| BackgroundNormal transparent cornea consists of corneal epithelia, Bowman’s membrane, stroma. Descemet’s membrane, and corneal endothelia. which presents one sixth part of ocular outer sphere fibrous membrane covered by lacrimal film. Corneal epithelia composes of4-6layers of noncomifying stratified pavement epithelium, which is0.05mm, holding5percent of the whole corneal thickness. Corneal limbus stem cells could transform into transient expansion cells and terminal differentiation cells gradually, which are the sources of corneal epithelial injury healing.Its lifecycle is7-14days. Corneal epithelia also consists of cell layer and basilar membrane, cell layer is composed of basilar cell, aliform cell and surface layer cell. Basilar membrane is composed of IV collagen fiber, laminin and other albumens.as a50nm thickness membrane. The tight junction of surface epithelial cells can prevent the water content from tears to get into stromal layer.Extensiveness defect can result in corneal edema, and corneal transparent, then lead to vision descent. Integrity corneal epithelia is the first barrier for eye ball to the pathogenic microorganism invasion, which is also the essential condition for maintaining satisfactory vision. Corneal epithelial is easily subjected to physical, chemical, and biological harmful factor. Clinical, trauma, infection, and operation and various kinds of disease could lead to corneal epithelial injury easily with the presence of corneal infection, ulcer, perforation, leucoma, neovascularization, haze, even blindness, and so on. A small areal corneal epithelial defect with ocular surface in good condition can cure spontaneously,if a large areal corneal epithelial defect accompanyed by protracted course of disease, the drug therapy for corneal epithelial wound healing is needed to avoid corneal infection and neovascularization. Therefore, the related chief growth factors play an important role in the process of corneal epithelial wound repair and the maintenance of corneal transparency.Clinically, recombinant human epithelial growth factor (rhEGF) and basic fibroblast growth factor(bFGF) are two major growth factors, involving in the process of corneal epithelial wound repair.Recent research indicates that they have different mechanism and emphasis on themselves particularly:EGF could bind to EGF acceptor on the basal cell membrance, promoting corneal limbus basal membrance cells to epithelium membrance migration,forming monolayer,then forming stratified corneal epithelium by mitosis, accelerating corneal wound epithelization. bFGF could promote collagen fiber secretion and blood capillary production magnify by mitosis effect and the hormonelike activity of non-mitosis,which could form corneal neovascularization to influence corneal transparency.Therefore, make sure whether rhEGF and bFGF eyedrops could induce corneal neovascularization and scar.and which has a better effect on corneal wound healing., to guide clinical medication more reasonable and safe, and to provide the best opportunity of clinical medication, comparative study of the effects of rhEGF and bFGF on corneal epithelial wound healing and neovascularization in vivo and vitro is necessary, which also makes for establishing groundwork for lucubrate.Objective1.To study the effects of rhEGF(Recombinant Human Epithelial Growth Factor) and bFGF(Recombinant Human Basic Fibroblast Growth Factor) on the proliferation of SD-HCEC1, RKCs, and HUVECs.and the migration capacity of HUVECs in vitro.2. To investigate the effects of rhEGF and bFGF on corneal epithelial wound healing and neovascularization in vivo.Methods.1. In vitro:(1).screening the concentrations of rhEGF and bFGF on the pr oliferation of corneal epithelial cell by3-(4.5-dimethylthiazol-2-yl)-2,5-diphenylt etrazolium bromide (MTT) method. SD-HCEC1seeded at1000cells per well i n96-well plates in corneal epithelial cell culture medium Supplemented with5ng/ml,10ng/ml,20ng/ml rhEGF.and2.5ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml bFGF. and negative control (neither10ng/ml rhEGF or10ng/ml bFGF). incubat ed for5day, respectively.The average number of cells was counted and a cell proliferation curve was generated according to the OD value measured at490nm.