The Effect Of CYP19A1 And Estrogen On Alzheimer’s Disease

Objective:To explore the effect of CYP19A1 gene expression and estrogen on the cell model of Alzheimer’s disease, further to provide experimental basis to illustrate the intrinsic relationships between Alzheimer’s disease and CYP19A1 and estrogen.Methods:1.The gestational age of 18 to 19 d C57BL/6J fetal mouse were taken to separate the hippocampus and use the trypsin digestion method for primary cell culture. The state of cells were observed under inverted phase contrast microscope. Tubulin related biomarkers microtubule atssociated protein 2(MAP2) and Hoechst33258 immunofluorescence staining were used to identificate the purity of hippocampal neurons.2. The cell model of Alzheimer’s disease was established by dealing hippocampal neurons with Aβ25-35.Different concentration gradient of Aβ25-35was used to interfere hippocampal neurons for 48 h, and then the cell activity and apoptosis were detected by CCK 8 and Hoechst 33258, respectively.3. Different concentration gradient of 17 β-estradiol was used to interfere the cell model of Alzheimer’s disease for 48 h, and then the cell activity and apoptosis were detected by CCK 8 and Hoechst 33258, respectively.4. The plasmid H4134 template were employed to amplify CYP19A1 gene by the polymerase chain reaction (PCR); Restrict ion enzymes Spe I-HF and XbaI enzyme were used to restriction enzyme digestion to cut off pLenti CMV-EGFP-P2A-MCS-3 flag (H138) expression vector; Seamless cloning kits were used to connect the purpose gene segments and linearization carrier;The PCR were employed to identify positive clones of pLenti CMV- EGFP P2A-CYP19A1 carrier. Lentivirus packaging was conducted by Obio trans fect ion reagent, the method of quantitative polymerase chain reaction (qPCR)was used to test the titer;human embryonic kidney cells(293 T) were transfect by recombinant lentivirus, the GFP expression was observed and the expression of objective plasmid was detected by Western Blot.5. Hippocampal neurons were transfected by lentivirus of CYP19A1 overexpression for 72 h, then interfered by Aβ25-35 for 48 h, and then the cell activity and apoptosis were detected by CCK 8 and Hoechst 33258. respectively.Results:1.The state of hippocampal neurons obtained from this culture method was very good. The purity of neurons was more than 95%and they could survive for 3 weeks stably.2. The hippocampal neurons cell model of Alzheimer’s disease was established stably, the optimum concentration of A β25-35 was determined to be 20uM.3. 10-4mol/L,10-5mol/L 17β-estradiol had protective effect on hippocampal neurons against injury and apoptosis caused by the A Aβ25-35.4. The positive clone pLenti CMV-EGFP P2A-CYP19A1 carrier was obtained successfully, and can transfect the 293 t cells successfully.The lentivirus of CYP19A1 overexpression was build successfully.5. The lentivirus of CYP19A1 overexpression can transfect hippocampal neurons cells successfully, CYP19A1 overexpression had protective effect on hippocampal neurons against injury and apoptosis caused by the Aβ25.Conclusion:CYP19A1 overexpression and 17β-estradiol had protective effect on hippocampal neurons against injury and apoptosis caused by the Aβ25-35.