| ObjectTo investigate the efficacy and mechanism of POSTN in regulating the immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells（hUC-MSC）and its therapeutic effect of acute graft-versus-host disease（aGvHD）.Contents1.Construction of sh-POSTN lentiviral vector for hUC-MSC transfection.2.In vitro experiments to explore POSTN in regulating the general biological characteristics and immunomodulatory effects of hUC-MSC.3.To explore the efficacy and mechanism of POSTN in regulating hUC-MSC for the treatment of aGvHD using mouse aGvHD model.Method1.hUC-MSCs were cultured in vitro.Stable knockdown POSTN（sh-POSTN）lentiviral vector and control（sh-NC）lentiviral vector were constructed and employed to transfect 293T cells.Inverted fluorescence microscope and flow cytometry were applied to detect the efficiency of lentivirus transfection.The expression level of POSTN in transfected cells was measured by4 real-time fluorescence quantitative PCR（q-PCR）.Stable knockdown POSTN（sh-POSTN）lentiviral vector and control （sh-NC）lentiviral vector were selected and utilized to transfect hUC-MSCs.The transfected hUC-MSCs were amplified and cultured.Giemsa staining and flow cytometry were used to compare the cell morphology and cell phenotype of hUC-MSC,sh-POSTN-MSC,and sh-POSTN-MSC.2.sh-POSTN-MSC and sh-NC-MSC were induced for adipogenetic and osteogenic differentiation.The number of lipid droplet and the expression level of alkaline phosphatase were detected by oil red O staining and alkaline phosphatase staining,respectively.During the process of adipogenetic and osteogenic differentiation,q-PCR was used to measure the expression levels of key transcription factors PPARγ,ADI,ALP,and OPN,while flow cytometry was used to analyze the cell cycle of the two groups of cells.The proliferation ability of the cells was qualified by CCK-8 method.3.Mouse T lymphocytes were co-cultured with hUC-MSC,sh-NC-MSC,and sh-POSTN-MSC in vitro.The proliferation ability of the co-culture system was detected by CFSE staining.After co-culture,the expression level of inflammatory factors TNF-α,IFN-γ,Foxp3,IL-2,and IL-17 and apoptosis-related genes PARP-1,CASP3（Caspase-3）,AIFM1 in T lymphocytes were identified by q-PCR;apoptosis of T lymphocytes was detected by Anniex V/PI staining.CMTMR staining was used to compare the adhesion ability of each group of cells to T lymphocytes.Furthermore,the expression of CCL2,CCL5,CXCL6,CXCL8,CXCL9,CXCL10,CXCL11,and IDO in each group of cells was detected by q-PCR.4.Mouse model of aGvHD was established using Co60γirradiation,and treated with UC-MSC,sh-POSTN-MSC,sh-NC-MSC and PBS via tail vein infusion espectively 48h later.The general physiological state was assessed by observing and ecording the body weight,survival rate,and Cook score of clinical manifestations of he treated mice.HE staining was used to compare the pathological changes and nflammatory cell infiltration in the liver,lung,small intestine and perianal skin of ach group of mice.The expression of inflammatory factors such as TGF-β,IFN-γ,IL-6 and Foxp3 in the spleen of each group of mice was measured by q-PCR.Result1.We successfully constructed sh-POSTN and control sh-NC lentiviral vectors and ransfected 293T cells.Green fluorescent cells could be observed under inverted luorescence microscopy,and the positive rate of the greens cells was>90% easured by flow cytometry.hUC-MSCs were further transfected with sh-POSTN r sh-NC lentivirus.The transfection rate was>90%,measured by fluorescence icroscopy observation combined with flow cytometry.q-PCR results showed that he expression of POSTN in the transfected cells was significantly lower than that in he control cells.Western Blot results confirmed that the protein expression level of OSTN was also significantly reduced compared with sh-NC-MSC.2.To investigate whether POSTN affects the general biological properties of h UC-MSC,we cultured sh-POSTN-MSC and sh-NC-MSC in vitro and characterized them in terms of cell morphology,cell phenotype,and induced differentiation ability.The results showed that the cultured cells showed fibrous-like long spindle shape and whirling growth.The flow cytometry results showed that the cells have high expression of mesenchymal cell surface markers CD73,CD90,CD105,low or no expression of monocyte surface markers CD14 and hematopoietic cell surface markers CD34,CD45.In vitro adipogenetic and osteogenic differentiation