Experimental Study For The Effects Of GBP2 On The Biological Properties Of Human Umbilical Cord Derived Mesenchymal Stem Cells

Objective:To investigate the effect of GBP2 on the biological properties of human umbilical cord-derived mesenchymal stem cell(hUC-MSC)and its efficacy of treating acute graft-versus-host disease(aGvHD).Contents:1.Construction of an overexpression GBP2 lentiviral vector and transfection of hUC-MSC.2.In vitro experiments to explore the effect of GBP2 on the biological properties and immunomodulatory ability of hUC-MSC.3.Construction of a mouse aGvHD model to investigate the effect of GBP2 on the efficacy of hUC-MSC in the treatment of aGvHD.Methods:1.hUC-MSC were amplified and cultured in vitro.The cell morphology and phenotype were identified by inverted microscopy and flow cytometry.The ability of hUC-MSC to be induced in adipogenic and osteogenic differentiation in vitro was examined.q-PCR was used to detect the expression of key transcription factors for adipogenic and osteogenic differentiation.2.The lentiviral vector for GBP2 overexpression and the control vector were constructed and transfected into hUC-MSC.The transfection efficiency was assessed by fluorescence microscopy and flow cytometry.The stable overexpression of GBP2 in hUC-MSC and(GBP2highhUC-MSC)and control cells(NC)was examined by real-time fluorescence quantitative PCR(q-PCR)and Western blot.Cell morphology and phenotype of hUC-MSC,NC,and GBP2highhUC-MSC were evaluated by Giemsa staining and flow cytometry.3.GBP2highhUC-MSC and NC were induced to adipogenic and osteogenic differentiating in vitro conditions.The adipogenic differentiation ability of the cells was examined by oil red O staining,and the osteogenic differentiation ability of the cells was examined by alkaline phosphatase(ALP)staining.q-PCR was used to detect the expression of key transcription factors for adipogenic and osteogenic differentiation PPAR-γ,ADI,OPN and ALP.The cell cycle,cell proliferation,and migration ability of hUC-MSC,NCs and GBP2highhUC-MSC were determined by flow cytometry,CCK8 method and cell scratch assay,respectively.4.The hUC-MSC,NC,and GBP2highhUC-MSC were co-cultured with mouse T lymphocytes,and the effects of the three MSC on the proliferation ability of T lymphocytes were detected by CFSE staining.Mouse monocytes were isolated and induced to differentiate into macrophages,which were co-cultured with three MSC to induce M1-type macrophages.The proportions of M1-type macrophages was assessed by flow cytometry.5.A mouse aGvHD model was established.hUC-MSC,NC,and GBP2highhUCMSC were injected into the mice by tail vein.The survival status of mice was assessed by monitoring and recording their body weight,survival rate and activity.HE staining was used to evaluate the pathological changes of liver,lung,small intestine and perianal skin.Immunohistochemistry assay and flow cytometry were used to detect M1 type macrophages in mouse liver tissues and peritoneal cavity.Results:1.The cultured hUC-MSC were identified.The results showed that hUC-MSC were visible as fibroblast-like,vortex-shaped,and adherent growth under inverted microscopy.The phenotype of hUC-MSC was examined by flow cytometry,which showed high expression of CD73,CD90,CD 105(>95%)and low expression of CD 14,CD34,CD45(<2%).The results of oil red O staining and alkaline phosphatase(ALP)staining showed that the cells in the induction group had a large number of red lipid droplets and a significant increase in ALP activity.q-PCR assay results showed that the expression of adipogenic and osteogenic differentiation-related transcription factors PPAR-y,ADI,OPN and ALP in the induction group was significantly increased.The above results indicated that the cultured hUC-MSC matched the MSC identification criteria.2.The lentiviral vector of overexpression GBP2 and the control vector were successfully constructed and transfected into the hUC-MSC.The transfection efficiency was examined by fluorescence microscopy and flow cytometry.The results showed that large amount of green fluorescence was observed in the lentivirus overexpressing GBP2 and the control virus transfected hUC-MSC,and the EGFP transfection efficiency of both cells reached over 95%.q-PCR and Western blot results showed that hUC-MSC transfected with the lentiviral