Establishment Of Cell Models For Parkinson's Disease In Vitro And The Neuroprotective Effect Of Citicoline On Dopaminergic Neurons

In the thesis, we set up cell models of degeneration of dopaminergic neurons by using mice mesencephalic dissociated culture in vitro. The toxicity of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPP*) and excititory animo acid (glutamate) were investigated and evaluated. The neuroprotective effect and possible mechanism of citicoline on the dopaminergic neurons were studied. It will provide a theoric base for its further application in the treatment of Parkinson's disease (PD) and to a better understanding of the possible mechanism of PD. 1 Cell models for PD in vitro1.1 The toxicity of MPP+ on dopaminergic neurons in mouse mesencephalic dissociated culture1.1.1 The morphological change of dopaminergic neurons induced with MPP*Our results showed that after incubation with different concentrations of MPP+ (0.1-15 umol/L), the density of the dopaminergic neurons was significantly reduced, and the procesesses became shorter and even lost. The numbers of the processes reduced significantly. Most of the processes were distort and swollen. 10 umol/L MPP+ induced the death of dopaminergic neurons in two different ways. The loss and degeneration of dopaminergic neurons in MPP+-treated groups were mainly in a relative chronic way that the loss and shortening of the processes were found first and then subsequently the cell bodies were lost. Only few dopaminergic neurons underwent death through another pathway in which the dopaminergic neurons bodies were lost before the processe lossing.1.1.2 The time-dependent effect of MPP+ on the loss of dopaminergic neuronsThe time course of dopaminergic neurons death induced with MPP+ was investigated.The numbers of TH-ir neurons in the control group, 2-, 4-, and 8-hour MPP+-treated group were 840+15.5, 791 +21.9, 849 + 31.7 and 790 + 31.7 respectively. The numbers of TH-ir cells in the latter three groups didn't decrease as compared with those in the control. After 24 hours incubation of MPP+, the loss of TH-ir neurons became appearant. The TH-ir cell numbers per well were 677 + 6.12,412 + 26.60,229 +20.31 and 191 + 15.23 in 24-, 48-, 72-and 96-hour MPP+-treated groups respectively. So the loss of dopaminergic neurons was related to the incubation time of MPP+. And there was a significant difference between any two groups which have been treated with MPP+ for 24, 48 and 72 hours. The numbers of dopaminergic neurons reduced by about 20% after 24-hour incubation with 10umol/L MPP+, 50% after 48 hours, and 75% after 3 days or 4 days. 1.1.3 the dose-dependent effect of MPP+ on the loss of dopaminergic neuronsIn order to clarify the dose effect of MPP+ on the loss of dopaminergic neurons, MPP+ of different final concentrations (0.1, 1, 10and 15 umol/L) were added into the cultures on the 10th DIV, and incubated for 48 hours. Our datas showed that the dopaminergic neuron loss induced with MPP+ was in a dose-dependant pattern. The numbers of dopaminergic neurons were 100% in the control group, while 70.3 + 6.38 %, 67.4 + 4.92 %, 51.7 + 2.95 % and 50.9 + 5.60 % in the 0.1,1,10, and 15 umol/L MPP+-treated groups respectively. The numbers of TH-ir cells were significantly lower than that in the control (p<0.05). The toxicity of MPP+ on the degeneration of dopaminergic neurons was dose-dependent. But there was no statistical significance between the 0.1umol/L group and 1 umol/L group, as well as 10 umol/L and 15 umol/L. But there was statistical difference between 1 umol/L and 10 umol/L. 1.2 The toxicity of MPP+ on the human neuroblastoma cell line (STA-NB-3)The cultures of human neuroblastoma cell line (STA-NB-3) were treated with different concentrations of MPP+ (62.5, 125, 250, 500 and 1000umol/L) and incubated for different days (1, 2, 3 and 4 days). Then the cell viability was determined by MTT assay. Our data showed that after treatment with 500 umol/L and 1000 umol/L of MPP+ for 1 day reduced the cell viability to 82.6 + 5.38% and 75.1 + 5.80% of the control respecrively (P<0.05).The cell viability decreased by 28% after treatment with 62.5 umol/L of MPP+ for 2 days. T...