The Experimental Study Of Bone Mesenchymal Stem Cell Conditioned Medium Promotes The Healing Of Corneal Alkali Burn In Rat

ObjectiveCornea alkali burn is a common and tough problem in clinical recently. The tissue reaction of alkali burn is different in different clinical stages. The treatment of corneal alkali burn including anti-inflammation, inhibiting the immune response and coreal transplant,but the effect is not ideal. At present, many studies have proved that MSCs can promotes corneal repair after alkali burn, but the effect of its paracrine cytokines should be further studied. This study investigated the effect of bone marrow mesenchymal stem cell comditioned medium?MSC-CM? on the cornea repair after alkali burn by establishing a rat corneal alkali burn model.Methods1. Established animal model, a single layer filter paper immersed in 1 mol/L NaOH solution with a diameter of 4mm was attached to the central corneal 10s. The treated group treated with MSC-CM and the control group treated with Phosphate-Buffer Saline?PBS?. The feeding period was 14 days;2. Observed and calculated the corneal epithelial defect area at 12 hours, 24 hours,48 hour, 3 days, 5days, 7 days, 10 days, 14 days after alkali burn by Slit lamp under fluorescein sodium staining; Observed the central corneal opacity and scored at 48 hour, 3 days, 5days, 7 days, 10 days, 14 days after alkali burn;3. Detected the corneal morphological changes and inflammatory cell infiltration by HE staining at 12 hour, 24 hour, 48 hour, 3 days, 7 days, 14 days after alkali burn; Detected the cell proliferation?PCNA?, stroma fibrosis?a-SMA? and cell apoptosis?Caspase-3? by immunohistochemical staining;4. Detect the gene expression of the inflammatory factors ?TNF-?, IL-6, IL-10?, cell proliferation factor?PCNA?, stroma fiberosis factors ?a-SMA, collegan-?,collegan-I?, cell apoptosis factors ?Bax, Bcl₂, Caspase-3? at 12 hour, 24 hour, 48 hour, 3 days, 7 days, 14 days by RT-PCR after alkali burn.Results1. Slit lamp examination: the percentage of epithelial defect area in treated group were less than control group at 24 hour, 48 hour, 3 day, 5day, 7 day after alkali burn, the difference was statistically significant?P<0.05?; The corneal opacity score was less than than control group at 48 hour, 3 day, 5day, 7 day, 10 days after alkali burn, no significant difference between the two groups?P>0.05?;2. HE and Immunohistochemical staining: 7 days after corneal alkali burn, the epithelial cell layers, the epithelial cell nuclear arrange, stroma edema, collegan fiber arrangement in treated group were better than control group; Inflammatory infiltration cells were less than the control group, PCNA positive cells count were more than control group, a-SMA and Caspase-3 positive cells count were less than control group, the difference were statistically significant ?P<0.05?;3. RT-PCR: 7 day after alkali burn the gexpression level of TNF-a ?P<0.01?, Bax?P<0.05?, Caspase-3 ?P<0. 001?,IL-6 ?P<0.05?, a-SMA ?P<0.001?, Collage-??P<0.01?, Bax ?P<0.01? in treated group were lower than control group, the expression level of IL-10 ?P<0.05?, PCNA ?P<0.001?, Collagen-? ?P<0.001?, Bcl₂?P<0.01? were higher than control group, the difference was statistically significan. At 14 days, IL-6 ?P<0.05?, a-SMA ?P<0.001?, Collage-? ?P<0.01? in treated group were lower than control group and the expression level of IL-10?P<0.05?, PCNA ?P<0.001?, Collagen-I ?P<0.001? was still higher than PBS group, the difference was statistically significant.Conclusion1. MSC-CM can promote the corneal epithelial healing and accelerate the healing speed of corneal epithelium;2. MSC-CM can inhibit the inflammatory response, stroma fibrosis and cell apoptosis of corneal after alkali burn, which may be related to the inhibition of gene expression of TNF-?, IL-6, ?-SMA, Collage-?, Bax and Caspase-3 and the promotion of the expression of IL-10, Collagen-I and Bcl₂.