The Establishment Of Neuroblastoma Aβ₄₂/neuro-2a Cell Line Stably Expressing Aβ₄₂

Objective：To construct recombinant plasmid pIRES2-EGFP-Aβ₄₂carrying Aβ₄₂gene，transfect the plasmid into murine neuroblastoma Neuro-2a cells. To establish a stableNeuro-2a cell line expressing Aβ₄₂, offer a new approach for the vitro study of Alzheimer’sdisease.Methods: Aβ₄₂gene digested from pGEMT-Aβ₄₂was ligated to pIRES2-EGFP genedigested from pIRES2-EGFP-LHX8to construct the recombinant plasmid. Use T4DNAligase ligated pIRES2-EGFP gene to construct the no-load plasmid. After enzyme digestionanalysis, the recombinant plasmid pIRES2-EGFP-Aβ₄₂and no-load plasmid were transfectedinto Neuro-2a cells in the mediation of liposome respectively. Use G418（800mg/L） to screenthe recombinant plasmid transfected cells for4weeks, the concentration of G418was kept at200mg/L in the subsequent course of cell culture. Neuro-2a cells were amplified and culturedin low concentration. Hematoxylin-eosin staining was utilized to inspect the morphology ofNeuro-2a cells. The transfection efficiency of target gene was detected by convertedfluorescence microscope. MTT test was used to examine the multiplication capacity ofNeuro-2a cells.The expression of Aβ₄₂and the distributional pattern of tau were analyzed byimmunohistochemistry staining.Results: Recombinant plasmid pIRES2-EGFP-Aβ₄₂and no-load plasmid pIRES2-EGFPconfirmed corrected by enzyme digestion analysis. The recombinant plasmid and no-loadplasmid transfected cells successfully expressed green fluorescent protein and presented greenfluorescence after amplifying and G418screening. Hematoxylin-eosin staining found that themorphology and proliferative capacity of Neuro-2a cells didn’t change significantly whencultured in low concentration. MTT test demonstrated that the multiplication capacity oftransfected cells didn’t impaired compared to N2a cells if cultured in low concentration.Immunohistochemistry staining confirmed the expression of Aβ₄₂in G418screened Neuro-2acells which transfected recombinant plasmid. On the contrary, Neuro-2a cells whichtransfected no-load plasmid or didn’t transfect gene didn’t express Aβ₄₂.Immunohistochemistry staining found that the distributional pattern of tau in cells whichtransfected recombinant plasmid was granular. Instead, tau in cells transfected no-loadplasmid or didn’t transfected gene is evenly distributed. Conclusions：Recombinant plasmid pIRES2-EGFP-Aβ₄₂was correctly constructed. Astable Neuro-2a cell line expressing Aβ₄₂was established. Demonstrated that the existence ofAβ₄₂lead to the aggregation of tau.