Therapeutic Study Of Adipose-derived Neural Stem Cell Transplantation On Rat Modals Of Parkinson Disease

Objective:To explore curative effects and mechanism of dipose-derived strom cell (ADSCs) induced into neural stem cells (NSCs) transplantation on Parkinson s disease (PD) rat model. Methods:1)ADSC were isolated and cultivated in vivo by collagenase typeⅠ. The third passage cells were identified by stem cells common marker CD29,CD44 and CD45 and were detected with flow cytometry. The nerve stem culture medium was established by the basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27. The third passage cells were induced to NSCs and neurocytes. Meanwhile observe the induced cellular morphology change, then cell expression of the neuron specific markers nestin,NF-200 and GFAP was determined with immunofluorescence.2) The 6-hydroxydopamine (6-OHDA) induced hemiparkinsonian rats with single eara double position injection strategy. APO induced rotation was quantities. The number change of TH in striatum detected by immunohistochemical staining.3)The successful Parkinson models were allocated randomly into two groups:the induced NSCs group(n=22), the physiological saline groups (n=22) and the normal groups (n=16). The cells were labeled by Brdu before transplanted 48 hours. The adipose-derived neural cells (1.2×106 cells/each model) with Brdu-labeled and physiological saline (12μl) were injected into right striatum of PD rat respectively. The APO-induced rotation test was taken at 2 week,4 week,8 week after transplantation and before transplantation. Brain frozen sections were carried out immunofluorescence double staining at 8 week of ADSCs to observe the live and differentiation of transplanted cells. The quantities of TH in striatum detected by immunohistochemical staining.The level of dopamine in nigrostriatal system were observed by High-performance liquid chromatography. The expression of TH mRNA in rat brain after transplantation by RT-PCR. Results:1) cell detection:①ADSCs detection:The morphology of ADSCs is uniform, monolayer and nemaline grow. Their common marker CD29 is (99.00±0.12%), CD44 is(95.62±0.68%), CD45 is (0.13±0.12%).②NSCs and nerve cell detection:The induced cells become sphere and can multiplication, their nestin positive express with immunofluorescen. when add serum, they can different nerve cells and neuroglial cells (NF-200, GFAP positive expressionl). 2)modal detection:There were 44 (44%) rats among 100 rats which were injected apomorphine (APO) showed steady rotation to the left (the rotation/time>280r/40min). So the PD model was regarded eligible. TH immunoreactive cells of striatum were lessened obviously on injured side.3) The detection after cells transplantation:①The rotation tests:Frequency of change rotation were significantly more in induced than physiological saline group at 4 week and 8 week after transplantation, but there are no difference at 2 week.②The immunofluorescence detectation:Some Brdu/nestin, Brdu/NF-200, Brdu/GFAP positive cells could be found in the implanted region of ADSCs induction group at 8 week after transplantation, but there are no Brdu/TH positive cells.③RT-PCR show:The expression of TH mRNA in rat brain of NSCs group more than physiological saline group.④High-performance liquid chromatography show:the DA content in rat brain of NSCs group more than physiological saline group. Conclusions:adipose-derived neural stem cell can therapy PD rat modal through increasing TH mRNA and DA in rat brain.