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Background Recent research showed that stromal cell-derived factor-1 (SDF-1) plays critical role in mediating inflammation,metastasis of tumour and neovascularization of tumour.However,There is still no report about the research of the effects of SDF-1 on alkali-burn-induced corneal neovascularization (CNV).Objective The aim of this study attempts to investigate the role of SDF-1 and chemokine receptor CXCR4 in alkali-induced corneal neovascularization in mice.Methods Alkali-induced-corneal neovascularization animal models were constructed of in 15 eyes of 15 clean C57/BL mice by placing the filter paper with 1 mol/L NaOH to the central cornea for 10 seconds.The animals were sacrificed and specimens of cornea were obtained in 3,7,14 days after alkali burn of cornea.The expression of SDF-1/CXCR4 protein in the corneal tissues was detected by immunohistochemistry and Western blot,and expression of SDF-1/CXCR4 mRNA was detected by reverse transcription PCR (RT-PCR).Results SDF-1 was absent expressed and CXCR4 was faintly stained in normal cornea.The expression level of SDF-1 and CXCR4 was significantly enhanced in different time points after alkali burn in comparison with normal control mice by RT-PCR(P＜0.05),and the same trend was seen in the expression level of SDF-1 and CXCR4 proteins by Western blot (P＜0.05).The expression level of SDF-1 and CXCR4 in cornea peaked in the seventh day and began to decline in the fourteenth day but was still higher than normal level.Conclusion The study indicates that SDF-1/CXCR4 plays an important role in the formation of corneal new vessel in alkali-burn-induced animal model.
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Objective The fluorescence-activated cell sorting (FACS) technique is a method for the identification and isolation of different cell populations.At present,the special surface marker for limbal stem cells has been not found yet.This study aimed to investigate the application of FACS technique in the research of rabbit limbal stem cells.MethodsCorneal limbal tissue was obtained from New Zealand white rabbits and cultured using the explant culture method in SHEM.Side population cells (SP cells) and non-SP cells were sorted from cultured rabbit limbal epithelium cells by FACS at a excitation wavelength 350 nm,and acquistion length 450 nm (blue light) and 675 nm (red light).The SP cells and non-SP cells were identified by detecting the expression of ABCG2 and K3/K12.The colony-forming efficiency of SP cells and non-SP cells were evaluated by the observation of cellular vitality with trypan blue staining.The percentage of colony formation was calculated as the colony number in various group/200×100%.ResultsIn 48-72 hours after primary culture,limbal epithelial cells migrated from the cultured tissue mass to form the mambrane-like structure and achieved 70%-80% confluence.The cells showed round,polygon and flattened shape.The proportion of SP cells in cultured limbal epithelial cells was 0.22%±0.09% with a colony-forming efficiency of 5.52±0.45% in SP cells and 0.78%±0.73% in non-SP cells,with a statistically significant difference between the two populations (t＝2.17,P＜0.01).After verapamil,an inhibitor of the expression of the ABCG2 protein,was added into the medium,the proportion of SP cells in the cultured limbal epithelial cells declined to 0.04%±0.006%.The SP cells presented a positive immunoresponse for ABCG2 and absence of immunoresponse for K3/K12,but a contradictory staining result was found in non-SP cells.ConclusionFACS can be applied in the research of limbal stem cells.