Study Of Guben-Jiannao Methods On Impact AD Cells And Caveolin-1,P-tau

ObjectivesWith the social problems exacerbated by aging, Alzheimer’s disease (AD) has become a common concern of the world’s problems, the prevalence increased with age, the incidence of exponential growth, while its high prevalence, not only the physical, psychological and economic affects patients, but also the medical staff, patients and medical and health system of friends and family, the entire society. Therefore, enhancing the impact of the cause, AD research, exploration and effective methods of prevention of drug, is very important in the pathogenesis the clinical value and practical significance of the study is to discuss the basic pathogenesis ADbased AD, treatment principles surveyed in cultured hippocampal neurons, successfully separated, vector pEGFP-C1-CAV1 carrier construction, CAV1-shRNA lentivirus-based AD in vitro model to establish currentlyrecognized, observe and reinforce the brain’s role in the treatment of AD method and mechanism for further research in Chinese medicine to treat AD provides the basis and reference.Methods(1)By separating the newborn rat brain hippocampus dissected, separating, planting neuronal growth, morphology observed with MAP-2, Tau as a marker of neurons cultured rat brain neurons and immunofluorescence Western blot test Primer. (2)Designed with restriction sites and protection bases, groping CAV-1 PCR reaction annealing temperature; the pEGFP-Cl vector for gene and CAV-1 EcoR I, Sal I double digestion, digestion products thereof were identified by electrophoresis and recovered;The positive identification of its gene and linear vector CAV-1 T4 ligase and competent DH5a transformed overnight, a small amount of the recombinant plasmids, and double digestion and electrophoresis identified plasmids were sequenced electrophoretic identification of positive verification; expand training sequence correct plasmid containing strains of bacteria and preserves, and then a lot of extract (inside to endotoxin) plasmid and detect its concentration, and finally hippocampal neurons transfected with pre-experiment. SiRNA target sequence. (3)Design CAV-1 and for two pairs of oligonucleotide sequences CAV-1 interference in the sense strand RNA interference and antisense strand Loop structures are connected to form hairpin structures, construct containing GFP reporter gene CAV1 gene shRNA lentivirus vector, and identified by sequencing; with the constructed shRNA-CAV1 lentivirus infected 293T cells, according to the expression after 72h of infection in each group and the number of GFP fluorescence of the dilution factor to calculate the virus titer, and hippocampal neurons Pre-infection experiments. (4) Preparation of drug-containing serum was measured by MTT assay serum containing different concentrations of the impact on the cells; Aβ1-42 diluted in sterile triple-distilled water to a final concentration of 2.5mmol/L,37℃ incubated 3d, system into a state of aggregation of Aβ1-42, when the rat hippocampal neurons, according to survival time differences induced neuronal cell morphology and p-Tau protein levels, select the best induction long. (5)Building a successful CAV1-shRNA lentivirus normal hippocampal neurons to silence CAV-1 gene; by building a successful pEGFP-C1-CAV1 plasmid normal hippocampal neurons to CAV-1 gene overexpression; containing serum intervene. Quantitative analysis by Western blot CAV-1, GSK-3β, p-Tau levels, used to determine/confirm CAV-1 protein in the regulation of GSK-3β, p-Tau levels is the key factor. (6)By building a successful pEGFP-C1-CAV1 hippocampal neurons transfected with a plasmid to overexpress CAV-1 gene; hippocampal neurons induced by Aβ1-42, build AD cell model; intervention with drug-containing serum. And cell morphology was observed by fluorescence microscopy, Western blot analysis of quantitative CAV-1, GSK-3β, p-Tau levels to explore and reinforce brain Act hyperphosphorylation of Tau protein effects.Results1. The primary cultured neonatal rat hippocampal neuronsNeuronal morphology:training four hours later, just adherent cells were round and transparency, refraction good, larger cell soma soma or about 1/3 of the smaller cells, evenly distributed. Training one day, visibility minority cell in the body cells with projections to generate projections length accounts for 1/2 of the cell body. Cultivation of two days, see an increase in the number of cells or about 1/3 of the projection of the total cell growth projections account for 1.5-3 times the length of the cell body. Training three days, see the cell body faster and increase the number of long processes of cells also increased, soma full, with pyramidal cells is more common, the protrusion length growth is not obvious. Training of four days, and a further increase in neurite extension of the initial formation of a sparse mesh. Training of five days, with time training, neurons increases, sudden trunk and sub-cellular technology significantly longer and thicker, more dense network formation. Cultivation of seven days, the cells slightly aggregation, cell processes significantly increase the length to form a more complete network.2. Identification of hippocampal neuronsImmunofluorescence