| Epidermal growth factor (EGF), combined by 53 aminoacids, was originally isolated from salivary glands of mice. Preliminary studies in humans suggest that topical EGF enhances healing of skin or cornea wounds. Recently the effect of EGF in treating the disease of gastrointestinal tract, where the concentration of EGF is the highest in the body, is a hotspot in this area. Therefore Exploiture of EGF is meaningful and worthwhile, lots of research works need to be done both on the mechanism of it's clinical effects and it's signaling transduction pathway(s) contributed to the effects.3 methods are used to produce EGF : Chemical synthesis, gene recombinant and biological synthesis. But their disadvantages , such as high investment, shortness of source etc, are hardly to be overcomed. In our study we purified EGF from the submaxillary glands of goats, tested it's biochemical activity and then used the goats' EGF in treating the animal model of ulceratic colitis and atrophic gastritis. The mechanism of the gene expression regulated by EGF was also studiedPart I The isolation, purification and identification ofEGF from goats' submaxillary glandsEGF , the prototype member of a family of growth factors ,shares structural and functional homology. Recently the effect of EGF in treating the disease of gastrointestinal tract, where the concentration of EGF is the highest in the body, such as atrophic gastritis, peptic ulcer, ulceratic colitis is a hotspot. 3 methods are used to produce EGF : Chemical synthesis, gene recombinant and biological synthesis. But their disadvantages such as high investment, shortness of source etc, are hardly to be overcomed.1. objectiveIsolate, purify and identify EGF from goats' submaxillary glands.2. material and methods2. 1 Crude isolation from the goats' submaxillary glands. Add HAC (4 c ) to thepowder of the goats' submaxillary glands, after centrifigated for 2 times,collected the supernatant, adjusted PH to 6.5 by 3mol/L NH4OH, added NaCLto 0. 4mmol/L overnight, centrif igated ISOOOrpm for 20mins. Added 20mmol/L trisand suspended with HCL PH5. 56.2.2 Chromatography analysis. Made 300*16mm Chromatography column with 20gSephadexG15 dry powder. Balanced with 10 column volume ofPH5. 56, 20mmol/Ltris. HCL solution. Add the suspended solution 5ml to SephadexG15 Chromatography column, collected the EGF flow peak which was the peakof EGF identified by the ELISA.2. 3 DEAE Chromatography column Made DEAE Sephadacel column with KX16/26(Phamacia), conneted P510 pump, linear flow rate 5ml/L. A solution: 20mM Tris.HCl PH5. 56. B solutin: 20mM Tris.HCL PH5. 5 with 1M NaCL .wavelength: 280nm. Injected the solution collected from SephadexGIS column and collected the EGF flow peak again.2.4 ELISA: added 80l samples to 5 time concentration of embedded solution 20l then embedded to ELISA react ion strip, for 37 2 hours, and 4 overnight. The next day blocked in 37 for 30mins with PBST. Added SIGMA rabbit anti-human EGF antibody 100ul/reaction, 37癈 for 1.5 hours, washed with PBS for 3 times , 3 mins every time, added enzyme labeled goat anti rabbit IgG 100ul/reaction 37 for 1hour. wash with PBS for 3 times , 3 mins every time, added benzodiamine -H202, 100 ul/reaction, 37 for 20mins then added 50ul2M sulphuric acid to stop the reaction, read the number on OD490nm. 2. 5 The identification of the bioactivity (1) MTT method: rats kidney cells were cultured in 1640 containing 10% FBS, incubated in 37, 5% C02 incubater for 48 hours, colleted the cells, wash with Hanks solution, dilluted to 5104 /ml cell number. Added 150/ul solution, 100ul samples to every reaction and repeated 5 times for every reaction. The other group was added Hanks solution only as control. 37, 5% C02 incubated for 12 hours, add MTT ( 5mg/ml ) 15ul and culured for another 4 hours, add isoacetonole lOOul to stop the reaction, the cell growth acceleration rate was calculated as follows acceleration rate= (Ai-Ao) /(Ao)100%(2)eyelid opening and incisor er...
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