ±1.85%,100 n M MGF group: 6.48% ± 1.26%;each group Th17/Treg ratio The proportion of 0n M group was: 0.46±0.10,10 n M group ratio was 0.40±0.09,100 n M group ratio was 0.29±0.06,100 n M group Th17/Treg ratio was significantly lower than 0n M group,the difference was statistically significant(P< 0.05).?Conclusions? After the intervention of peripheral blood PBMC by mechano growth factor MGF,the proportion of Treg in peripheral blood PBMC increased,the ratio of Th17 decreased,and the ratio of Th17/Treg decreased.