动物造模,帕金森病大鼠模型 z-bio 2021-01-14 330

　　Purpose: To establish a Parkinson's disease model of human α-synuclein (α-synuclein, α-SYN) A30P mutant transgenic rat.

　　Methods: The lentivirus system was used to construct the overexpression 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 After transfecting 293FT cells and transiently transfecting for 24 hours, the expression level of α-SYN was detected by WB. After that, it was packaged with lentivirus and concentrated, and the virus diluent was injected into the substantia nigra dense part of the rat by stereotactic technology 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 compact area of the midbrain. Rotatingrod experiment evaluates the behavioral changes of rats injected with A30P lentivirus.

　　Results: The wild-type and mutant A30P gene expression vectors can highly express α-SYN protein in 293FT cells. The results of TH immunofluorescence show that the over-expressing α-SYN wild-type and A30P mutants are larger than the virus dilution group. Mice can cause a decrease in the number of dopaminergic neurons in the substantia nigra compact part of the midbrain, while the α-SYNA30P lentiviral injection group has more midbrain substantia neurons missing, which has a significant difference. For the brains of A30P transgenic rats Immunofluorescence of the neuron deletion site found that TH staining in this area was almost negative, and a large amount of α-SYN protein aggregates. This result indicates that the disappearance of brain neurons is accompanied by the aggregation of α-SYN protein and a sharp decrease in TH expression. It further reveals that overexpression of α-SYN A30P can lead to the reduction and degeneration of the number of dopaminergic neurons. In addition, the results of the rotatingrod experiment show that the overexpression of α-SYNA30P rats show obvious progressive motor impairment.

　　Conclusion: The PD model of human α-SYNA30P mutant transgenic rats was established through the lentiviral system, laying a foundation for the research of PD pathogenesis and drug development.