| Keratinocyte growth factor-2 (KGF-2), also known as fibroblast growth factor-10 (FGF-10) and a member of the fibroblast growth factor family. KGF-2 is highly specific to epithelial tissues and has rare physical, chemical, and biological stabilities. It can encourage healing to corneal epithelial injury, while it will not promote new angiogenesis in cornea. Reference data suggested that KGF-2 was specifically beneficial to treat corneal injury. Because cornea has no blood vessel, the process of repair for basal matrix is rather slow when compared to other tissues. Thus, it becomes essential to speed up this healing process. KGF-2 can boost the repair of epithelial cells and stromal cells, but retain the properties of not exceeding overgrowth of collagen. This has great significance in reducing post-traumatic scar. Although corneal cells can secrete fixed quantity of KGF-2, supplementation of KGF-2 from extrinsic source is still required for faster repair, in order to promote proliferation and migration of corneal epithelial cells and stromal cells. Therefore, the study and development of KGF-2 product to understand the effect on corneal injury imply great significance to expand clinical application of KGF-2.The study would extract total RNA from pulmonary fibroblasts of human fetuses, where cDNA could be obtained by process of RT-PCR. With cDNA as a template, primer was designed and PCR was run to amplify genetic fragment of KGF-2. Carrier of pET3c was selected, where cutting points for Ndel and BamHI enzymes were designated at the upstream and the downstream of primer. The acquired KGF-2 gene was then annexed to the pET3c carrier. Recombinant carrier, after verified by both enzymes, by PCR, and by genetic sequencing, would be confirmed that KGF-2 gene was accurately integrated. BL21 (DE3) was selected as host bacteria. By using 1mmol/L IPTG-induced expression of target protein, KGF-2 proteins, which are mostly in soluble expression, would be obtained. Expression of soluble proteins would take up approximately 15% of the total bacterial proteins.Based on the criteria for manufacturing of genetically engineered product, KGF-2 would be used for process study on fermentation and purification.Glucose would be the source of carbon, while yeast extracts and peptone would be the source of nitrogen. Micro-elements, such as 2.5g/L Dipotassium hydrogen phosphate, 1.0g/L monobasic potassium phosphate, and 4.0g/L NaCl were added into fermented culture media. 10% inoculation ratio would be employed when transferring primary bacterial strain to secondary bacterial strain. 5% inoculation ratio would be used when transferring secondary bacterial strain to the fermented culture media. Culturing temperature for fermentation of bacterial strains in the genetic engineering of KGF-2 would be set at 37℃.During the process of fermentation, by controlling the flow acceleration rate of glucose, soluble oxygen concentration would be controlled at 30% or above. By adding 2N HCl or 4N NaOH, pH-value would be adjusted between 6.8 and 7.0. OD600 for bacteria reached 16 to 20, where addition of IPTG was conducted to induce expression.According to the affinity of KGF-2 to heparin and its electronegativity in buffer solution of pH 6.0, the study selected affinity chromatography for heparin and exchange chromatography for weak positive ions, where product of more than 95% purity would be acquired in a two-step process. By using immunological blotting, sequencing of N-terminal, and molecular weight analysis of mass spectrum, KGF-2 proteins were differentiated. By employing two methods, HPLC and non-reducing SDS-PAGE, purity of KGF-2 bulk could reach above 95.0%. Tested sample by SDS-PAGE, via electrophoresis, showed up as a single band. In examination of reverse-phase HPLC, a single spectral peak appeared within 15 minutes of duration. By Lowry method, the quantity of KGF-2 bulk was measured.The study would use clarity and activity as measuring indices, where the KGF-2 eye drops were confirmed to be stable at pH of 6.0 and the most appropriate buffer system would be 20mM phosphate. By orthogonal test, the best protective agents for KGF-2 eye drops were 1% albumin and 2% mannitol.Based on properties and pharmacokinetic characteristics of KGF-2, NIH3T3 cells, keratinotic epithelial cells, corneal epithelial cells, corneal stromal