(2). SD-HCEC1seeded at1000cells per well,RKCs seeded at1000cells per well, HUVECs seeded at5000cells per well in96-well plates. Twenty-four hours after seeding, the cells were treated with10ng/ml rhEGF,10ng/ml bFGF and negative control (neither10ng/ml rhEGF or10ng/ml bFGF). every5wells was measured per day for7days,respectively. The average number of cells was counted and a cell proliferation curve was generated according to the OD measured at570nm,490nm (Power Wave XS; Bio-Tek, Winooski, VT). The MTT proliferation assay was performed as previously described. All samples were assayed in three duplicates.(3). Colony forming efficiency (CFE).To evaluate proliferative capacity of SD-HCEC1, RKCs, HUVECs from the four groups(Negative Control,10ng/ml rhEGF,10ng/ml bFGF,20ng/ml bFGF), the CFE was assessed in cultures,using a previous method with modification. SD-HCEC1,RKCs in the four groups were seeded at100,200cells/well into six-well culture plates without3T3fibroblasts or any other cells as a feeder layer. HUVECs were seeded at1500cells/well into six-well culture plates with1%gelatin(sigma,St.Louis,Mo) as a feeder layer. Colonies with more than eight viable cells were counted manually under an inverted phase microscopy.and cultured cells were stained with Giemsa on day7. Experiment was repeated at least three times. The CFE was calculated by dividing the number of colonies by the number of viable cells seeded on day0.(4). Ki67flow cytometry analyses. To determine the percentage of Ki67-positive cells, we digested SD-HCEC1from the four groups(Negative Control,10ng/ml rhEGF,10ng/ml bFGF,20ng/ml bFGF),RKCs and HUVECs from the three groups (Negative Control,10ng/ml rhEGF,10ng/ml bFGF) was performed as previously described. Data analysis was conducted using Cell Quest software.(5).Cell migration assay. The cell migration assay was performed as previously described. The HUVECs from the Negative Control,10ng/ml rhEGF,10ng/ml bFGF groups were washed with PBS. The size of the acellular area was photographed at0,8and24h after cell scraping and analyzed with the Image J software.Every group has four fixation observation points.2.Animal experiments procedure in vivo. A central corneal epithelial wound mode was made as previously described.Postoperative rabbit eyes were treated with topical administration of rhEGF,bFGF,DCBE,and NS drops, respectively,four times per day. The areal variety of corneal epithelial wound healing and neovascularization were observed by fluorescein staining and photograph and analyzed with the Image J software. The areal variety of corneal epithelial wound healing and neovascularization were observed.Results1. The optimal concentrations of rhEGF and bFGF on the proliferation of corneal epithelial cell were10ng/ml(1.356±0.218,2.056±0.210).2.The results of Cell proliferation experiment.by MTT assay.(1). About SDHCEC1, in the rhEGF, bFGF, and Negative Control (neither rhEGF or bFGF) group,the proliferation capacity of SD-HCEC1was various(F=51.800,P=0.000).The proliferation capacity in the1Ong/ml bFGF(P=0.000)and10ng/ml rhEGF(P=0.048) was better than Negative Control.(2).About RKCs, In the rhEGF, bFGF, and Negative Control (neither rhEGF or bFGF) group.the proliferation capacity of SD-HCEC1was various (F=9.604,P=0.000).From day1to day3the proliferation effect in the bFGF group was not obvious.from days4, the proliferation effect in the bFGF group speed up, whereas, the proliferation effect in the10ng/ml rhEGF group was not better than Negative Control constantly (P=0.263), which indicated the proliferation effect of rhEGF on RKCs was not obvious, the proliferation effect of bFGF on RKCs was obvious in the advanced stage.(3).About HUVECs, The proliferation capacity in the bFGF group, rhEGF group and Negative Control was obvious (F=958.247,P=0.000). the proliferation effect in the10ng/ml bFGF group and in the10ng/ml rhEGF group is stronger than Negative Control (P=0.000).from days2, the proliferation effect in the10ng/ml bFGF group lasted strong, even stronger than the10ng/ml rhEGF group, whicn indicated bFGF had a strong proliferative effect on HUVECs.3. Effects of rhEGF and bFGF on the Colony forming efficiency (CFE) of SD-HCEC1, RKCs.and HUVECs.