The Experiment Research Of EGF Nanometer Particle Promoting Ulcer Healing In Diabetic Rat

Objective: To prepare epidermal growth factor (EGF) nanometer particle andevaluate its shape, particle diameter, drug loading efficiency, encapsulated ratio andrelease. To carry out cell research with EGF nanometer particle in vitro, throughobserving reproductive activity of mouse fibroblast, to evaluate if the EGF losesactivation in the EGF nanometer particle preparing progression, to study if theparticulate carrier has toxicity. To cure the diabetic rats' ulcers with EGF nanometerparticle, and to compare ulcer healing difference with EGF nanometer particle andEGF, to investigate the mechanism of promoting healing with EGF nanometerparticle.Methods: 1. To utilize the modified multiple emulsion way withpoly(lactic-co-glycolic acid(PLGA) as carrier to prepare EGF nanometer particle.2.To watch exosyndrome of EGF nanometer particle with transmission electronmicroscope(TEM). 3. To analyze particle diameter distribution of EGF nanometerparticle with laser particle size appearance/Zeta electric potential appearance. 4. Todetermine drug loading efficiency, encapsulated ratio and release with ELISA. 5. Tocultivate L929 cell in culture flask, control cell density in 1.0×10~5/ml, then inoculatein cell culture plate. 6. To add suspension with EGF nanometer particle of 1μg/L, 5μg/L, 10μg/L, 50μg/L, 100μg/L into cell culture plate, and establish control holes,culture 24 hours, determine the OD density at 490nm with MTT. To research theinfluence of cell proliferation with different concentration EGF nanometerparticle.7.To add EGF nanometer particle, EGF stock solution of 10μg/L into cellculture plate, and establish empty nanometer particle control holes, culture 24 hours,determine the OD density at 490nm with MTT. To study the influence of cellproliferation with same concentration but different dosage form EGF.8. To addsuspension with corresponding empty nanometer particle of 0.01μg/L, 0.1μg/L, 1μ g/L, 10μg/L, 100μg/LEGF into cell culture plate, and establish control holes,culture 24 hours, determine the OD density at 490nm with MTT. To research if theparticulate carrier has toxicity for cell and influence cell proliferation. 9. To preparediabetic rat model through vena caudalis with streptozocin(STZ). 10. To preparediabetic rat ulcer model with hole puncher. 11. To divide these rats into 4 groups: EGFnanometer particle group(A group), EGF stock solution group(B group), emptynanometer particle group(C group) and PBS menstruum control group(D group). Wegive drug once a day. 12. To take photographs at 3, 7, 14, 21days and calculate thewound healing rate. 13. To draw the materials from rats' ulcer wound at 3, 7, 14, 21-daysand watch HE slide and immunohistochemistry(epidermal growth factorreceptor, EGFR; proliferating cell nuclear antigen PCNA). 14. Draw the materials fromrats' ulcer wound at 14 days and to watch with TEM.Results: 1. The particle diameter of EGF nanometer particle is 150nm, particlediameter distribution is relative uniform, there isn't conglutination among EGFnanometer particles, and their dispersibility is good. Drug loading efficiency is 0.02％;encapsulated ratio is 85％. Releasing drug accord with Higuchi release kinetic model.EGF nanometer particle rapidly releases EGF to reach the effective concentration infast infiltration phase, then it slowly releases EGF within effective concentration inmidvelocity infiltration phase and polymeride degradation phase, it lasts 24hours.2.Different concentration EGF nanometer particles all promote cellproliferation. Among them, 10μg/L is optimal concentration.(Vs, control group,P＜0.01). 3. Different dosage form EGF but same concentration 10μg/L hasdifference. EGF nanometer particle group is better than EGF stock solution group(P＜0.01). They are both better than empty nanometer particle group and control group(P＜0.01). Empty nanometer particle group dosen't have difference withcontrol(P＞0.05). 4. Different concentration empty nanometer particles don't have toxicity, don't effect cell proliferation(P＞0.05). 5. Succeeded prepare diabetic ratmodels, first forming model rate is 80.7％, second addition reaches 100％. 6. Healingrate shows: A group is better than B group at the 14th day(P＜0.01). 7. EGFR shows: Agroup is better than B group after the 7th day(P＜0.01). PCNA shows: A group is betterthan B group after the 14th day(P＜0.01). 8. TEM results show: There are manyendoplasmic reticulums in fibroblast, and endoplasmic reticulum expands, includemany collagen proteins. The others there are a few endoplasmic reticulums infibroblast and a few collagen proteins.Conclusion: Succeeded prepare EGF nanometer particle. Cell experiment showsEGF keeps activity after preparing EGF nanometer particle, EGF nanometer particlecan better promote cell proliferation than EGF stock solution. Particulate carrierdoesn't have toxic action to cell. EGF nanometer particle can better promote ulcerheal than EGF stock solution in diabetic ulcer rat experiment. So EGF nanometerparticle should be more fit for clinical application.