Study On The Effect Of Adipose Derived Stem Cells On Corneal Injury Healing And Its Mechanism

PurposeCorneal blindness caused by corneal injury is one of the main causes of blindness in China.For severe comeal injury,corneal transplantation is the most important treatment at present,but the lack of corneal donor and postoperative immune rejection limit the implementation of transplantation.The development of stem cell therapy may provide a new approach.Adipose derived stem cells(ADSCs),as a well-sourced seed cells,have been found to benefit the healing of injured cornea,which might involve the paracrine phenomenon of stem cells.However,the influence and function mechanism are still unknown.This study intends to explore the effect of ADSCs in repairing corneal stroma injury,and conduct a preliminary study on the repair mechanism,analyzing the role of its exosomes.Moreover,a preliminary study on corneal endothelial repair was attempted.MethodsAdipose derived stem cells were isolated from the adipose tissue of the groin of New Zealand white rabbits for culture,and their ability of multidirectional differentiation was evaluated.Corneal stromal cells(CSCs)were isolated from rabbits and co-cultured with ADSCs,and cell proliferation was detected by CCK-8,while cell apoptosis was detected by flow cytometry.The levels of metalloproteinase(MMPs)and collagen-related protein were detected by western blot,so as to evaluate the effect of ADSCs on corneal stromal cells.Exosomes of ADSCs were extracted,and nanoparticle tracking and analysis(NTA)was used to determine the diameter of exosomes.The expression of markers such as CD9,CD81,flotillin-1 and HSP70 was confirmed by flow cytometry and protein imprinting to detect the purity of exosomes.Exosomes were co-cultured with corneal stromal cells,and were measured with CCK-8,Annexin v-FITC/PI,western blot and morphological features to evaluate the effect of exosomes on corneal stromal cells.The effect of CoCl2(0,50,100,150,200,400μm/L)culture at 12h,24h,48h,and 72h on the proliferation capacity of adipocyte stem cells was tested by chemical anoxia method,and appropriate concentration values were selected for treatment.BMP4 and VEGF in different concentration were added to the culture medium to induce ADSCs,and the results were observed.ResultsThe ADSCs isolated from rabbit groins had the ability of multidirectional differentiation.CD90+ cells selected by flow with the immune markers on the surface of stem cells,were more capable of cell proliferation and had a enhanced surface antibody expression.Also,stronger osteogenic and chondrogenic differentiation were found in CD90+ cells than CD90-and unclassified ADSCs.Co-culture of rabbit ADSCs and CSCs can promote the proliferation of corneal stromal cells,inhibit cell apoptosis and maintain the transparency of corneal stromal.I,Ⅱ Ⅲ,Ⅳ,Ⅴ type collagen and Fibronectin protein expression were up-regulated,while MMP1,MMP2,MMP3 protein expression were suppressed,which manifested the promotion of synthesis of extracellular matrix.Exosomes were extracted from ADSCs and validated.The treatment of ADSCs derived exosomes promoted the proliferation and inhibited the apoptosis of corneal stromal cells.MMPs were down-regulated,and the collagen fibers in corneal stromal cells are upregulated.Besides,50μmol/L CoCl2 showed no damage on the proliferation capacity of ADSCs at the treatment time of 12-48h.But only a small number of cells similar to the multilateral morphology of endothelial cells were observed after the combined induction of BMP4 and VEGF.ConclusionAdipose derived stem cells significantly promoted the growth and metastasis of corneal stromal cells,in which paracrine played an important role in the co-culture of ADSCs and CSCs.The extracellular matrix synthesis of corneal stromal cells was accelerated by the ADSCs derived exosomes,which promoted the proliferation and inhibited the apoptosis of corneal stromal cells.The clinical application of exosomes might be superior to the direct application of stem cells,which were more stable,convenient to transport and more efficient.Patients with clinical corneal injury may benefit from this study and further research results.