A Modified Method For Preparation Of A?1-42 Oligomers,Protofibrils And Fibrils,and Comparisons On Their Neurotoxicity

ObjectiveBetween different experimental studies,there is a considerable difference in the reference value of A?1-42 using concentration.The preparation methods of A?142 oligomers,protofibril and fibre in different experiments were not complete,mainly including the following six steps:hexafluoropropanol(HFIP)monomerization A?1-42 lyophilized powder,drying removal HFIP,DMSO solubilization,dilution by culture medium,removing impurities in the A?1-42 solution and incubation.The main differences of the methods in the literature are the steps of drying removal HFIP and removing impurities in the A?1-42 solution.The purpose of this study was to modify A?1-42 oligomers,protofibrils and fibrils preparation method and to identify the finished products for evaluating the feasibility of the modified method.The differences of neurotoxicity of A?1-42 oligomers,protofibrils and fibrils were compared to provide the basis for AD experimental study.Methods and materials1.According to the principle and mechanism of vacuum freeze-drying instrument and ventilation cabinet to remove HFIP,a simple negative-pressure low-temperature drying device was designed.2.Before and after removing impurities in the A?1-42 solution,three groups of liquid were left.The following are three groups of liquids:Group A,liquid before removing impurities.Group B,liquid after removal of impurities by filtration.Group C,liquid after removal of impurities by ultra-fast centrifugation.3.Western blot(WB)analysis of A?1-42products prepared by ventilation cabinet drying(+4? 24h),and simple negative-pressure low-temperature drying incubated methods(+4? 24h),for comparing of the differences between the ventilation cabinet drying and the simple negative pressure low temperature drying.4.The A?1-42 liquor prepared by simple negative-pressure low-temperature drying device was incubated at 4? 24 h,37? 24h and 37? 7d temperature and time conditions.WB analysis and electron microscope observation of the obtained A?1-42 oligomers,protofibril and fibre finished products to evaluate the feasibility of the modified method.5.The primary neurons were cultured in vitro and the primary neurons were identified by immunofluorescence to evaluate the primary purity of neurons,6.The morphology of neurons was observed under ordinary optical microscope after 24 h of action on neurons by A?1-42 oligomers,protofibril and fibre finished products at concentrations of 1,5 or 25?mol/L respectively.The cell activity of primary neuronal cells was detected by CytoTox 96(?)Non-Radioactive Cytotoxicity Assay and Cell Counting Kit-8 to evaluate the biological titer of A?1-42 oligomers,protofibrils and fibrils finished products prepared by the modified method,and to compare on neurotoxicity of different state A?1-42.Results1.A simple negative-pressure low-temperature drying device was designed.The conditions of drying removal HFIP can be effectively controlled by the simple negative-pressure low-temperature drying device,which consists of a sealed container with a one-way air valve,a negative pressure suction machine and a constant temperature refrigerator.2.ELISA results showed that the A?1-42 was lost after filtration or centrifugation during the preparation of A?1-42 oligomers,protofibrils and fibrils,and the amount of filtration loss was less than that of centrifugation.3.The semiquantitative analysis of WB bands of A?1-42 finished products prepared using ventilation cabinet drying incubated methods(+4? 24h)or using simple negative-pressure low-temperature drying incubated methods(+4? 24h).By comparing and analyzing the percentage of gray value of monomers,oligomers,protofibrils and fibrils region in the whole region,it is found that using simple negative pressure low temperature drying+4? 24h incubated methods has higher percentage of oligomers.4.The A?1-42 product prepared by using simple negative-pressure low-temperature drying method was tested by WB and observed under transmission electron microscope.A?1-42 oligomers,protofibrils and fibrils finished product conforms to the morphological characteristics of A?1-42 oligomers,protofibrils and fibrils.A?1-42 oligomers,protofibrils and fibrils were prepared successfully by the modified method.5.Neuron primary identification results,the purity of neurons?95%,the original neurons prepared in this study have high purity and meet the requirements of subsequent in vitro experiments.6.LDH and CCK8 results suggest that A?1-42 oligomers,protofibrils and fibrils are neurotoxic and lazy in concentration.Oligomers toxicity is greater than protofibrils.Protofibrils toxicity is greater than fibrils.Of the preparation methods used in this study,the recommended A?1-42 use concentration is 5?mol/L.Conclusions1.The use of a simple negative-pressure low-temperature drying device instead of a ventilation cabinet during in preparation of A?1-42 oligomers,protofibrils and fibrils can better control and record the conditions of drying removal HFIP to increase the repeatability of the experiment.2.During the removing impurities in the A?1-42 solution,the use of filtration instead of the ultracentrifugation method can effectively reduce A?1-42 loss and save cost.3.A?1-42 oligomers,protofibrils and fibrils can be successfully prepared by modified method,which is using a simple negative-pressure low-temperature drying device instead of a ventilation cabinet and using filtration instead of the ultracentrifugation4.A?1-42 oligomers,protofibrils and fibrils have a concentration-dependent toxicity to primary neuronal cells.It is proved that A?1-42 oligomers,protofibrils and fibrils obtained by modified method have biological effects.Oligomers toxicity is greater than protofibrils.protofibrils toxicity is greater than fibrils.Of the preparation methods used in this study,the recommended A?1-42 use concentration is 5?mol/L.