The Purification Of Nerve Growth Factor From Venom Of Vipera Russelli Siamensis And Possible Neuroprotcctive Mechanisms On SH-SY5Y Cells Apoptosis Induced By Aβ_25-35.

Objective In order to find a simple,fast and highly efficient method for the separation and purification of nerve growth factor(NGF)from venom of vipera russelli siamensis ,the combination process of several different kinds of chromatographic media was studied. To investigate the change of viability of SH-SY5Y cells acted by soluble Aβ25-35 and the possible protective effect of venom NGF on this change.Methods: 1.According to the properties of NGF and the character of different chromatographic media, a novel two-step chromatographic purification method,consisting of chromatography of crude venom on CM52 cation-exchange medium followed by a size exclusion on a Sephadex G-75 column is presented.2.All kinds of PAGE was used on NGF relative molecular mass determination;PC12 cell cultured with low blood serum was used on the activity of NGF identification.3. SH-SY5Y cells by Aβ25-35–induced damage were used as the AD model of neurons for observation. SH-SY5Y cells were divided into 3 groups:blank group,Aβgroup(treated with 40μmol/L soluble Aβ25-35),and NGF intervention group(treated with 40μmol/L soluble Aβ25-35 plus 2.5μg/ml NGF). Cell viability was detected by MTT method and degree of injury by lactate dehydrogenase(LDH) method at 12,24,48 and 72h respectively.Results: 1.Through two-step chromatographic purification process,the obtained NGF was proved to be homogeneous on all kinds of PAGE and its relative molecular mass was estimated to be approximately 32000. 2. the obtained NGF had the activity of speed neuron fiber growth. 3. cells viability was detected by using MTT assay. Compared to blank group ,Aβ25-35 group decreased significantly (P<0.01)and leakage concentrations of LDH increased significantly(P<0.01)at every time point and dynamically in a time and dose-dependent manner. 4.For Venom NGF intervention groups,MTT reduction increased significantly compared to Aβ25-35 group(P<0.01 or P<0.05)at every time point in a time and dose-dependent manner,and leakage concentrations of LDH decreased significantly(P<0.01)at every time point in a time and dose-dependent manner.Conclusion:1.Ultra-physiologic dose Aβ25-35 is of toxic action early and time-dependently on.2.SH-SY5Y cells injured by soluble Aβ25-35 can be used as cell model that represents earlier pathological 1esion of AD.3.Venom NGF has neuroprotective effect in a time and dose -dependent manner and anti-Aβ25-35-induced viability cutting down and membrane integrity damage.