| Objective:To investigate the mechanisms of protective effects of bone marrow mesenchymal stem cells(BMSCs)on hypoxic injuries of human brain microvascular endothelial cells(HBMECs)via urokinase-type plasminogen activator(uPA)and uPA receptor(uPAR)mediated stromal cell-derived factor-1α(SDF-1α)and chemokine C-X-C motif receptor-4(CXCR4)signaling pathways by the cell experiments in vitro.Methods:To simulate in vitro ischemic stroke,HBMECs were sequentially deoxygenated,glucose-deprived,and reoxygenated to construct the Oxygen and Glucose Deprivation followed by Re-oxygenation(OCG/R)model,which was subsequently co-cultured with BMSCs.1.To verify the protective effects of the intervention of BMSCs on hypoxic injuries of HBMECs:(1)Groups arrangement: HBMECs subcultured without intervention comprised the control group,HBMECs treated with OGD/R comprised the OGD/R group,and HBMECs treated with OGD/R and co-cultured with BMSCs thereafter comprised the OGD/R+BMSCs group.(2)Performing flow cytometry to detect apoptosis,MTT test to evaluate cell viability,Brd U test to detect cell proliferation,and tube formation assay to detect angiogenesis.2.To verify the effects of knockout of uPA on certain indices of HBMECs:(1)Implementing Western blot to detect the protein expression and RT-q PCR to detect the gene expression of uPA and uPAR in the control group,the OGD/R group,and the OGD/T+BMSCs group.(2)HBMECs sequentially treated with OGD/R,co-cultured with BMSCs,and transfected with sh-NC as the knockout control group(OGD/R+BMSCs+sh-NC group),HBMECs sequentially treated with OGD/R,co-cultured with BMSCs,and transfected with sh-uPA to knock out the expression of uPA as the uPA knockout group(OGD/R+BMSCs+sh-uPA group).Western blot was conducted to detect the protein expression and RT-q PCR was performed to detect the gene expression of uPA and uPAR in the control group,OGD/R group,OGD/R+BMSCs group,OGD/R+BMSCs+sh-NC group,OGD/R+BMSCs+sh-uPA group.(3)Performing flow cytometry to detect apoptosis,MTT test to evaluate cell viability,Brd U test to detect cell proliferation,and tube formation assay to detect angiogenesis in the aforementioned five groups.3.To verify the effects of knockout of SDF-1α on certain indices of HBMECs:(1)Investigate the effects of knockout of uPA on the protein and gene expression of SDF-1 α and CXCR4 of HBMECs.Using Western blot to detect the protein expression and RT-q PCR to detect the gene expression of SDF-1α and CXCR4 in the control group,OGD/R group,OGD/R+BMSCs group,OGD/R+BMSCs+sh-NC group,OGD/R+BMSCs+sh-uPA group.(2)HBMECs were treated with OGD/R,co-cultured with BMSCs,and transfected with sh-SDF-1 α to knock out the expression of SDF-1α as the SDF-1α knockout group(OGD/R+BMSCs+ shSDF-1α group).To assess the effect of knockout of SDF-1α,we implemented Western blot to detect the protein expression and RT-q PCR to detect the gene expression of SDF-1α and CXCR4 in the control group,OGD/R group,OGD/R+BMSCs group,OGD/R+BMSCs+sh-NC group,OGD/R+BMSCs+sh-SDF-1α group.(3)Performing flow cytometry to detect apoptosis,MTT test to evaluate cell viability,Brd U test to detect cell proliferation,and tube formation assay to detect angiogenesis in the aforementioned five groups.4.To verify if uPA/uPAR exert their effects via the SDF-1α/CXCR4 signaling pathway.(1)Using cell transfection to overexpress SDF-1 α in HBMECs to construct the SDF-1α overexpression group(OGD/R+BMSCs+SDF-1α group).Transfected with sh-NC in HBMECs as the knockout negative control,transfecting Vector as overexpression negative control to construct the knockout and overexpression control group(OGD/R+BMSCs+sh-NC+Vector group).Knocking out uPA and overexpressing SDF-1 α to construct the knockout of uPA and overexpression of SDF-1α group(OGD/R+BMSCs+sh-Upa+SDF-1α group).(2)Performing flow cytometry to detect apoptosis,MTT test to evaluate cell viability,Brd U test to detect cell proliferation,and tube formation assay to detect angiogenesis in the OGD/R group,OGD/R+BMSCs group,OGD/R+BMSCs+sh-NC+Vector group,OGD/R+BMSCs+sh-uPA group,OGD/R+BMSCs+SDF-1α group,and OGD/R+BMSCs+sh-uPA+SDF-1 α group.(3)Using Western blot to detect the protein expression and RT-PCR to detect the gene expression of uPA,uPAR,SDF-1α,and CXCR4 in the aforementioned six groups.Results:1.(1)The apoptosis level was significantly increased in the OGD/R group than that in the control group.The apoptosis level was decreased in the OGD/R+BMSCs group compared with the OGD/R group.