| Part1.The cell culture and characteristic of HUVEC and EPCObjective:To study the isolation, culture and identification of human umbilical vein endothelial cells (HUVECs) in vitro from umbilical vein and to establish a method of endothelial progenitor cells (EPCs) isolated from human umbilical cord blood in vitro, and to provide a sufficient amount of source for endothelial and endothelial progenitor cell experimental study.Methods:HUVECs were isolated from umbilical cord by enzyme digestion and were cultured in vitro. Immunohistochemistry were performed to analyze the expression of CD31, CD34, vWF and a-SMA. Immunofluorescence was performed to detect dual binding of ac-LDL and UEA-I. RT-PCR, Western blot were performed to detect the expression of D31, CD34, vWF, a-SMA. Tube formation assay was performed to test the tube formation ability of primate-cultured HUVEC. Isolated mononuclear cells from human umbilical cord blood with density gradient centrifugation, and cultured in EGM-2medium. EPCs were identificatd by immunofluorescence, RT-PCR and Western blot.Results:Cultured HUVECs were displayed cobble-stone morphology, which were positively stained for CD31, CD34, vWF, but negative for a-SMA. Cultured HUVECs could endocytose ac-LDL and UEA-1, RT-PCT and Western blot showed that primate cultureed HUVEC could express CD31, CD34, vWF, but negative for a-SMA.Tube formation assay indicated that primate cultured HUVEC could form tube-like structure on rat tail collagen type I. The mononuclear cells cultured in EGM-2differentiated into EPCs7-14days later. The rate of CD31immunofluorescence staining was97.52±1.49%, the rate of CD34immunohistochemical staining was20.9±2.47%, the rate of CD45immunohistochemical staining was0.27±0.10%, the rate of CD14immunohistochemical staining was0.15±0.08%, the rate of CD105immunohistochemical staining was99.26±3.24%, the rate of ALDH immunohistochemical staining was43.75±1.52%, the rate of KDR immunohistochemical staining was79.09±2.32%, the rate of vWF immunohistochemical staining was98.17±0.47%, the rate of CD146immunohistochemical staining was96.23±3.40%. Cultured EPC could endocytose ac-LDL and UEA-1, RT-PCT and Western blot showed that primate cultureed HUVEC could express CD31, CD34, CD105, ALDH, KDR, vWF, CD146, but negative for CD14, CD45,a-SMA. Single clone formation assay showed that EPC had hight proliferation ability.Conclusion:We can obtain a sufficient amount of HUVECs and EPCs using this culture method. Part2. The effectof propranolol on HUVEC, EPC and EOMA.Objective:To explore the effect of propranolol on the HUVEC, EPC and EOMA.Methods:The murine hemangioendothelioma endothelial cells (EOMA), human umbilical vein endothelial cells (HUVECs), and endothelial progenitor cells (EPCs) were treated with propranolol at different concentrations and time. Cell proliferation was assessed by MTT assay, cell migration was determined by wound healing and Boyden’s chamber assay, and tube formation was performed by seeding those cells on rat-tail collagen type I coated24-well plate and treated with propranolol for24h. The expression of VEGF, FGF-2, MMP-2, MMP-9, CXCR4and eNOS were measured by reverse transcriptase PCR and Western blot. Results:Propranolol inhibits angiogenesis by suppressing the migration and tube formation of EOMA in a dose and time-dependent manner without affecting cell viability. It also inhibits vasculogesis by suppressing the migration and tube formation of EPCs. Propranolol inhibits angiogenesis by suppressing the migration and tube formation of HUVECs in24h. These phenomena were accompanied by inhibiting the expression of VEGF, MMP-9, eNOS and CXCR4in EOMA and EPC, but only VEGF and MMP-9were down regulated in HUVEC.When HUVECs exposed to propranolol for a longer time, propranolol could induce apoptosis of HUVECs by up regulating the expression of Bax and Caspase-3and down regulating the pression of Bcl-2.Conclusion:Propranolol inhibits angiogenesis and vasculogenesis by inhibiting the expression of VEGF, MMP-9, eNOS and CXCR4. Part3. The anti-angiogenesis and anti-vasculargenesis molecular mechanism of propranolol.Objective:To explore the anti-angiogenesis and anti-vasculargenesis molecular mechanism of propranolol.Methods:The murine hemangioendothelioma endothelial cells (EOMA), human umbilical vein endothelial cells (HUVECs) and endothelial progenitor cells (EPCs) were treated with propranolol at different concentrations for24h, the expresses of Akt, ERK, phosphorylated Akt, phosphorylated ERK were measured by Western blot. To explore the mechanism, the Akt kinase inhibitor LY294002and MEK kinase inhibitor PD98059were used to counteract the activation of Akt and ERK pathways, respectively. The expresses of VEGF, CXCR4, MMP-9and eNOS were measured by Western blot.Results:Propranolol inhibits the expression of Akt, p-Akt, ERK, p-ERK in a dose and time dependent manner. LY294002and PD98059inhibits the expression of VEGF, CXCR4, MMP-9, and eNOS in EOMA and EPC, also inhibits the expression of VEGF and MMP-9in HUVEC. Combined with propranolol further inhibits the expression of VEGF, CXCR4MMP-9and eNOS in EOMA and EPC and inhibits the expression of VEGF and MMP-9in HUVEC. These phenomena were accompanied by deactivating the Akt and ERK pathways.Conclusions:Propranolol inhibits angiogenesis and vasculogenesis by down regulating the expression of VEGF, MMP-9, CXCR4, eNOS via dual suppressing Akt and MAPK signaling. |
PDF Full Text Request