results showed that showed a large number of red lipid droplets presented after oil red O staining in the cells,while the cells turned blue-purple after alkaline phosphatase staining.The expressions of the adipogenetic differentiation key transcription factors PPARγ,ADI and osteogenic differentiation key transcription factors OPN and ALP were all significantly up-regulated detected by q-PCR,confirming that all cultured MSCs had the adipogenetic and osteogenic differentiation,but this ability was significantly downregulated in sh-POSTN-MSC,suggesting that POSTN affects the osteogenic differentiation of MSCs.Furthermore,the cell cycle of cultured cells was analyzed by flow cytometry,and the results showed that the G0/G1 phase ratio of sh-POSTN-MSC was relatively down-regulated;the proliferation ability of the cells was also significantly reduced than that of the control group,showed by CCK-8 results（P<0.05）.These results confirmed that sh-POSTN-MSC and sh-NC-MSC had the general biological characteristics of MSC,but POSTN affected the osteogenic ifferentiation and cell proliferation ability of h UC-MSC.3.To investigate whether POSTN affects the immunomodulatory effect of MSC,we co-cultured sh-POSTN-MSC and sh-NC-MSC with mice T cells and measured the proliferation ability of T cells by flow cytometry.sh-POSTN-MSC group showed a significantly higher proliferation ability of T cells（65.1±7.36%）compared with sh-NC-MSC group（49.6± 4.12%）and h UC-MSC group（46.7±6.14%）（P<0.001）,suggesting that the ability of sh-POSTN-MSC to inhibit T cell proliferation was significantly reduced.The expression level of related inflammatory factors was detected by q-PCR,and its results showed that only the expression of TNF-α in T cells co-cultured with sh-POSTN-MSC was significantly up-regulated compared with the control group（P<0.05）.The apoptosis of T cells in the sh-POSTN-MSC group was significantly lower than that in the h UC-MSC and sh-NC-MSC groups（P<0.001）.Adhesion assay results proved that the number of T cell adhesion in the sh-POSTN-MSC group was significantly lower than that in the h UC-MSC and sh-NC-MSC groups（P<0.05）.The q-PCR detection results of the expression of related immune regulatory factors showed that the expression of CXCL10 in the sh-POSTN-MSC group was significantly down-regulated compared with the control group（P<0.05）,indicating that its chemotaxis ability to T cells was reduced.4.To investigate the efficacy and mechanism of POSTN-regulated MSCs in the treatment of a Gv HD,we applied them mouse a Gv HD models.sh-POSTN-MSC,sh-NC-MSC,h UC-MSC,and PBS were infused into each group of mice for treatment.The body weight of mice in all groups decreased first and then increased slowly,while the body weight of mice in h UC-MSC group,sh-NC-MSC group,and sh-POSTN-MSC groups were significantly higher than those in the PBS group.Also,the body weight of mice in h UC-MSC group and sh-NC-MSC groups were higher than that of mice in sh-POSTN-MSC group,but the weight difference was not large enough to have statistical significance.From the 21 st day after treatment,the survival rate of mice in the sh-POSTN-MSC group was significantly lower than that in the h UC-MSC and sh-NC-MSC groups,but significantly higher than that in the PBS group.At the 14 th and 21 st day after treatment,the clinical Cook score of mice in the sh-POSTN-MSC group were significantly higher than those in the h UC-MSC and sh-NC-MSC groups,but significantly lower than those in the PBS group,suggesting that the general survival state of mice in the sh-POSTN-MSC group was worse than the others.HE staining was employed to detect the pathological changes of mice in each group and showed that mice in the sh-POSTN-MSC group had more severe pathological damage and inflammatory cell infiltration in the liver,lung,small intestine and perianal skin compared with those in the h UC-MSC and sh-NC-MSC groups.Preliminary mechanism exploration results showed that the expression level of TGF-β in spleen T lymphocytes of mice in sh-POSTN-MSC group was significantly decreased compared with other groups.Conclusion POSTN affects the osteogenic differentiation ability of h UC-MSC and the proliferation ability of T lymphocytes,which may influence the efficacy of h UC-MSC in treating a Gv HD in mice by regulating the chemotaxis and adhesion of T cells. |
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