(1).The Colony forming efficiency (CFE) of SD-HCEC1s in the10ng/ml rhEGF group,10ng/mlå'Œ20ng/ml bFGF groups, and Negative Control was obvious (χ2-=8.538, v=3, P=0.036).The Mean rank of four groups were5.33,7.17,11.00, and2.50, which indicated that the capacity of the CFE in the20ng/ml bFGF group was the strongest among the four groups, the shape of monoclonal formation in the Negative Control was the smallest,with the comparion of the rest three groups.(2). The Colony forming efficiency (CFE) of RKCs in the10ng/ml rhEGF group,10ng/mlå'Œ20ng/ml bFGF groups, and Negative Control was obvious (χ2=9.100, v=3,P=0.028).The Mean rank of four groups were5.33,7.17,11.00, and2.50, which indicated that the capacity of the CFE in the20ng/ml bFGF group was the strongest among the four groups.(3). The Colony forming efficiency (CFE) of HUVECs in the10ng/ml rhEGF group,10ng/mlå'Œ20ng/ml bFGF groups, and Negative Control was obvious (χ2=8.390, v=3,P=0.039) The Mean rank of four groups were5.00,9.17.9.50, and,2.33, which indicated that the capacity of the CFE in the10ng/ml and20ng/ml bFGF groups was much stronger.4. Effects of rhEGF and bFGF on the Ki67flow cytometry analyses of SD-HCEC1, RKCs. and HUVECs.(1).The ki67expression levels of SD-HCECls in the10ng/ml rhEGF group,10ng/ml and20ng/ml bFGF groups,and negative control were obvious (χ2=10.385, v=3, P=0.016). The Mean rank of four groups were5.00,8.00,11.00, and2.00. which indicated that the ki67expression levels of SD-HCEC1s in the10ng/ml bFGF and20ng/ml bFGF group were higher.(2). The ki67expression levels of RKCs in the10ng/ml rhEGF group,10ng/ml bFGF group.and negative control were obvious (χ2=7.200, v=2, P=0.027). The Mean rank of three groups were5.00,8.00, and2.00, which indicated that the ki67expression levels of RKCs in the10ng/ml bFGF group were higher than in the10ng/ml rhEGF.(3). The ki67expression levels of HUVECs in the10ng/ml rhEGF group,10ng/ml bFGF group, and negative control were obvious (χ2=6.489, v=2,P=0.039). The Mean rank of three groups were5.33.7.67,and2.00, which indicated that the ki67expression levels of HUVECs in the10ng/ml bFGF were higher than that in the10ng/ml rhEGF group and Negative Control.5. Effects of rhEGF and bFGF on HUVECs Cell migration assay.The unhealing areal rates in the10ng/ml bFGF,10ng/ml rhEGF group, and Negative Control were obvious (F=1138.752,P=0.000),which was different in different hours (F=959.377, P=0.000).The unhealing areal rates of10ng/ml bFGF and10ng/ml rhEGF groups were less than Negative Control at8hour and24hour (P=0.000). The unhealing areal rate in the10ng/ml bFGF group was also less than the10ng/ml rhEGF group (P=0.000).All these indicated that bFGF and rhEGF both promoted the capacity of HUVECs Cell migration, however.bFGF had a stronger capacity than that of rhEGF.6. Effects of rhEGF, and bFGF on the healing rate of rabbit cornea.The healing rates in the bFGF group, the rhEGF group, and Negative Control were obvious (F=5.022, P=0.026). on days1,2. the healing rate in the rhEGF group was obviously higher than that in the bFGF group(P=0.022)and NS group(P=0.015). On days3.the healing rate of bFGF, rhEGF, and NS was100±0%,100%±0%.99.50%±0.70%, respectively.7. Effects of rhEGF,and bFGF on rabbit corneal neovascularization.The corneal neovascularization area was various in the rhEGF, bFGF, and NS groups (F=4.173, P=0.042).The corneal neovascularization area in the rhEGF group was not larger than that in the NS group (P=0.666),whereas.the corneal neovascularization area in the bFGF group was larger than in the NS group (P=0.020).They indicated that bFGF had promoted corneal neovascularization with the corneal sutural chronic stimulus more obviously than rhEGF.Conclusions rhEGF and bFGF all had the promotive effects of corneal epithelial wound healing.but rhEGF emphasised on the role of corneal epithelium.which had the less promotive effect of corneal neovascularization than bFGF. bFGF could promote both the proliferation of corneal epithelial and stromal cell.It suggested that either rhEGF or bFGF eyedrops could be selectd for the cure of simple corneal epithelial wound in short-term, bFGF eyedrops should be selected for corneal stromal wound healing, rhEGF eyedrops should be selected for chronic corneal epithelial erosion with the long-term aplication of growth factor drug,such as dry eye.and so on. |
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