Experimental Study Of Intracerebral Co-graft Of Neural Stem Cells And Olfactory Ensheathing Cells For Treating Parkinson Disease

In vitro:To investigate the effects of olfactory ensheathing cells(OECs) on proliferation and differentiation of neural stem cells(NSCs). NSCs and OECs were separated from ventral midbrain and olfactory bulb of embryonic rats aged 14.5 days. Divide the cells into two groups: group A :NSCs co-cultured with OECs group ;group B :NSCs cultured solely. Observe the proliferation and differentiation of NSCs and OECs by P75 stained immunochemistry and P75, Brdu/Nestin,GFAP,NF,P75/Nestin single or double-stained immunofluorescence respectively . Most of OECs on the 7th day tested by P75 stained immunochemistry appears to be positive. NSCs formed typical floaty neurospheres on the 7th day, the results of immunofluorescence double stained by Brdu/Nestin were positive .On the 14th day, some neurospheres in group B were proned to differentiation. Most of the cells were GFAP positive cells marked with red fluorescein . Few of them were NF positive cells marked by green fluorescein .The neurospheres in group A grew accreting on OECs, stretched its tubcles began differentiation from the 12th day. On the 14th day , both kinds of cells clustered together intensively. P75/Nestin immunofluorescence showed Nestin positive NSCs marked by red fluorescein and P75 positive OECs marked by green fluorescein . GFAP and NF stain showed the majority are NF positive cells marked by green fluorescein , the minority are GFAP positive cells marked by red fluorescein. Not only percentage of the NF positive cell was 69.5% more than 47.2% of control group but also percentage of the NF/TH positive cell was 36.7% more than 6.34% of control group.In vivo:To observe the effect of neural stem cells and olfactory ensheathing cells co-graft in the rat model of Parkinson disease. The singel cell suspensions derived from E14.5 rat embryonic mesencephlon were prepared and plated into the serum-free medium containing DMEM/F12(1:1),EGF,bFGF,and B27supplement.After the primary neurosphere were formed,the single cell suspension of primary neurosphere were diluted by limiting dilution method,and single cell clone sphere of neural stem cell were obtained with successive proliferation. The neurospheres were labled by using immunofluorescence detection of Brdu and Nestin.The rat models of PD were established with injecting 6-OHDA into the SNC and VTA of the rat brains and divided into three groups:NS(0.9%saline)tranplatation group,NSCs tranplatation group,OECs tranplatation group and NSCs+OECs tranplatation group.The concentration of cells for tranplatation was1.0x105cells/μl.The change of rotation behavior was measured compared with three groups at different time point(7d,14d,30d,60d) after tranplatation.The survived cells were detected by HE and Brdu immunocytochemical stain;the differentiated cells were marked by TH immunocytochemical stain. 1,Single cell neuropheres consisting lots of cells were isolated from E14.5 rat embryonic mesencephlon after two weeks culturing.Large amounts of single cell clone spheres derived from one NSCs were obtained by successive passage.The neuropheres exhibited both Brdu and Nestin positive(90%).2,Compared with control groups,there was no obvious changes in the first three weeks after transplantation.But at 30d and 60d time point,the rotation of rat groups of NSCs ,groups of OECs and groups of NSCs + OECs transplatation were distinctly improved( P<0.05). 3,The brain tissue were stained HE one day later after transplantation.The cells were gathered around the transplatation channel. 4,Brdu immunocytochemistry:Brdu+ cells body were small and karyons were snuff color .The cells shape were roundness,oblong,polygon.A large amount of cells were found in the core of the transplantation.And the Brdu+ cells were decreased quickly as time went through.5,TH immunocytochemistry: The emergence of TH+ cells was at 14d after transplantation.TH+ cells shape were roundness or oblong and there were no perimeters on the cells.The positive cells were filemot.TH+ cells were gradually increased at 30d after transplantetion.We also found small perimeters on these cells.Compare with NSCs group,there are more TH+ cells in the NSCs+ OECs group. 1,Our cell line belong to NSCs and is multipotent, which has the ability to undergo self-renew. So there are the stem cells of the central nervous system. 2,The results indicated OECs can promote the proliferation and differentiation of NSCs. 3,The transplantation of NSCs has a little efficiency on the treatment of PD.4,OECs may promote the NSCs derived from E14.5 rat embryonic mesencephlon to turn into dopaminergic neurons.The rotation behavior were obvious meliorated in the PD rats by using NSCs+ OECs.