vector showed highly expressed GBP2(P<0.001),which indicated that NC and GBP2highhUC-MSC were obtained.The biological properties of the two cells were assessed by Giemsa staining and flow cytometry.The results showed that the cell morphology and cell phenotype of NC and GBP2highhUC-MSC remained unchanged.3.The adipogenic and osteogenic differentiation was induced in NC and GBP2highhUC-MSC,and the results showed that both MSC could be induced to differentiate into the adipocytes and osteocytes in vitro.Both adipogenic and osteogenic key transcription factors were highly expressed.Compared with the NC group,the ALP activity of GBP2highhUC-MSC was significantly increased,and the expression of osteogenic differentiation marker genes OPN and ALP by q-PCR was also significantly higher than that of the NC group(P<0.01),suggesting that overexpression GBP2 could effectively promote osteogenic differentiation of hUC-MSC.4.The hUC-MSC,NC,and GBP2highhUC-MSC were incubated into 6-well plates.The cell cycle,cell proliferation,and migration ability of the three MSC were examined.The results showed that the cell cycle was not altered in any of the three MSC.CCK-8 assay showed that the proliferation ability of GBP2highhUC-MSC was significantly higher than that of hUC-MSC and NC.Cell scratch assay results showed that the migration ability of GBP2highhUC-MSC was also significantly higher than that of hUCMSC and NC.5.hUC-MSC,NC,and GBP2highhUC-MSC were co-cultured with mouse T lymphocytes.The results showed that the proliferation of T cells co-cultured with GBP2highhUC-MSC was significantly enhanced(P<0.01).The expression of the proinflammatory factor IL-17A in T cells was significantly decreased by q-PCR,suggesting that the ability of GBP2highhUC-MSC to inhibit T cell proliferation was diminished.The three MSC were co-cultured with mouse macrophages and induced macrophages to M1 type polarize,and the expression of CD80,a surface marker of M1 type macrophages,was detected by flow cytometry.The results showed that macrophages co-cultured with GBP2highhUC-MSC highly expressed CD80,suggesting that the ability of GBP2highhUC-MSC to inhibit M1 polarization of macrophages was significantly reduced.These results suggest that overexpressing GBP2 could significantly reduce the inhibitory effects of hUC-MSC on T cell proliferation and M1 polarization of macrophages,which in turn affected the immunomodulatory ability of MSC.6.To investigate whether GBP2 affects the efficacy of hUC-MSC in the treatment of aGvHD,we constructed a mouse aGvHD model and treated it by tail vein injection of hUC-MSC,NC,and GBP2highhUC-MSC.aGvHD group was injected with PBS as a positive control group,and normal mice was taken as a negative control group.The results showed that the body weight of mice in all groups except the negative control group decreased significantly and began to increase slowly after one week of treatment,with the GBP2highhUC-MSC group recovering the least body weight compared with hUC-MSC and NC groups,but there was no significant difference in body weight between the groups.After 3 weeks of treatment,the survival rate of mice in the hUCMSC and NC groups was significantly higher than that in the GBP2 highhUC-MSC.The Cook score of the hUC-MSC and NC groups was also significantly lower than that in the GBP2highhUC-MSC groups.The pathological changes of liver,lung,perianal skin and small intestine in each group were detected by HE staining.The results showed that the tissue edema and inflammatory infiltration of mice in the GBP2highhUC-MSC group were significantly higher than those in the hUC-MSC and NC groups.Immunohistochemical assay and flow cytometry were used to detect macrophage M1 polarization in mouse liver tissue and mouse peritoneal macrophages.The results showed that the proportion of macrophage M1 polarization was significantly increased in both the GBP2highhUC-MSC groups.The above results indicated that overexpression of GBP2 could significantly reduce the therapeutic effect of hUC-MSC on aGvHD.Conclusion:Overexpression of GBP2 could effectively promote osteogenic differentiation of MSC,reduced the inhibitory effect of MSC on T-lymphocyte proliferation,and promoted M1-type polarization of macrophages,which in turn reduced the efficacy of MSC in the treatment of aGvHD.