Sestrin2 Promoting The Angiogenesis Of Prevascularized Cell Sheets Under Hypoxia Microenvironment

Objective:This thesis investigates the effect of Sestrin2(SESN2)on the vascularization ability of prevascularized cell sheets in vitro and in vivo in a hypoxia microenvironment to provide new ideas for successful engineered tissue transplantation.Methods:In this study,HUVECs with stable and high expression of SESN2 were firstly obtained by lentiviral transfection and labeled with green fluorescent protein(GFP).The transfection efficiency and SESN2 protein expression were further examined using fluorescence microscopy and Western-blot(WB).Meanwhile,r BMSCs were extracted by the whole bone marrow apposition method and cultured to the second generation,which were inoculated into six-well plates at a density of 2.0×10~5/hole and replaced with high-sugar DMEM medium containing 10%fetal bovine serum(FBS),1%penicillin-streptomycin solution(PIS),1%L-glutamine,and 50 mg/L ascorbic acid on alternate days,and r BMSCs cell sheets were formed in continuous culture.Next,HUVECs transfected with SESN2(SESN2 group)were inoculated into r BMSCs cell sheets at a density of 5.0×10~4/cm~2 and incubated under normoxia and hypoxia conditions for 1,3 and 7 days,respectively,and then detect by the following methods:Cell arrangement was observed under an inverted fluorescence microscope;Cell counting kit(CCK-8)was used to detect the effect of SESN2 on the viability of HUVECs;CD31 immunofluorescence assay was used to observe vascular network formation;The enzyme linked immunosorbent assay(Elisa)and quantitative reverse transcription-polymerase chain reaction(q RT-PCR)were used to detect vascular endothelial growth factor-A(VEGF-A)and platelet-derived growth factor subunit-BB(PDGF-BB).Finally,the prevascularized cell sheets transfected with SESN2 were transplanted to the subcutis of nude mice,and angiogenesis was detected by gross observation,hematoxylin-eosin staining(HE staining),and CD31 immunofluorescence at 1,7,and 14 days,respectively.Results:(1)After lentivirus transfection,the stably transfected HUVECs with high expression of SESN2 can be successfully obtained,and the transfection efficiency reaches more than 90%;The proliferation experiment of CCK-8 cells showed that the high expression of SESN2 reversed the inhibitory effect of hypoxia on HUVECs proliferation,and slow virus transfection did not affect the cell proliferation.(2)The whole bone marrow apposition method can successfully obtain rabbit bone marrow mesenchymal stem cells,and the flow assay showed 95.5%positive expression of CD29,96.6%positive expression of CD44,0.57%positive expression of CD34,and0.58%positive expression of CD45.(3)High-density inoculation of r BMSCs and continuous culture in a specific medium for 14 days resulted in the formation of cell sheets.(4)CD31 immunofluorescence staining showed that SESN2 significantly promoted endothelial cell rearrangement and vascular network structure in hypoxia environment.High expression of SESN2 significantly increased the release of angiogenesis-related cytokines VEGF-A and PDGF-BB from prevascularized cell sheets,and VEGF-A was released in early angiogenesis,while PDGF-BB was released in late angiogenesis.(5)In vivo experiments showed that high expression of SESN2could promote vascular network formation,and HE staining showed that the network structure pre-constructed in vitro anastomosed with host vessels to form a functional vascular network.Conclusion:SESN2 can effectively improve the adaptability of endothelial cells to the hypoxia microenvironment and promote the formation of vascular network structures of prevascularized cell sheets.