identification results:green fluorescence under a fluorescence microscope is the dendrites of hippocampal neurons, red fluorescence that is hippocampal axons, the two superimposed figure is a projection of hippocampal neurons (dendrites and axons). Western blot identification results:1st day due to fewer cells, only a small number of neurons with projections, so the MAP-2, Tau protein expression levels rarely; the first three days neuron dendrites, axons number has increased, the MAP-2, the expression of Tau protein has increased; day 5 dendrites, axonal elongation and thickening to form a network, so the MAP-2, the expression of Tau has more.3.pEGFP-C1-CAV1 expression plasmidThe recombinant plasmid unidirectional sequencing results with NCBI’s Gene Bank CAV-1 rat gene sequence BLAST homology of 100%, and the front and rear cleavage site is consistent with the plasmid vector sequences, no mutation, deletion, confirmed the correct construction of the plasmid sex. Plasmid liposome ratio 5μl: 7.5 μl transfection, GFP fluorescence can get a better expression (transfection efficiency), and good cell state.4. shRNA-CAVl Construction and expression of lentiviralLentivirus sequencing results showed that the structure of CAV-1 shRNA sequence is correct, without missing its base peak figure, no overlapping peaks. Virus titers (averaged) for 9×108TU/ml, containing per milliliter represents able to infect and enter the number of the viral genome into the target cells was 9x108 months, sufficient appeal. Preliminary experiments obtain optimal virus MOI of 10, good expression in target cells in hippocampal neurons.5. Preparation and evaluation of AD cell model containing serumSerum containing different concentrations of hippocampal neurons inhibition rate, increasing the concentration of relations with western medicine containing serum drug concentrations in serum at 10% comparing with the blank indifference, in order to maintain the consistency of the experiment, so choose containing 10% Serum intervention. Aβ1-42 induced hippocampal neurons with time, the cell viability decreased at 12h survival rate was 79%,60% survival rate at 24-36h below; cells after 12h fuzzy boundaries, began to gather,retraction, part cell processes decreased, there projections broken at 24-48h, cell necrosis; p-Tau-Ser396 protein expression levels over time increments,12h at the highest, but decreased in 24h,24h which is due to cell necrosis resulting in reduced protein expression. To ensure the viability of neurons better, get a better shape and lead to abnormal phosphorylation of Tau protein we chose induction length of 12h.6. The impact of law and restoring brain hippocampal neurons CAV-1, p-Tau levelsIn normal hippocampal neurons CAV-1 gene silencing that CAV-1 level down will cause reduced GSK-3β Ser9 phosphorylation sites, phosphorylation at Tyr216 point rise, the activation of GSK-3β activity, so that at Thr231 and Ser396 Tau protein phosphorylation in elevated; overexpression CAV-1 gene, i. e. upregulated CAV-1 levels will result in GSK-3β phosphorylation at Ser9 point rise, at Tyr216 site reduced phosphorylation, inhibits GSK-3β activity, so that Tau protein reduced Thr231 and Ser396 phosphorylation sites.7. The method of influence and reinforce AD brain hippocampal neurons CAV-1, p-Tau levelsModeling after 12h, under an inverted microscope mode (+) group of neurons in a large number of protruding broken, soma disintegration serious, very poor state, indicating the ideal modeling results; and solid (+) group, although there are a small number of dead cells appear neurons partially protruding broken, but the overall growth of the state yet it is good. Vouchers visible brain processing solution containing serum on Aβ1-42 induced hippocampal neurons have a protective effect, can greatly improve their growth state, delaying its decline and apoptosis. Hippocampal neurons in AD, Aβ1-42 after modeling cause GSK-3β phosphorylation sites Ser9 decreased phosphorylation at Tyr216 point rise, the activation of GSK-3 P activity, so that Tau protein in Thr231 and abnormal phosphorylation of Ser396 sites. And with a liquid containing serum brain consolidate intervention and over-expression of CAV-1, the increase in the p-GSK-3β Ser9, reducing the p-GSK-3β Tyr216, inhibits GSK-3β activity, so that Tau protein in Thr231 and Ser9 abnormal phosphorylation sites is reduced.Conclusions1.AD is the basic pathogenesis of days has this deficiency, caused by virtual reality, phlegm blind orifices. Guben-Jiannao methods is basic and effective therapies.2.Guben-Jiannao methods can promote and reinforce brain hippocampal neurons and improve CAV-1 protein levels in AD cells.3.Adjust the CAV-1 levels may cause changes in hippocampal neurons GSK-3β activity, thereby causing changes in the level of p-Tau, during CAV-1 protein is a key factor.4. Guben-Jiannao methods can increase brain consolidate CAV-1 expression, so that p-GSK-3βSer9 expression levels increased and p-GSK-3β Tyr216 lower expression levels, thereby reducing the activity of GSK-3β, serve to reduce abnormal phosphorylation of Tau role in the formation CAV-1/GSK-3β signaling pathway.5. Guben-Jiannao methods can better protect law brain hippocampal neurons.