Separation And Identification Of Exosomes From STAT3 Gene-modified Mesenchymal Stem Cells Of Rats

Objective:Previous studies found that bone marrow mesenchymal stem cells(MSCs)derived exosomes are rich in STAT3 protein.However,it remains unknown whether MSCs derived exosomes can repair a post-infarct heart through delivering STAT3 protein.Here,we established STAT3-modified exosomes derived from bone marrow MSCs of rats by using STAT3 overexpression and interference lentivirus vector with enhanced green fluorescent protein for preparation to test this hypothesis.Methods: 1.Primary cell isolated culture:after arrow cavities of bilateral femur and tibia from rats washed with culture medium,flushing fluid collected for obtaining MSCs by using a method of adherent culture.2.Flow cytometer: to identify the MSCs with specific surface markers including antibodies of CD29/CD34/CD44/CD45/CD90/CD105.3.Construction of STAT3-modified lentivirus:to build the STAT3-overexpression and siRNA-STAT3 lentivirus vectors with enhanced green fluorescent protein,and packaging STAT3-modified lentivirus with 293 T cells.4.Lentivirus transfection:to transfect the MSCs of rats with STAT3-modified lentivirus at a concentration of 8 MOI.5.Immunofluorescence microscopy:to detect GFP expression in STAT3-modified MSCs by transfected with STAT3-modified lentivirus.6.Exosomes isolate kit: to isolate and acquiring STAT3-modified exosomes after BMSCs treated by serum-free hypoxia culture for 48 hours.7.Western blot:to detect the amount of STAT3 protein and exosomes specific surface markers with specific antibodies of CD9/CD63/CD81.8.Transmission electron microscope:to observe the morphology of those isolated exosomes.Results: 1.Bone primary mesenchymal stem cells extracted is long spindle which have a long protrusions on both ends and uniform distribution of growth.When the cell confluence reach up to 90%,spire-like distribution of cells will be observed.2.The 4th generation BMSCs were analyzed with flow cytometer.The results showed the positive rate of CD29/CD44/CD90/CD105 reach up to 90%.Only extremely infrequent positive expression of CD34 and CD45 were observed.These results indicated that the cells extracted had typical stem cells surface markers and high purity.3.After the BMSCs transfected with STAT3-modified lentivirus,green fluorescence was emmittted from those BMSCs under immunofluorescence microscopy.This result showed that BMSCs were transfected by STAT3-modified lentivirus,successfully.4.After the STAT3-modified BMSCs were treated by serum-free hypoxia culture for 48 hours,the exosomes were extracted and purified with exosomes purification kits according to the instruction,and then saved in the condition of-80 ° C.5.The result from WB showed that the extracted exosomes express CD9/CD63/CD81, which are used to confirm exosomes.6.The result from WB found that BMSCs-exosomes express STAT3.After the BMSCs were modified by overexpression of STAT3,STAT3 expression of the exosomes ncreased remarkably.After the BMSCs were modified by interference expression of TAT3,STAT3 expression of the exosomes was inhibited significantly.7.BMSCs-derived exosomes is round or oval with a diameter of 30-100 nm.The typical ample has a cup bottom groove structure.Conclusion:1.BMSCs-derived exosomes are rich in STAT3 protein.2.After BMSCs modified by overexpression or interference expression of STAT3, TAT3 expression of the exosomes increased and inhibited significantly,respectively.3.STAT3-modified exosomes from BMSCs acquired successfully will be beneficial to ext study on the mechanism that the BMSCs-derived exosomes repair the post-infarct heart.