动物造模,帕金森病大鼠模型 z-bio 2022-09-01 289

　　Objective: To establish a Parkinson's disease model in human α-synuclein A30P mutant transgenic rats.

　　Methods: The lentivirus system was used to construct the wild-type α-SYN vector pLKO-CMV-α-SYN-WT-P2A-GFP and the α-SYNA30P mutant vector pLKO-CMV-α-SYN-A30P-P2A-GFP, respectively. 293FT cells were transfected into 293FT cells, and the expression level of α-SYN was detected by WB 24 hours after transient transfection. After lentivirus packaging and concentration, the virus dilution was injected into the substantia nigra pars compacta of rats by stereotaxic technique to overexpress α-SYN. Wild-type and A30P mutant lentiviral particles. Immunofluorescence staining was used to detect the distribution of α-SYN and tyrosine hydroxylase (TH), and to observe the changes in the number of dopaminergic neurons in the substantia nigra pars compacta. The Rotatingrod test was used to evaluate the behavioral changes of rats injected with A30P lentivirus.

　　Results: The gene expression vectors of wild-type and mutant A30P could highly express α-SYN protein in 293FT cells. The results of TH immunofluorescence showed that compared with the virus dilution group, the wild-type and A30P mutant overexpressed α-SYN were significantly higher than those in the virus dilution group. The number of dopaminergic neurons in the substantia nigra pars compacta decreased in all rats, while the substantia nigra neurons in the α-SYNA30P lentivirus injection group were lost more, with a significant difference. Immunofluorescence at the site of neuron deletion showed that TH staining was almost negative in this area, and a large number of α-SYN protein aggregated in this area. It was further revealed that overexpression of α-SYN A30P can lead to the reduction and degeneration of dopaminergic neurons. In addition, the results of rotatingrod experiments showed that overexpression of α-SYN A30P rats exhibited obvious progressive motor impairment.

　　Conclusion: The PD model of human α-SYNA30P mutant transgenic rats was established by lentivirus system, which lays a foundation for the research on the pathogenesis of PD and drug development.