Effect Of Combined With HADSCs And Salvia Miltiorrhiza On The Survival Rate Of Granule Adipose Tissue Transplantation

Objective:To explore the effect of combined with human adipose-derived stem cells?hADSCs?and Salvia miltiorrhiza on the survival rate of granule adipose tissue transplantation.Methods: Isolation,culture and identification of hADSCs in vitro.Adipose tissue derived from a 25 year old healthy woman.Adipose tissue derived from her belly fat.The centrifugal method was used to purify the fat granules.Removal of impurities from the upper layer of grease and the lower layer.Part of the adipose tissue was used to prepare hADSCs.Another part of the fat particles are purified and then placed in a syringe.We selected 24 female nude mice.They are all 8 weeks old.Body weight?23±3?g.They were randomly divided into four groups?n=6?.Group A: Adipose tissue 0.5ml;Group B: Adipose tissue 0.5ml+hADSCs;Group C:Salvia miltiorrhiza injection(0.5g·kg^-1·d^-1): Adipose tissue 0.5ml;Group D: Salvia miltiorrhiza injection(0.5g·kg^-1·d^-1): Adipose tissue 0.5ml+ h ADSCs.hADSCs: adipose tissue=6×105:0.5ml.Nude mice of group C and group D were injected with the Salvia miltiorrhiza injection.Nude mice of group A and group B were injected with the same dose of normal saline.We transplanted the adipose tissue into the back of each nude mouse.There are two transplant sites on the back of each nude mouse.And the two transplant sites were left and right.Adipose tissue was transplanted into subcutaneous tissue of nude mice.In each group,3 nude mice were randomly sacrificed at fifteenth days.Remove the graft from the back of the nude mice.First of all,the general observation of the specimens.To observe the morphological characteristics of the tissues and cells.The specimens were fixed with 10% formaldehyde and then paraffin embedded and sliced.Make 5 slices of each specimen,1 slice on a HE dyed,another four tickets on a immunohistochemical dyed for counting the number of capillaries.30 days after the surgery,collecting specimens from each group by random,measuring the volume of body fat grafts by hand-made.The specimens were fixed with 10% formaldehyde and then paraffin embedded and sliced.Make 1 slice on a HE dyed of each specimen.Results:1 Isolation,culture and identification of hADSCs in vitro: The cell extracts were cultured in DMEM medium.The cell suspension was placed in a sterile cell culture flask of 75 cm2.4-7 days after culture,we can see a large number of fusiform cells and polygonal cells,the polygonal cells gradually deformed into fusiform cells.A large number of spindle cells appeared 14 days or so,These cells were vortex or bundles.Because there is a small amount of blood vessels and other tissues in the primary culture of adipose tissue,so the culture flask will produce individual red blood cells and other impurity cells,after subculture,the h ADSCs become more and more pure.When the cultivation of 13^-14 days,the original cells can be passaged and achieve 80% fusion.The cell will growth faster after the passage and every 4-5 days could pass 1 generation.2 Flow cytometry assay : The results showed that h ADSCs positive expression of CD29,CD44,CD90,And hADSCs almost does not express CD31,CD45,CD146.It showed that the cultured cells were more purified mesenchymal stem cells.3 Histological observation of specimens: Fifteenth days after transplantation,graft removal showed complete capsule formation,visible capillaries in the membrane surface shape,comparison of several groups of results,new capillaries is the most abundant group D graft,new capillaries are the least in group A,B,C two groups of capillary quantity between A,D between the two groups;thirtieth days after transplantation,the graft volume of the largest group D A group is the smallest,B,C two group size between A and D two groups of size between.4 The number of micro vessels in the specimen section was detected by immunohistochemistry: On the 15 day after operation in each group of fat grafts immunohistochemistry microvessel density values were 22 compared with LSD-t method,A group and B group,A group and C group,A group and D group,B group and D group,the difference was statistically significant between group D and group C?P <0.05?.Between B group and C group,the difference was statistically significant?P> 0.05?,can be considered the highest vascular density in D group,A group was the lowest of microvessel density,vascular density of B group and C group value between A group and D group.5 Effect of Salvia miltiorrhiza combined with h ADSCs on the fat volume of transplanted particles: After 30 days of each volume were 22 fat transplantation compared with LSD-t method,A group and B group,A group and C group,A group and D group,B group and D group,the difference was statistically significant between group D and group C?P <0.05?,while there was no significant difference between B group and C group?P> 0.05?,that fat volume of transplantation group D was the highest,fat volume of transplantation group A the lowest fat volume of transplantation group B and group C value between A group and D group.Conclusion:Salvia miltiorrhiza combined with hADSCs can promote the survival of adipose tissue transplantation,is helpful to the reconstruction of its early vascular system,improve the survival rate of the transplantation of adipose tissue.