Studies Of Biological Significance And Regulation Of PDL2Expressed On Human Placenta Derived Mesenchymal Stem Cells

I. The biological significance of PDL2expressed on human placenta derived mesenchymal stem cellsObjective:To investigate the effects of PDL2, expressed on human placenta mesenchymal stem cells (hPMSCs), on the adhesion, migration capacity and the immunoregulation on T cells of hPMSCs, via silencing the expression of PDL2by siRNA.Methods:(1) Cells were isolated from healthy and term human placenta by the method of digestion, cultured in vitro and used after the third passage. The morphology of hPMSCs was observed under the inverted microscope, the surface markers on the hPMSCs were identified by flow cytometry (FCM) and the cells were induced to differentiate into osteoplast, adipose cells and neural like cells.(2) The expression of PDL2was detected by reverse transcription polymerase chain reaction (RT-PCR), laser scanning confocal microscopy (LSCM) and FCM.(3) Specific siRNA of PDL2was transfected into hPMSCs via liposome method. Then the expression level of mRNA and protein of PDL2was detected by RT-PCR and FCM respectively, after being transfected for48h.(4) The adhesion rate of hPMSCs was determined by cell count as well as the morphology of hPMSCs was observed after being stained with hematoxylin-eosin (HE); the migration rate of hPMSCs under the culture conditions of DMEM-LG complete medium, DMEM-LG complete medium with SDF-la and hPMSCs culture supernatant were evaluated using a transwell cell culture system.(5) T cells were isolated from healthy human peripheral blood by gradient centrifugation and cultured with hPMSCs, then stimulated with phytohemagglutinin (PHA) or phorbol12-myristate13-acetate (PMA).(6) FCM was used to analyze the expression of the early activation phenotype CD69on T cells and the cell cycle of T cells; the proliferation of T cells was detected by FCM after CFSE staining and IL-17secretion of T cells was assayed by intracellular staining.Results:(1) MSCs derived from human placenta showed plastic adherence and fibroblastic morphology; hPMSCs expressed CD44, CD29, CD105, and CD166, but did not express CD34, CD14, or CD45; in an ex vivo conditional culture system, hPMSCs could be induced to develop into osteoplast, adipose cells and neural like cells.(2) hPMSCs highly expressed PDL2both on mRNA and protein levels; the specific siRNA-FAM could be effectively transfected into hPMSCs and the siRNA of PDL2could efficiently suppress the expression of PDL2on hPMSCs.(3) The difference of hPMSCs adhesion rate between PDL2silenced groups and control group was no statistical significant after0.5h inoculation, but after1h or3h inoculation, the adhesion rate of hPMSCs was higher, in PDL2silenced groups, than that in the control group (p<0.05); Transwell cell culture system assay indicated that hPMSCs migration rate in silenced group was significantly reduced under the conditions of DMEM-LG complete medium, DMEM-LG complete medium (with SDF-1a) or hPMSCs culture supernatant compared with the control group (p<0.01).(4) The expression of the early activation marker CD69of T cells was inhibited in the presence of hPMSCs, but there was no significantly difference in the silenced groups and the control groups; the proliferation of T cells in PDL2silenced groups, which was significantly increased compared with control groups, remained less than that in only PMA stimulated group; compared with the control groups, the number of T cells in G0/G1phase was decreased, while the number of T cells in S phase was increased in the silenced groups; hPMSCs caused a sharp increase of IL-17secretion which was further up-regulated in PDL2silenced groups.Conclusion:(1) hPMSCs highly expressed PDL2;(2) The expression of PDL2on hPMSCs could be efficiently silenced by PDL2siRNA.(3) PDL2took part in the immunoregulation of hPMSCs on T cells proliferation, cell cycle and secretion of cytokine, as well as the adhesion and migration ability of hPMSCs. Ⅱ. The expression and regulation of PDL2on hPMSCsObjective:To investigate the expression of PDL2on different generation hPMSCs, as well as to study the role of IFN-y in regulating the expression of PDL2on hPMSCs.Methods:(1) The expression of PDL2on hPMSCs in different generation was detected by real-time PCR and FCM respectively.(2) RT-PCR and FCM were used to analyzed the expression PDL2on hPMSCs treated with IFN-y or/and INCB018424.(3) The level of phosphorylation of STAT1and STAT3hPMSCs treated with IFN-y or/and INCB018424was determined by Western blot.Results:(1) There was a significant difference of the expression of PDL2both at mRNA and protein level in different generation of hPMSCs, and the highest expression level of mRNA and protein were determined on20th passage and12th passage respectively.(2) The expression of PDL2on hPMSCs was up-regulated after being treated with IFN-y, as well as the level of phosphorylation of STAT1and STAT3was also increased.(3) The expression of PDL2and phosphorylation of STAT1and STAT3in hPMSCs stimulated or unstimulated with IFN-y were decreased after being treated with INCB018424.Conclusion:The expression level of PDL2was variable in different generation, and that could be up-regulated by IFN-y through JAK-STAT signal path way.