cells, and BaF3 cells with transfection of FGFR2IIIb were selected for this study, to analyze in vitro biological activities of KGF-2, as well as the influence by extrinsic heparin. KGF-2, to the five types of cells above, showed in vitro promoting proliferation effect, in which NIH3T3 cells and corneal stromal cells became fibroblasts. Hacat cells and corneal epithelial cells were of epithelial origin, while BaF3 cells were the transfected cells with FGFR2IIIb. KGF-2 promoted NIH3T3 cells and the effect zone on corneal stromal cells was between 1μg/ml and 100μg/ml. Under induction of extrinsic heparin, KGF-2 promoted NIH3T3, where the proliferation of corneal stromal cells was significantly increased. KGF-2 promoted keratinotic epithelial cells and the effect zone for corneal epithelial cells was between 1ng/ml and 100ng/ml. The effect zone for proliferation of transfected BaF3 cells by KGF-2 was between 0.1ng/ml and 37.5ng/ml. Laboratorial results revealed that epithelial cells had high affinity receptors for KGF-2. Under low concentration of KGF-2, it could induce proliferation. Fibroblasts had low affinity receptors for KGF-2, where induction of signal transduction required higher concentration of KGF-2. Primary cultured corneal epithelial cells, due to its inability to passage culturing, corneal stromal cells required induction by extrinsic heparin to achieve obvious proliferative activities. Therefore, these two types of cells were not suitable for evaluation of activities of KGF-2 in the manufacturing process. Other three types of cells could be used for activity assessment of KGF-2. The study, based on pharmacokinetic characteristics of KGF-2, selected transfected BaF3 cells for activity assessment of final product KGF-2.Models of alkaline burn and laser injury were used to evaluate the therapeutic effect of KGF-2 on corneal injury. Results showed that after application of different concentrations of KGF-2 and in 1 week, the manifestations of various injuries were much lighter than the burns in control group, where the thickness of cornea had restored to near normal, edema had receded, and no obvious inflammatory response was observed. In 2 weeks, no recurrence of inflammation was seen. Corneal epithelial cells were growing and arranging in order. Stromal layer contained more collagen fibers and laminar fibers were in order and parallel to corneal epithelium. There was no difference from normal cornea. It suggested that KGF-2 could significantly reduce edema in corneal stroma, inhibit overgrowth of fibroblasts in corneal stroma, and inhibit infiltration of inflammatory cells, which would provide a good environment for regeneration and repair of corneal epithelial cells. The group of bFGF with treatment of positive control drug, in 2 weeks after therapy, showed wave-like overgrowth in corneal epithelium, where the surface was rough and more fibroblasts were seen in stroma. Also, laminar fibers were also in waves.Measurement of OD-value suggested that KGF-2 was beneficial to restoration of corneal transparency. Also, drug concentrations at 25μg/ml and 50μg/ml had similar therapeutic effect, without statistical significance. Morphological quantitative statistics on in vitro corneal burn showed that KGF-2 was beneficial to restoration of corneal transparency and allowed reduction in size of opaque plague after corneal burn. Also, in 7 days after burn, the average gray-value and the burn surface area of cornea in the group with 25μg/ml KGF-2 were significantly smaller than the group with 2.5μg/ml bFGF.MTT results revealed that KGF-2 had promotional effect on restoration to activities of corneal epithelial cells after alkaline or laser burn, where one week of administration showed promising results.Overall, the study used prokaryotic expression system of E. coli to successfully establish KGF-2 engineered bacterial strain. Also, the processes of fermentation and purification for KGF-2 were studied, where method of measurement for in vitro biological activities of KGF-2 was established. Models of alkaline and laser injury on Japanese rabbits were used to mimic and study the in vivo promotional effect on repair of corneal injury by KGF-2. This study would provide a basis for clinical application of KGF-2 in the Ophthalmology Department.
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