Experimental Research On Influence Of Rubbit Epithelial Flap In Epi-LASIK For The Expression Of TGF-β₁, BFGF And Apoptosis In Corneal Stroma

Background:It has good safety,efficacy,predictability and stability to accept Epi-LASIK. Compared with LASIK,the patients with Epi-LASIK have a long recovery time,pain and haze.Complications,especially haze,confine the generalization of Epi-LASIK.Corneal wound healing cause haze.Researchs about corneal wound healing indicated:injury of epithelial cells cause apoptosis in corneal stroma,apoptosis is the main factor of influencing corneal wound healing,and cytokines such as TGF-β₁,bFGF are also factors.Another study indicated:division growth of stem cells in limbus corneae is the way of epithelial healing after LASEK.So,epithelial flap maybe result in apoptosis in corneal stroma which cause corneal wound healing.The effect of epithelial flap in Epi-LASIK for corneal wound healing should be further studied. Part 1 Cell Culture and Detection for Reproductive Activity of Epithelial Flap in Epi-LASIKPurpose:To discuss the main way of corneal epithelial wound healing in LASEK and Epi-LASIK.Methods:Twenty human corneal epithelial flaps samples and 12 rabbit coneal epithelial flaps samples were collected through operation of LASEK and Epi-LASIK. Twelve rabbit corneal limbus samples were gathered as control.Human and rabbit epithelial flap cells and rabbit corneal limbus cells were cultured using cell culture or tissue culture method.The cell reproductive activity was analyzed by cell culturing and the WST-8.Results:The growth energy in epithelial flap cells with different cultural methods is poor.The optical density value of cultrued cells was 0.582±0.113 in human Epi-LASIK group,0.449±0.101 in human LASEK group with a significant difference between them(t=3.907,P=0.000).The optical density value of rabbit cltured cells was 0.429±0.092 in Epi-LASIK group and 0.380±0.081 in LASEK group(P=0.255).Cell reproductive activity of rabbit LASEK and Epi-LASIK groups were lower than that of rabbit control group(0.557±0.129),showing a evident difference among them(P=0.005;P=0.000,respectively).Conclusions:Reproductive activity of epithelial flap cells in vitro was poor.The reproductive activity of keratocytes in LASEK was lower than that in Epi-LASIK. Corneal epithelial wound healing in LASEK and Epi-LASIK mainly depends upon the division growth of corneal limbus stem cells rather than epithelial flap cells. Part 2 Experimental Research on Influence of Rabbit Epithelial Flap in Epi-LASIK for the Expression of TGF-β₁,bFGF and Apoptosis in Corneal StromaPurpose:To discuss the influence of corneal epithelial flap in Epi-LASIK for corneal wound healing from the effect of epithelial flap for the expression of TGF-β₁,bFGF and apoptosis in corneal stroma.Methods:21 rabbits were operated in traditional Epi-LASIK with epithelial flap,and another 21 rabbits in Epi-LASIK without epithelial flap.3 rabbits were as negative control.To gain 6 cornea per group for the detection of TGF-β₁,bFGF and apoptosis in corneal stroma in postoperative 4h,1d,3d,5d,7d,15d,30d respectively. Immunohistochemistry and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling) were used.Corneal epithelial healing and haze were observed postoperatively.Results:The expression of TGF-β₁,bFGF and apoptosis in corneal stroma of group with epithelial flap was higher than that without epithelial flap.The difference of apoptosis between two groups was statistically significant(F=5.452,P=0.042).The expression of TGF-β₁,bFGF between two groups didn't show a evident difference(F=0.552,P=0.475;F=1.602,P=0.234,respectively).Average epithelial healing time was 4.05d in group with flap and 3.88d in group without flap(t=0.640, P=0.526).Haze of grade 1 appeared in 3d postoperatively in two groups.The extent of haze increased in 5d and 7d,decreased in 15d,and disappeared in 30d postoperatively.The rate of haze in group without flap was lower than that with flap, showing a evident difference(x²=4.269,v=1,P=0.039).Conclusions:The way without epithelial flap in Epi-LASIK could reduce apoptosis in corneal stroma and haze,which improve the corneal epithelial wound healing.