The Experimental Study Of Guangxi Cobra Venom Nerve Growth Factor

Objective To isolated and purified nerve growth factor and the materials that had NGF's activities from Guangxi cobra venom, and studied their physical and chemical properties and biological activities.Method (1)To separate cobra venom and got NGF and components II (we called it NGFx here) which had NGF's activities, we used gel filtration chromatography and ion-exchange chromatography method.We identified their molecular weight and purity by SDS-PAGE.(2)Used PC12 cell culture method to measure NGF and NGFx's biological activity, and observed the growth of synaptic by inverted phase contrast microscope and electron microscope.(3)By the mothod of serum pharmacology, the rats were i.g administration of NGF, and then made of NGF-containing serum.Then we detected the anti-tumor activities of the serum by MTT method in vitro.(4)Used capillary electrophoresis colum to make a further purity identification of NGFx, MALDI-TOF MS to determine its molecular weight.And used MALDI-TOF-TOF MS to identify what kind of material it was. Result (1)By gel filtration chromatography and ion-exchange chromatography method, we had isolated NGF and NGFx.After running SDS-PAGE, we calculated that NGF's molecular weight was 24 547, NGFx's molecular weight was 19 588.Both of them had reached electrophoretically purity. (2)After PC 12 cells exposed to NGF and NGFx, compared to control group, we had observed that two groups of cells grew a large number of protuberances in inverted phase contrast microscope and electron microscope.But compared to NGFx group, NGF group's cells grew more longer, more concentrated.In electron microscope, besides protuberances, we found that in the cytoplasm organelles disappeared and there were more of the lipid droplets.(3)Different concentrations of NGF-containing serum had different inhibition rate on tumor cells in vitro.25.00%,12.50%,6.25% of NGF-containing serum to human gastric cancer MGC-803 cells' growth inhibition rate were 86.95%,30.38%,10.32%, to human gastric cancer SGC-7901 cells'were 78.50%,56.78%,47.20%, to human hepatic carcinoma BEL-7404 cells'were 46.36%,41.17%,34.53%, to human cervical cancer Hela cell's were 77.31%,70.19%,37.48%, compared to the control group P<0.05.(4) NGFx had passed capillary electrophoresis colum, and we just saw one peak.Its molecular weight was 19 892.818 that determined by MALDI-TOF MS.After determined by MALDI-TOF-TOF MS, and then aligned with NCBI database, the last matching score was only 62.Because the score was too low that we can't identify what kind of material it was.Conclusion (1)Both NGF and NGFx that we purified can induce PC 12 cells differentiating into neural-like cells and grow a large number of protuberances.Although NGFx can induce PC 12 cells differentiating, its activities and wether it was a new material and what kind of material need to make a further affirmance.(2)NGF-containing serum had the growth inhibiting capacity to human gastric cancer MGC-803 cells and SGC-7901 cells, human hepatic carcinoma BEL-7404 cells and human cervical cancer Hela cells, appeared dosage dependence.