Neuroprotective Effects Of BDNF And Noggin Modified BMSCs In Focal Cerebral Ischemia In Rats

Objecitive:1. To establish the method of the isolation, cultivation and gene transfection of bone mesenchymal stem cells (BMSCs).2. To evaluate the influence of transfection of Ad-GFP、Ad-GFP-BDNF. Ad-GFP-Noggin to BMSCs and the expression of BDNF and Noggin.3. To observe the effects of BMSCs with BDNF and/or Noggin on the promotion of recovery after middle cerebral artery occlusion (MCAO) in rats.4. To analyze the mechanism of BMSCs with BDNF and/or Noggin on the promotion of recovery after MCAO in rats.Method:1. BMSCs were identified by their phenotypical properties and their ability to differentiate into osteocyte and adipocyte. Ad-GFP、Ad-GFP-Noggin gene and Ad-GFP-BDNF gene were transfected to the BMSCs. The target proteins were measured with Western blot.2. MCAO model was esteblished by nylon strand and identified by TTC staining.3. BMSCs modified with different genes were injected into MCAO rats through femoral vein at24th hours after MCA occlusion. mNSS was used to evaluate the neurological severity. The distribution of survival transplanted cells was observed through immunofluorescence.4. The influence of transplantation to the expression of VEGF, BCL-2/Bax, GSK3β/p-GSK3β, Akt/p-Akt and TLR4/MyD88around isehemie area was studied through Western blot and immunohistochemical assessment. The levels of MMP-9and ROS around isehemie area were assayed by ELISA method.Results:1. The cells selected by their adherence ability had basic phenotypical properties of BMSCs, and could differentiate into osteocyte and adipocyte.2. BMSCs without transfection and those transfected with Ad-GFP expressed BDNF at low level, and did not express Noggin. BMSCs modified by Ad-GFP-Noggin and Ad-GFP-BDNF could express Noggin and BDNF respectively with high efficiency.3. The MCAO operation resulted in contralateral hemiparesis. The infarct area could not be stained by TTC. The neurological function of all rats was improved in different level one week after BMSCs transplantation (modified with or without Noggin and BDNF), and rats which got BMSCs modified with both BDNF and Noggin had the lowest mNSS level.4.1week after the transplantation, survival BMSCs could be observed both in and around the infarct area.3weeks after the transplantation, survival BMSCs could be observed only around the infarct area. There was no BMSCs in the infarct area, and a few BMSCs could been seen in the contralateral corpus striatum and subcortex.5. All the transplanted groups had more expression of VEGF、BCL-2、p-GSK3β and p-Akt around the infarct area than the vehicle group, and animals got gene modified BMSCs had more expression of VEGF、BCL-2、 p-GSK3β and p-Akt than those without (P<0.01). The expression of Bax、TLR4and MyD88was less in all the therapeutic groups, especially the combinating transfected group(P<0.05). There was no difference in expression of GSK3β and Akt between any group. All the transplanted groups had lower level of MMP-9and ROS, especially the combinating transfected group.Conclusion:1. BMSCs could be isolated and purified from rat bone marrow through the adherence ability and subculture.2. Ad-GFP、Ad-GFP-Noggin and Ad-GFP-BDNF could transfect BMSCs safely, and the transfected cells could express BDNF and Noggin persistently and efficiently. The transfection of both Ad-GFP-Noggin and Ad-GFP-BDNF to BMSCs is safe and efficient.3. The injection of different gene modified BMSCs could improve the neurological function of MCAO animals, and the transplanted cells could survive around the infarct area.4. The transplantation of BMSCs promotes the expression of VEGF、BCL-2、 p-GSK3β and p-Akt around the infarct area, decreases the expression of Bax、TLR4and MyD88. In addition, the transplantation of BMSCs could decrease the level of MMP-9and ROS. Thus, our results demonstrate that BMSCs transplantation supresses MCAO focal ischemia-induced apoptosis and inflammation, possibly through Akt/GSK3β and TLR4/MyD88pathway. Moreover, BDNF and/or Noggin transfected BMSCs transplantation could increase this effect. The transplantation of BMSCs modified with BDNF and Noggin is a promising therapeutic method in the treatment of ischemic stroke.