| Objective:To confirm that Guiling Pa'an Pill can promote the proliferation,migration and differentiation of the neural stem cells(NSCs)transplanted in the brain of rat models of Parkinson's Disease(PD).To explore the underlying action mechanism of Guiling Pa'an Pill by observing its impact on the behavior of NSCs transplanted PD rats,the survival of the transplanted NSCs in the substantia nigra of rat brain and the protein expression of Nurr1 and Shh.Methods:12?g of 6-hydroxydopamine(6-OHDA)was injected into the right substantia nigra compacta of the rats to establish PD rat models.14days after model establishment,gave apomorphine(APO)by intraperitoneal injection(0.15mg/kg);models constantly rotating to the left with in total rotating laps more than 210 times in 30 minutes were regarded as successful.Divide PD rats into six groups(high dose group of Traditional Chinese Medicine(TCM),medium dose group of TCM,low dose group of TCM,Madopar group,model group,control group).Took 14-day old pregnant rats,opened the abdominal cavity after anesthesia,removed fetal rats under sterile conditions,isolated the hippocampus of fetal rats,and prepared single cell suspension.Prepared primary and passaged cultures with the technique of serum free cultivation.Identified the protein expression of Nestin through immunocytochemical staining to check whether the cultured cells were the NSCs.Prepared passaged NSCs cell suspension with a concentration of 1×10~6/L.Marked the NSCs with Brd U 24 hours before inject.The control group rats were inject 5?l of saline solution into the right striatum.The other five groups were inject with same amount of the suspension into the right striatum of the rats.The high dose group,medium dose group and low dose group were orally gavaged with Guiling Pa'an Pill(3.6g/kg,1.8g/kg and 0.9g/kg respectively);Madopar group were orally gavaged with Madopar(50mg/kg);the model group and control group were orally gavaged with saline in same volume.4 weeks later,still injected APO to induce rotation of the rats,observed the change of rotation laps and then executed the rats.Paraffin embedded sections were prepared with substantia nigra.Detected the number of Brd U marked cells and the protein expression ofNurr1andShhinsubstantianigrabyimmunohistochemistry.Semi-Quantitative Study was conducted with LEICA micrograph system and digiscope-analysis microscopic image acquisition and analysis system.Data analysis was done with SPSS18.0 statistical analysis software.Results:The cells cultured in vitro were NSCs with the evidence that they were Nestin positive.The rotation laps per minute of rats reduced significantly in both the treatment groups and the model group.Compared with the model group and the control group,all the groups treated with Guiling Pa'an Pill had higher survival rate of the transplanted NSCs in the substantia nigra of rat brain and protein expression of Nurr1 and Shh(P<0.05).Conclusion:NSCs transplantation can improve the symptoms of PD rats,and its efficacy can be enhanced by Guiling Pa'an Pill.The underlying action mechanism of Guiling Pa'an Pill might be that it up regulates the expression of Nurr1 and Shh.High level expression of Nurr1 and Shh,provides beneficial microenvironment for cells,increases their apoptosis resistance and promotes the secretion of related neurotrophins.These improve the survival rate of transplanted NSCs and induce them to differentiate into mature functional dopaminergic neurons. |
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