Zuogui Pill In Vitro To Induce Mscs Differentiation And Transplantation In The Treatment Of Mice With Spastic Cerebral Palsy

ObjectivesNewborn period is the most important period for the development of brain and brain demage to recovery which commanly seen in cerebral palsy patients. Only in this period with proper measures can we repair premature brain tissue injury partly or completely. In this study we applied traditional Chinese medicine Zuoguiwan and mNGF to enhance the differenciation of NSCs from MSCs, to increase the amount of NSCs and possibly find new mentod to treat cerebral palsy (CP). Mouse spastic cerebral palsy models were established and NSCs transplanted to examine the effects. The mechanism of Zuoguiwan on the differenciation of MSCs, which was a representative medicine of Bushenyisui theory in traditional Chinese Medicine was also explorated. The final aim of this study was to establish new method to treat CP in the early period after it happened.MethodsThe study included four parts.Part One was the collection, cultivation, separation and expansion of mouse MSCs. The first step was the collection of bone marrow liquid from SD mouse femour. The technique of gradient density centrifugation was applied to separate the MSCs.Part Two was an in vitro study on the effects of Zuoguiwan and mNGF on MSCs proliferation and differentiation. Then the MSCs were divided into 4 groups to conduct control studies. Zuoguiwan, mNGF, and both of them were used as influnence factors for different groups. The traditional protective agents against oxidant in the differentiation process such as DMSO,β- Mercaptoethanol was not applied.Part Three was the establishment of spastic CP model with 49 mice.The technique applied was continued electric stimulation on the pyramidal nerve for 8 minutes to destroy its function. The mouse models appeared as hemiplegia and keep stable condition for a long time.Part Four was an in vivo controled study on the effects of NSCs after local transplantation into brain tissue through the same approach as in Part Three. BBB (or Basso, Beattie, Bresnahan) locomotor rating scales was applied to evaluate the treatment results. Two models in each group were excuted at 0,1,2,4,6 weeks to have pathologic study on the repairment of brain damage by NSCs transplantation by HE staining. ResultsThe collection, cultivation, sepatation and expansion of mouse MSCs was ralatively quick and easy. Flowcytometry analysis of the surface antigens of the differentiated cells had typical MSCs phenotype and showed highly homogeneity.Control study in Part Two showed that both Zuoguiwan and mNGF could enhance the differenciation of NSCs fron MSCs, and the effect of mNGF was better than Zuoguiwan. When both of them were applied, synergy effect was clear. Histochemistry study showed that the NSCs had possitive Nestine, NSE, GFAP and other landmark of NSCs.In Part Three the technique applied was continued electric stimulation on the pyramidal nerve for 8 minutes to destroy its function.48 qualified mice models were acquired.In vivo transplantation of NSCs showed that neuron division and proliferation happened in local tissue. Three weeks after transplantation, movement of the hemiplegia mice improved obviously.As the representative prescription of Bushenyisui theory, Zuoguiwan and many other similar traditional Chinese medicines could enhance the differenciation of NSCs from MSCs. We believe that Bushenyisui theory has close relationship with stem cells. We postulate that the genital essence in traditional Chinese medicine might be the multipotent stem cells, which could differentiate into many kinds of specific cells. Applying the Bushenyisui method, the multipotent stem cells was activated and differentiated into specific cell groups, repaired tissue damage, the function and clinical appearances improved.The results of this primary study encourage us to continue our efforts in this field.We wish that one day the technique will be perfect and could be applied in clinic.Then, millions of patients could benefit from this technique.Conclusions●The cultivation, sepatation and expansion of mouse MSCs were ralatively quick and easy. Zuoguiwan and mNGF could enhance the differenciation of NSCs from MSCs, and the effect of mNGF was better than Zuoguiwan.●It is the first report about the effect of mNGF on the differentiation of MSCs. When both of them were applied, synergy effect was clear. Protective agents against oxidant in the differentiation process were not applied. Continued electric stimulation on the pyramidal nerve for 8 minutes to destroy its function successed in acquiring qualified spastic hemiplegia mouse models. ●In vivo transplantation of NSCs showed that cell division and proliferation happened in local brain tissue. Three weeks after transplantation, movement of the hemiplegia mice improved obviously. Further experiments with Primates are necessary before the technique could be applied in clinic.