Effect Of HIF-1α Transfection On Adipose Mesenchymal Stem Cells And Isolation And Identification Of Exosomes

Objectives:Adipose tissue of C57BL/6 male mice was isolated to obtain mouse adipose mesenchymal stem cells.HIF-1α lentiviral vectors were constructed and transfected into ADMSCs.The effects of normal oxygen and hypoxia environment on ADMSCs were observed,and exosomes of ADMSCs were isolated and identified.Methods:After abdominal anesthesia,4-week-old C57BL/6 male mice were disinfected and fixed.The ADMSCs were obtained from bilateral groin adipose tissue of the mice through culturing medium,digestion of collagenase,centrifugation,filtration,and recentrifugation.Transfer to the third generation,when the cell fusion degree reached90%,frozen reserve.Defrost quickly.The morphology and growth characteristics of cells were observed by inverted microscope.Special markers on the cell surface were detected by flow cytometry.The lentivirus vector was constructed by a foreign company,and HIF-1α was transfected into the third generation of ADMSCs by Lipofectamine2000 to obtain HIF-1α-overexpressed ADMSCs.The expression of HIF-1α at the protein level after HIF-1α transfection was detected by Western blot.CCK-8method was used to observe the effect of HIF-1α transfection on the viability of ADMSCs in normal oxygen and low oxygen environment.Flow cytometry and Mod Fit software were used to evaluate the effect of HIF-1α transfection on apoptosis rate of ADMSCS in normal oxygen and low oxygen environment.EXOs were isolated and extracted with Exo Quick-TC,and identified by transmission electron microscopy and Western blot.Results:Under the inverted microscope,the obtained ADMSCs showed a long spindle shape with irregular morphology and spiral growth.When the cells were passed to the third generation,the cells showed a short spindle shape with protrusions at both ends,regular morphology and size,with a certain growth direction and high degree of integration.The surface antigens of the 3rd generation ADMSCs were detected by flow cytometry,and CD29,CD44,CD90 and Sca-1 were positive,while CD34 and CD45 were negative.We transfected ADMSCs with HIF-1α and detected HIF-1α protein expression by Western blot.The results showed that HIF-1α protein expression was significantly increased after HIF-1α transfection.When HIF-1α was transfected into ADMSCs,the viability of ADMSCs was not significantly affected by CCK-8 assay.In low oxygen environment,its cell viability has an effect,but the effect is not significant;Flow cytometry and Mod Fit software were used to evaluate the apoptosis rate of cells under normal and hypoxia conditions.EXOs were isolated and extracted with Exo Quick-TC kit.The EXOs showed oval shape and regular morphology under transmission electron microscope.Western blot analysis showed positive expressions of CD63 and CD81 in EXOs.Conclusions:The cell morphology of C57BL/6 mouse adipose mesenchymal stem cells was uniform,the proliferation rate was stable and the degree of fusion was high when cultured in vitro to the third generation.HIF-1α transfected ADMSCs did not affect the cell viability and apoptosis rate in normal oxygen environment.In hypoxic environment,its cell viability was affected,but the effect was not significant,and the apoptosis rate was not affected.In addition,exosomes were isolated and identified successfully.