The Effection And Machanism Of HASCs And HASCs-CM In Burned Rattus Treatment

Obejective:To analyze the effection and machanisms of burn treatment by humanadipose-derived stem cells and conditioned medium(ASCs-CM) in rats.Methods:(1) After digesting subcutaneous fat tissues with collagenase P, cells werecultured with medium containing DMEM&10%FBS, cell growth curves were drawnto evaluate the population doubling times, characteristics of ASCs were identified byflow cytometry; when induced into adipocytic, chondrogenic, Osteoblastic lineage,Oil red O, Alcian blue and Alizarin red staining were conducted respectively. Theexpression of Oct4and Sox2in P1, P2, P5, P6ASCs were detected byimmunofluorescence.(2) ASCs were cultured under different conditions including normal, hypercapnicenvironment and10ug/L TGF-β1, then ASCs-CM were collected and tested byELISA to detect VEGF, bFGF, IGF, EGF. Results were analyzed by One-WayANOVA.(3)After digesting with collagenase P, rat skin cells were cultured in mediumcontaining H-DMEM&10%FBS.(4) Full-thickness skirn burn model was made on the selected adult rats and wererandomly divided into saline control group, DFs group, hASCs-CM group and hASCsgroup. The groups were observed dynamically at different phases by grossobservation and HE staining.(5) We used anti-mitochondrial antibody to test human-mitochondria expression,then evaluate distribution of hASCs in rat tissue damage. The groups were observeddynamically at different phases by Immunohistochemical test of collagens andMMPs. The datas of OD values were analysed by One-Way ANOVA.Results:(1) ASCs were spindle-like and passaged stably, population doubling time were31h. The phenotype feature of ASCs were CD29+, CD44+,CD105+, CD34-, CD45-.After induction, ASCs could differentiate into adipocytes, chondrogenic cells andosteoblast. P1and P2ASCs expressed Oct4in the nucleus, but the expression of Oct4in P5, P6ASCs were located in the cytoplasm; Sox2were expressed in cytoplasm ofP1and P2ASCs, but hardly expressed in P5and P6ASCs. (2) The hypercapnic environment and TGF-β1conditions of ASCs-CM hadhigher level of the growth factors VEGF, EGF, bFGF and IGF-1.(3) After digesting with collagenase P, rat back skin fibroblast cells werecultured with medium containing DMEM&10%FBS, Cells were short spindle,polygonal or irregular shape. After2~3d DF cells increased rapidly into75%-90%confluence.(4) Compared with control groups, DFs, ASCs-CM and ASCs promoted skinrepair significantly, ASCs groups showed better effection than ASCs-CM, such asmore matured granulation tissue, less capillary and clearer skin structure layer.These results suggest ASCs were able to promote burn repair effectively.(5) Human ASCs were detectedmainly in the wound space. After14d and21d,the expression of collagen type III in ASCs group were higher than other groups,but there were no statistically signifant difference at7d. Similarly, The expression ofMMP1in ASCs group was higher than other groups after14d and21d, but showedno statistically signifant difference at7d. Collagen type Ⅳ were expressed at lowlevels in different groups, but there was no statistically significant difference. MMP9were not detected in all groups.Conclusion:(1) ASCs have some common features of mesenchymal stem cells and thepotential of multi-leneage differention. ASCs express pluripotent markers Oct4andSox2in different position, reflecting that ASCs of different passages have differentpotential.(2) The hypercapnic environment has promote paracrine effect of ASCs, andTGF-β1affects ASCs more apparently.(3) ASCs groups showed best effection, such as more matured granulation tissue,less capillary and clearer skin structure layer. These results suggest ASCs were ableto promote burn repair effectively.(4) As the expression of collagen type III and MMP1were elevated, and therelations of collagen type Ⅰ and collagen type III in scarring were reversed, ASCscould promote the restoration of burn skin by regulate collagen type balance throughhigher MMP1expression.(5) In burn repair, since collagen type Ⅳ has an important role in reepidermaland scarring, MMP9and collagen type Ⅳ may promote burn healing in longer repair.So we should prolong the observation time to evaluate the effections of MMP9inhASCs repairing.