The Pharmacology Study Of Recombined Human Keratinocyte Gorwth Factor Variant-2(rhKGF-2) On Corneal Wound Healing

Corneal is an important transparent tissue occupying the anterior pole of the globe. The injury because of mechanical injury, operation and chemical burn are very common in superficial injury of eye. The processes of wound healing and scarring are essential means by which the body maintains its structural integrity, but tissue repair in and around the eye frequently compromises vision. In fact, fibrosis and contraction play a part in the development of most major blinding eye condition and in the failure of surgical treatments.BACKGROUNDKeratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In 1998 Emoto isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung- keratinocyte growth factor-2(KGF-2) .It's specificity reporter is PTK reporter: FGFR2â…¢b(KGFR) and FGFR. KGF-2 can protect and promote epithelial cells from damaging effects induced, informal with FGF, KGF-2 has little effect on fibrosis and endothelial cells. so it can lessen tissue repair reactive. The certain treatment effect include:â‘ accelerate the tissue repair of skin wound healing;â‘¡the energetic effect of ulcer;â‘¢activation corneal epithelial stem cells, accelerate it's proliferation and differentiation, at last accelerate the wound healing of epithelial defect. In privions research, we gained the mutation form of KGF-2,named as rhKGF-2, patent number(03101156),we maintained it's accelerate effect on epithelial cells, changes it's accelerate effect to be suppressor effect on fibrosis cells. And had a satisfaction treatment result on hepatic fibrosis.OBJECTIVEWe have two parts of experimentation: in vivo and in vitro. In the in vivo experimentation, we choice rabbit to be the text animal and construct two analogs. Observation the effect of 3 different concentrations eye drops of rhKGF-2 topical application on eyes after alkali burn and mechanical injury on epithelial repair, CNV and cloudiness of cornea. .In the in vitro, we primary culture rabbit corneal epithelial cell and rabbit corneal fibroblast cell. Use MTT and RT-PCR technique, text whether rhKGF-2 have this opposite effect on corneal epithelial cell and fibroblast cell. And initial approach it's mechanism of action. METHODSRabbit corneal alkali burn models were made by applying a filter paper of 8 mm in diameter soaked of 1 mol/L NaOH solution to the center of corneal for 60s.And then respectively give 0.05 %,0.025 %,0.0125 % rhKGF-2 eye drop. bFGF eye drops as the positivecontro1.rhKGF-2 eye drop solvent as the negative control 4 times per day. The corneal reepithelialization and neovascularization was observed by slit-lamp biomicroscope. The rate of corneal reepithelialization and the area CNV coveraged the corneal were analyzed and compared with those in negative control.Rabbit corneal mechanical injury models were made by a surgical way. And then respectively give 0.05 %,0.025 %,0.0125 % rhKGF-2 eye drop . bFGF eye drops and Dexamethasone sodium phosphate eye drops as the positivecontrol .r hKGF-2 eye drop solvent as the blank control 4 times per day. Corneal haze, histopathologic response, electron microcopy were investigated.Primary culture rabbit corneal epithelial cell and rabbit corneal fibroblast cell. Use MTT and RT-PCR technique, text whether rhKGF-2 have this opposite effect on corneal epithelial cell and fibroblast cell. And initial approach it's mechanism of action.RESULTS1.The alkali burn model on rabbit was succeeful. In first 24 h, the corneal epithlialization overall healing rate of rhKGF-2 eye drop group is 1.52 mm²Â·h^-1,1.57 mm2·h^-1 and 1.46 mm²Â·h^-1 and that in negative group is 0.98 mm²Â·h^-1,respectively(P<0.05). CNV was observed in each group after 3 days, rhKGF-2 eye drop can advance its happen at a high concentration(0.05 %), but suppressed it at a lower concentration (0.025 % and 0.0125 %) .The negative and bFGF eye drop group can significantly induced CNV occur , the induce ratio in day 7 is 100%.Much higher than other groups.2. The mechanical injury on rabbit was succeeful.3 mouths after the operation, the corneal haze and the average number of cornea cells in shallow 50μm corneal basement in rhKGF-2(0.05%)was significantly lower than bFGF group and blank group. The histopathologic response and electron microcopy shows the epithelial incrassation, disorder of cell and collages arrangement in rhKGF-2(0.05 %)are much lighter than that in bFGF group and blank group.3.We culture rabbit primary corneal epithelial cell and rabbit primary corneal culture fibroblast cell succeeful. The result of MTT shows rhKGF-2 has a significant accelerate effect on rabbit corneal epithelial cell and a significant inhibition effect on rabbit corneal fibroblast cell. The result of MTT shows in the fibroblast cell treatment with rhKGF-2,the express of TGF-β1 and CAGF cytokine mRNA all down regulated. At the same time, the express of internal reference:β-actin didn't change. CONCLUSION1.Local application of rhKGF-2 eye drops at dosages ranging from 0.05 % to 0.0125 %could enhance the cornea wound healing process, in dosages of 0.025 % and 0.0125 % it can also suppressed the CNV.2.Local application of rhKGF-2 eye drops could reduce the number of stroma keratocye, then effectively inhibition early stage corneal haze after rabbit corneal mechanical injury.3. rhKGF-2 has a significant accelerate effect on rabbit corneal epithelial cell at the same time has a significant inhibition effect on rabbit corneal fibroblast cell. rhKGF-2 produce a marked effect thought down regulated the mRNA express of TGF-β1 and CAGF cytokine.