Research On PARK2 Gene Expression In Rat Striatal Neurons On Exposure Of Manganese After Silenced By Lentivirus-mediated RNAI PARK2

Objective: Research on the PARK2 expression and its product level—Parkin in the rat striatal neuron, transfected by lentiviral vectors with p GMLV-SC1 RNAi, after exposure to doses of manganese with the same exposure time. And collecting basic data for the further study about the mechanism of manganese on the neurons.Methods: sh RNA targets of PARK2 were designed, and four pairs of sh RNA lentiviral vectors with p GMLV-SC1 RNAi were constructed, which were transfected into striatal neuron. Western blotting and real-time fluorescence quantitative(Real Time PCR) were performed to examine the silence effect. Rat striatal neurons infected with lentivirus group and lentivirus RNAi group were exposed to manganese at concentration of 0, 100,300 and 500 μmol / L for 24 h respectively, the levels of PARK2 m RNA and Parkin were detected by Western blot and real-time fluorescence quantitative(Real Time PCR).Results: Four pairs of lentiviral vector were successfully constructed which were identified by enzyme digestion and sequencing, and rat striatal neurons, infected with lentiviral vectors, were identified by observing the expression of GFP fluorescence. Levels of Parkin and PARK2 m RNA were decreased significantly(P <0.01) compared with the control group and negative control group by Western Blot and Real Time PCR. Although four pairs of sh RNA showed positive silence effect, PARK2 sh RNA4 showed the most significant with inhibition rate(> 81%), while the difference between control group and the negative control group was not significant(P>0.05). Real Time PCR and Western Blot showed the following results: com pared with the control group, the levels of PARK2 m RNA and Parkin decreased signifi cantly(P <0.01) in lentiviral group after exposure to 300 and 500 μmol/L of Mn for 24 h, while the decreases were significant(P <0.05, P <0.01) in Lentiviral RNAi group after exposure to 100, 300 and 500μmol/L of Mn for 24 h..Conclusion: Lentiviral vector of PARK2 sh RNA was successfully constructed, which deceased PARK2 m RNA and Parkin levels in rat striatal neuron. And doses of Mn exposure for 24 hours furthered the decrease.