Effects Of NGF, Noggin Modified RBMSCs On Experimental Cerebral Infarct Rats

Objective:1.The method for the isolation and cultivation of rat bone mesenchymal stem cells(rBMSCs)was established to offer an experimental foundation for the further research.2.The transfect efficiency of 3 kinds of recombinant adenovirus including Ad-GFP,Ad-GFP-NGF and Ad-GFP-Noggin was studied.The influence of transfection and the expression of NGF and Noggin was observe.3.The effects of chemical supplement,NGF and/or Noggin in inducing rBMSCs into neuron-like cells were discussed.4.The effects and mechanisms of rBMSCs with NGF and/or Noggin on the promotion of recovery were studied after middle cerebral artery occlusion(MCAO)in rats.Methods:1.rBMSCs were isolated from SD rat bone marrow and purified through adherence ability.These cells were identified as rBMSCs by their phenotypical properties and their ability of differentiating into osteocyte and adipocyte.2.The proper MOI was chosen through recombinant adenovirus density gradient,rBMSCs were divided into 5 groups:rBMSCs,rBMSCs transfected with Ad-GFP,rBMSCs transfected with Ad-GFP-NGF,rBMSCs transfected with Ad-GFP-Noggin,rBMSCs transfected with Ad-GFP-NGF combined with Ad-GFP-Noggin.Detecting the influence of transfection to rBMSCs and the expression of NGF and Noggin through immunofluorescence and Western blot.3. Inducing rBMSCs differentiate into neuron-like cell,and observing their expression of neuron specific marking proteins NF(H).4.Establishing MCAO model by nylon strand,rBMSCs modulated with different genes were injected into MCAO rats through femoral vein at the 5th day after MCA occlusion.Modified neurological severity score(mNSS)was used to study the recovery of neurological deficits The distribution of survival transplanted cells was observed,and their differentiation into neurons and astrocytes was detected through immunofluorescence.The influence of transplantation to the expression of VEGF and Synaptophysin around ischemic area was studied through immunohistochemical assessment.Results:1.The cells selected by their adherence ability had basic phenotypical properties of rBMSCs,and could differentiate into osteocyte and adipocyte.The rBMSCs with good activity and high purity could be obtained by subculturing.2. When MOI=50 Ad-GFP,Ad-GFP-NGF and Ad-GFP-Noggin could transfect rBMSCs with high efficiency and low toxicity,rBMSCs transfected with recombinant adenovirus had green influorescence autonomically.BMSCs without transfection and those transfected with Ad-GFP expressed NGF at low level,and did not express Noggin.After transfected with Ad-GFP-NGF and Ad-GFP-Noggin respectively, rBMSCs had positive reaction to NGF or Noggin correspondingly through immunofluorescence and Western blot using antibodies against NGF and Noggin. BMSCs transfected with both Ad-GFP-NGF and Ad-GFP-Noggin had positive reaction to both to NGF or Noggin at the same time.3.rBMSCs had neuron-like shape after 5 hours in chemical supplement.NF(H),but astrocytic marking protein GFAP,was positive in them.And these cells died after 48 hours.The shape of rBMSCs transfected with Ad-GFP was not change.In Ad-GFP-NGF transfected group,Ad-GFP-Noggin transfected group and combinating transfected group,the cells got the shape of neuron,some of them were positive to NF(H)and negative to GFAP.These cells grew in good state.The ratio of NF(H)+ cells in combinating transfected group was the highest(p＜0.05).4.The MCAO operation resulted in contralateral hemiparesis.The infarct area could not be stained by TTC,and got ischemic pathological changes.At the 3rd week after injected rBMSCs modified with gene,the neurological function of all rats was improved in different level,and the rats which got rBMSCs transfected by both Ad-GFP-NGF and Ad-GFP-Noggin had the greatest recovery(p＜0.05).3 weeks after the transplantation,survival rBMSCs could be observed around the infarct area.Some of them were positive to NF(H),some others to GFAP.The rats transplanted with rBMSCs modified with two genes had more survival cells and higher ratio of NF(H)+ or GFAP+ cells than other groups (p＜0.05).The ratio of GFAP+/GFP+ cells was always higher than NF(H)+/GFP+ cells in all animals(p＜0.05).3 weeks after the transplantation,all transplanted rats had more VEGF and Synaptophysin positive cells around the lesion than rats with PBS or with no transplantation(p＜0.05).Among them,animals with rBMSCs modified with two genes had the greatest expression of VEGF and Synaptophysin (p＜0.05).Conclusion:1.rBMSCs could be isolated and purified from rat bone marrow through their adherence ability and subculture.This method was simple,and would not influence the activity of cells,rBMSCs could be identified effectively by phenotypical properties and multipotention.2.Ad-GFP,Ad-GFP-NGF and Ad-GFP-Noggin could transfect BMSCs safely.The transfected cells could express GFP,NGF and Noggin persistently and efficiently.The transplantation of Ad-GFP-NGF combined with Ad-GFP-Noggin to rBMSCs is safe and efficient.3. Chemical supplement,NGF and Noggin could induce rBMSCs into neuron-like cells in vitro.But chemical supplement interrupted the survival of cells,while NGF and Noggin could improve their proliferation.When two proteins exist simultaneously, this differentiation could be further enhanced.4.MCAO model made by nylon strand was relevant to human situations of cerebral infarction.The transplantation of rBMSCs modified with different genes,especially with both NGF and Noggin, intravenously could improve the neurological deficits of MCAO animals.The transplanted cells could survive around the infarct area.Some of them could differentiate into cells with phenotype of neuron or astrocyte.The expression of VEGF and Synaptophysin increased after the transplantation,and this indicated that the transplantation could improve the angiogenensis and neuronal plasticity in ischemic area.