ABSTRACT
Objective To study and prepare the monoclonal antibody library against human FXYD6 functional region,to screen the hybridoma cell lines secreting the monoclonal antibodies against intracellular or extracellular region of human FXYD6,and to identify the biological function of monoclonal antibody against extracellular domain.Methods FXYD6 functional region recombinant protein which did not contain the transmembrane region was prokaryotically expressed,purified,and FXYD6 recombinant protein was used to immunize BALB/c mice.Then splenocytes after immunization were fused with myeloma cells SP2/0.After several rounds of screening and cloning,the hybridomas which secreted the antibodies against the extracellular domain or the intracellular domain of human FXYD6 were established.The antibody specificity and subtype were identified with indirect ELISA,western blot and immunohistochemistry.The monoclonal antibodies against the extracellular domain which recognized the native conformation were screened with flow cytometry.The antibody against extracellular region was prepared with the ascites revulsion method and purified.The affinity constants were measured with indirect ELISA.The function of extracellular monoclonal antibody was detected by HepG2 cell line with high expression of FXYD6.Results The hybridoma cell library which secreted the monoclonal antibody against extracellular domain or the intracellular domain of human FXYD6 was successfully obtained,and extracellular region monoclonal antibodies with the functional blocking were prepared.Conclusion The prepared anti-human FXYD6 extracellular monoclonal antibodies could inhibit HepG2 cell proliferation.
ABSTRACT
Objective · To investigate the effect of poly (L-lactic acid caprolactone) (PLCL)/gelatin electrospinning on the angiogenesis differentiation of endothelial progenitor cells (EPCs).Methods· Rat bone marrow-derived EPCs were isolated and cultured,then identification was performed.After preparation of PLCL/gelatin blend electrospun scaffold,scanning electron microscopy and water contact angle test were carried out.EPCs were grown on PLCL/gelatin electrospinning and CCK8 was used to detect cell proliferation.The expression of vascular endothelial growth factor (Vegf) and kinases insert region receptor (Kdr) was observed by RT-PCR and the expression of VEGF protein was observed by Western blotting.Results· The density gradient centrifugation combined with differential adherence method could effectively isolate EPCs.PLCL/gelatin electrospun nanofibers were porous,and the hydrophilic properties were favorable for cell adhesion,and EPCs grew well on the scaffold.The expression of Vegfand Kdr gene in PLCL/gelatin group was higher than that in control group (P=0.000),and the expression of VEGF protein was also increased (P=0.000).Conclusion · PLCL/gelatin is an ideal scaffold for tissue engineering,and it can promote the angiogenesis differentiation of EPCs.
ABSTRACT
Objective: To investigate the effect of poly (L-lactic acid caprolactone) (PLCL) /gelatin electrospinning on the angiogenesis differentiation of endothelial progenitor cells (EPCs). Methods: Rat bone marrow-derived EPCs were isolated and cultured, then identification was performed. After preparation of PLCL/gelatin blend electrospun scaffold, scanning electron microscopy and water contact angle test were carried out. EPCs were grown on PLCL/gelatin electrospinning and CCK8 was used to detect cell proliferation. The expression of vascular endothelial growth factor (Vegf ) and kinases insert region receptor (Kdr) was observed by RT-PCR and the expression of VEGF protein was observed by Western blotting. Results: The density gradient centrifugation combined with differential adherence method could effectively isolate EPCs. PLCL/gelatin electrospun nanofibers were porous, and the hydrophilic properties were favorable for cell adhesion, and EPCs grew well on the scaffold. The expression of Vegf and Kdr gene in PLCL/gelatin group was higher than that in control group (P=0.000), and the expression of VEGF protein was also increased (P=0.000). Conclusion: PLCL/gelatin is an ideal scaffold for tissue engineering, and it can promote the angiogenesis differentiation of EPCs.