Effect Of GUI Ling Pa'an Wan On The Expression Of TH,GDNF And Ptx3 In The Substantia Nigra Of Rats With PD After Transplantation Of NECs

Objective:Based on the neural stem cells(NSCs)as the donor cell,the cell replacement therapy is currently a hot research of Parkinson's disease,and the cell replacement therapy is also considered to be the greatest potential in Parkinson's disease treatment,and the best strategies for clinical application.But with further study,scientists find that: the survival rate of these neural stem cells is very low,and can seriously affect the long-term effect of Parkinson's disease.Traditional Chinese medicine has its unique advantages in the treatment of Parkinson's disease,so we choose Parkinson's disease rat model based on 6-OHDA as the research object,and observe the effect of Gui ling pa'an Wan on the expression of TH,GDNF and Ptx3 in the substantia nigra of rats with Parkinson's disease after transplantation of NECs,and explore the possible mechanism of effect of Gui ling pa'an Wan on NSCs after transplantation into the substantia nigra of rats with Parkinson's disease,and find a new method to improve the long-term clinical efficacy of NSCs transplantation for the treatment of Parkinson's disease.Methods:We chose male and female SD rats which were mature,and caged them by 2:1 to obtain Ed16 pregnant rats,and isolated the hippocampal cells from the fetal rats,and dissociated them to single-cell suspension by mechanical trituration.And then these cells were inoculated into the culture medium of neural stem cells(DMEM/F12,B27,EGF,FGF,Penicillin-Streptomycin)to be cultured and passaged,and identified with Nestin by immunofluorescence.With the differentiation medium of neural stem cells(DMEM/F12,FBS,B27,EGF,FGF,Penicillin-Streptomycin),these cells were induced to differentiation adherently.Finally,made the identified NSCs with Nestin marked by BrdU and identified the BrdU.Part two:firstly,6-OHDA was injected stereotactically into the substantia nigra of the rats to establish the rat model of PD,and then APO was injected intraperitoneally into the rats one week later to observe the behavioral changes of the rats.Randomly these successful rat models of PD were divided into 6 groups: the high dose of traditional Chinese medicine(TCM)group,middle dose of TCM group,low dose of TCM group,Madopar group,NSCs transplantation control group and blank control group.What's more,NSCs tagged by BrdU were injected stereotactically into the striatum of these rat models of PD according to different groups,intragastric administration and normal feeding.And then observed the behavioral changes of rats respectively before the transplantation and 4 weeks after transplantation,intragastric Administration for 30 days,and then took the brains of these rats,BrdU was detected by immunohistochemical method in rat striatum and substantia nigra of TH,GDNF,Ptx3,in situ hybridization in substantia nigra compact THmRNA HE staining observation.Statistical methods: SNK test was used to compare the differences among different groups,and Pearson correlation analysis was performed between different indices.Results:1.The primary cultured cells were mostly spherical in shape,and had different sizes and strong refractivity.The cells began to agglomerate 24 hours later,several or dozens cells,after 7 days of culture hundreds of cells gathered into nerve bulbs,compact arrangement and the regular edge.2.Passage cells began to agglomerate into the cell ball with dozens of cells after culture for 24 hours,compared with the primary cells,it was faster for the passage cells to agglomerate into the cell ball,and the cell ball was more regular.3.The results of Nestin immune fluorescence showed that the primary cells and passage cells almost all showed positive expression of Nestin,showing green fluorescence,and scattered Nestin positive cells were also observed.Nestin positive cells were also found in the F4 cells,but the positive expression of Nestin was weakened.4.24 ?g 6-OHDA was injected at a single point into the substantia nigra of SD rats,and the success rate of rats model of PD group was 71%.5.After analysis of SNK of the ratio of the number of rotating ring(K),the results showed that the K of all the three groups of Guiling pa'an Wan were higher(P<0.01),showing that the three groups of Guiling pa'an Wan improved the symptoms of PD rats more obviously.6.All the three groups of Guiling pa'an Wan had more BrdU positive expression in rat striatum than other groups(P<0.01),that was to say that the three groups of Guiling pa'an Wan in striatum had more surviving NSCs,showing that Guiling pa'an Wan could have the effect to promote the survival of exogenous NSCs.7.Compared with the NSCs transplantation control group,all the three groups of Guiling pa'an Wan had more TH and TH-mRNA in substantia nigra(P<0.01),that was to say that the Chinese medicine group had more DA neurons than the NSCs transplantation control group,showing that Guiling pa'an Wan could promote the exogenous NSCs differentiation into DA neurons.8.Compared with the NSCs transplantation control group,all the three groups of Guiling pa'an Wan had more GDNF and Ptx3 in substantia nigra(P<0.01),showing that Guiling pa'an Wan could increase the expression level of Ptx3,GDNF,and could indirectly inhibit neuronal apoptosis to promote its survival.9.Pearson analysis of TH and GDNF,TH and Ptx3 respectively,the results showed that TH and GDNF were significantly positively correlated(r=0.879,P=0.000),and TH and Ptx3 were significantly positively correlated(r=0.943,P=0.000)Conclusions:The NSCs can obtained mechanically from the hippocampus of Ed16 fetal rats,and NSCs can be cultured,passaged and identified with Nestin successfully.After transplantation into the striatum of the rats,Guiling pa'an pill combined with neural stem cells can effectively improve the symptoms of PD rats,what's more,the middle dose of TCM group improves more obviously.On the one hand,Guiling pa'an Wan can promote the survival of exogenous NSCs,which can directly promote the exogenous NSCs differentiation into DA neurons.On the other hand,Guiling pa'an Wan can promote the expression of GDNF and Ptx3 in the brain of PD rats,and can indirectly inhibit neuronal apoptosis to promote its survival.