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Purpose isolated and purified cobra venom nerve growth factor (nerve growth factor, NGF) and NGF active substances, and to study their physical and chemical properties and biological activity. Method (1) using gel filtration chromatography and ion exchange chromatography separation cobra venom NGF and NGF activity component II (we will name NGFx) by sodium dodecyl sulfate - polypropylene acrylamide gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) to determine their molecular weight and identified purity. (2) the growth of rat adrenal pheochromocytoma cell line PC12 cell culture method, determination the NGF and NGFx biological activity, respectively, under the inverted phase contrast microscope and electron microscope observation projections. (3) using the method of serum pharmacology, the separation and purification of NGF in rats gavage, made the NGF containing serum, the serum containing anti-tumor activity in vitro and then measured by MTT. (4) further identified using capillary electrophoresis NGFx purity, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and its molecular weight, and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify the kind of substance. Results (1) determined by gel filtration and ion exchange method, isolated NGF and having NGF activity NGFx, calculated after SDS-PAGE, the molecular weight of the out of NGF is 24 547, a molecular weight of NGFx 19588, have reached the electrophoretically pure. (2) NGF and NGFx PC12 cells, respectively, compared with cells in the control group, the inverted phase contrast microscope and electron microscope to see the cells in the body grow protrusions, but compared role group of NGFx, the grow after NGF role projection longer and more intensive. In the electron microscope, also visible in addition to see protruding organelles in the cytoplasm gradually disappear, appear more lipid droplets. (3) different concentrations of NGF-containing serum inhibition rate of tumor cell growth in vitro of NGF-containing serum concentration of 25.00%, 12.50%, 6.25% of human gastric cancer MGC-803 cells inhibition rates were 86.95%, 30.38%, 10.32%, the rate of inhibition of human gastric cancer SGC-7901 cells were 78.50%, 56.78%, 47.20%, on human hepatoma BEL-7404 cells inhibition rates were 46.36%, 41.17%, 34.53%, of the people Hela cells, inhibition rates were 77.31%, 70.19%, 37.48%, and the control group compared to the average P lt; 0.05. (4) NGFx further by capillary electrophoresis column, I saw a peak; matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine its molecular weight 19892.818, after MALDI-TOF/TOF tandem time-of-flight mass spectrometry identification, the resulting peptide sequence with NCBI database sequence alignment, the match score was only 62, a lower score, can not determine what the substance. Conclusion (1) was isolated NGF and NGFx, can induce differentiation of PC12 cells into neuron-like cells to grow synapses. NGFx can promote the differentiation of PC12 cells, but its activity, whether new substances as well as what kind of material needed to further define. (2) Guangxi cobra venom NGF serum containing human gastric cancer cell line MGC-803 and SGC-7901 human hepatoma cell line BEL-7404 and Hela cells have growth inhibition was dose-dependent manner.
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