(2)The cell viability,cell proliferation,and angiogenesis of HBMECs were substantially decreased in the OGD/R group than those in the control group.The cell viability,cell proliferation,and angiogenesis were considerably improved in the OGD/R+BMSCs group compared with those in the the OGD/R group.2.(1)The protein and gene expression of uPA and uPAR were significantly downregulated in the OGD/R group.The protein and gene expression of uPA and uPAR were significantly upregulated in the OGD/R+BMSCs group compared with those in the OGD/R group.The difference between the protein and gene expression of uPA and uPAR in the OGD/R+BMSCs+sh-NC group and those of the OGD/R+BMSCs group was not statistically significant.The protein and gene expression of uPA and uPAR in the OGD/R+BMSCs+sh-uPA group were significantly downregulated compared with the OGD/R+BMSCs+sh-NC group.(2)The difference between the apoptosis level of the OGD/R+BMSCs+sh-NC group and that of the OGD/R+BMSCs group was not statistically significant.The apoptosis level of the OGD/R+BMSCs+sh-uPA group was significantly increased compared with the OGD/R+BMSCs+sh-NC group.(3)The differences between cell viability,cell proliferation,and angiogenesis of the OGD/R+BMSCs+sh-NC group and those of the OGD/R+BMSCs group were not statistically significant.Cell viability,cell proliferation,and angiogenesis of the OGD/R+BMSCs+sh-uPA group were significantly decreased than those of the OGD/R+BMSCs+sh-NC group.3.(1)The protein and gene expression of SDF-1α and CXCR4 of the OGD/R group were significantly downregulated than those of the control group.The protein and gene expression of SDF-1α and CXCR4 of the OGD/R+BMSCs group were were significantly upregulated than the OGD/R group.The differences between the protein and gene expression of SDF-1α and CXCR4 of the OGD/R+BMSCs+sh-NC group and those of the OGD/R+BMSCs group were not statistically significant.The protein and gene expression of SDF-1α and CXCR4 of the OGD/R+BMSCs+sh-uPA group were significantly downregulated than the OGD/R+BMSCs+sh-NC group.The protein and gene expression of SDF-1α and CXCR4 of the OGD/R+ BMSCs+sh-SDF-1α group were significantly downregulated compared with those of the OGD/R+BMSCs+sh-NC group.(2)The apoptosis level between the OGD/R+BMSCs+ sh-NC group and the OGD/R+BMSCs group was not statistically significant.The apoptosis level of the OGD/R+BMSCs+sh-SDF-1α group was significantly increased than that of the OGD/R+BMSCs+sh-NC group.(3)The differences between cell viability,cell proliferation,and angiogenesis of the OGD/R+BMSCs+sh-NC group and the OGD/R+BMSCs group were not statistically significant.The cell viability,cell proliferation,and angiogenesis were significantly decreased in the OGD/R+BMSCs+sh-SDF-1α group than those in the OGD/R+BMSCs+sh-NC group.4.(1)The differences between the protein and gene expression of uPA,uPAR,SDF-1α,and CXCR4 as well as apoptosis,cell viability,cell proliferation,and angiogenesis of the OGD/R+BMSCs+sh-NC+Vector group and those of the OGD/R+BMSCs group were not statistically significant.(2)The protein and gene expression of SDF-1α and CXCR4 of the HBMECs were significantly upregulated;cell viability,cell proliferation,and angiogenesis of the HBMECs were significantly increased;apoptosis was significantly decreased in the OGD/R+BMSCs+SDF-1α group than those in the OGD/R+BMSCs+sh-NC+Vector group.(3)The protein and gene expression of uPA and uPAR were significantly upregulated;cell viability,cell proliferation,and angiogenesis of HBMECs were significantly decreased;and apoptosis was significantly increased in the OGD/R+BMSCs+sh-uPA group than those in the OGD/R+BMSCs+sh-NC+Vector group.(4)The protein and gene expression of uPA,uPAR,SDF-1α,and CXCR4 were significantly downregulated;apoptosis was significantly increased;and cell viability,cell proliferation,and angiogenesis of HBMECs were significantly decreased in the OGD/R+BMSCs+sh-uPA+SDF-1α group than those in the OGD/R+BMSCs+SDF-1α group.(5)The protein and gene expression of SDF-1α and CXCR4 were significantly upregulated;apoptosis was significantly decreased;and cell viability,cell proliferation,and angiogenesis of HBMECs were significantly increased in the OGD/R+BMSCs+sh-uPA+SDF-1α group than those in the OGD/R+BMSCs+sh-uPA group.Conclusions:BMSCs could exert protective effects on OGD/R induced hypoxic injuries of HBMECs via upregulating uPA/uPAR and the SDF-1α/CXCR4 pathway mediated by them. |
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