Adipocy-derived Stem Cell Transport MiR-31-5p Promotes The Survival And Mechanism Of Fat Grafting

Objective:1.Study the mechanism of adipose-derived stem cell transport miR-31-5p to promote the survival of fat grafting.2.Cells transfect adipose-derived stem cells to explore the transcription or expression of miR-31-5p to understand its mechanism of action and its impact on organisms.Methods:(1)In vitro cytology experiment I.Adiposite-derived stem cells(ASCs)were cultured,placed in an incubator at37°C,5% CO2,saturated humidity,and replaced with mesenchymal stem cell culture medium after 24 h to remove unwalled cells.Replace the culture medium every 2～3days thereafter.Passage when the cells grow to 80%～90% fusion.Identification of adipose stem cells by flow cytometry.(2)In vitro cytology experiment II.a.Build a packaged eukaryotic expression plasmid against miR-31-5p(chemically synthesized by the company).b.Fat stem cells were transfected with miR-31-5p and divided into three groups:miR-31-5p transfected fat stem cells(group A),empty vector fat stem cells(group B)and fat stem cells(group C).c.Expression of three groups of miR-31-5p was detected by PCR.d.The cell proliferation and migration ability of the three groups were detected by CCK8 and cell migration assay.(3)Animal experimentation.All animal experiments were approved by the Animal Protection Committee of Gannan Medical University.After 7 days of adaptive rearing in the animal room,all animals were randomly divided into miR-31-5p transfected adipose stem cells(group A),empty carrier adipose stem cell group(group B),and adipose stem cell group(group C).Healthy male BALB/c mice were anesthetized by intraperitoneal injection.Adipose tissue was injected into the subcutaneous back tissue of the mice at a dose of0.5m L/mouse,and then 5×106 adipose stem cells were injected into the above groups,with 1 spot for each.Fat stem cells were supplemented once a week in each group to observe the growth of adipose tissue in each group.After 5 weeks of feeding,the mice were killed,and the transplanted adipose tissue masses in each group were stripped away and their sizes were photographed.HE staining: The adipose tissue mass was stained with HE to observe the angiogenesis.Immunohistochemistry: The protein expression of CD31,perilipin,FIH-1,HIF-α and VEGFR2 was detected by immunohistochemistry.The expressions of CD31,FIH-1 and VEGFR2 proteins were detected by WB.Results:3.1 The cell morphology and positive expression of CD29,CD44 and negative expression of CD34 of mouse adipose stem cells.3.2 miR-31-5P was successfully transfected with adipose stem cells.3.3 miR-31-5P promotes adipose stem cell proliferation.3.4 Cell Transwell experiment to detect the migration ability of three groups of cells.3.5 Effect of miR-31-5p on fat graft retention in mouse models.3.6 HE staining to evaluate the survival effect of three groups of fat grafts.3.7 Western blot measured the protein expression of CD31,FIH-1 and VEGFR2 in three groups of grafts.3.8 Immunohistochemical staining of fat grafts in three groups to evaluate the retention effect of grafts.Conclusion:1.miR-31-5p successfully transfected adipose stem cells and promoted the survival of fat grafts.2.miR-31-5p transfected adipose stem cells have better cell proliferation and migration ability than the control group and empty vector group,and miR-31-5p can improve the proliferation ability of adipose stem cells and promote adipocyte cell migration.3.miR-31-5p promotes the transformation of adipose stem cells to normal adipose morphology,which is conducive to the growth of fat grafts to normal branching direction.