The Effect Of NSCs On The Expression Of Neurotrophins And Apoptosis Gene At The Transected Spinal Cord And The Related Sites In Rats

【Objective】We investigated effects of transplanted neural stem cells(NSCs),which derived from the hippocampus of green fluorescent protein(GFP)transgenic pregnant mouse,on the hind limb motor as well as sensory function recovery.And the survival and differentiation of the transplanted NSCs were also observed as well as the expression ofneurotrophins and apoptosis gene.【Methods】NSCs,derived from hippocampus of GFP transgenic pregnant mouse, were transplanted into injured site of spinal cord of rats in acute phase.The motor functional recovery of hind limb was evaluated using Basso-Beattie-Bresnahan(BBB) scoring at each weekend in 4 weeks after injury or cells transplantation respectively. The CSEP and BDA tracing experiment were performed to observe the regeneration of motor and sensory fibers.After the spinal cord was obtained and done frozen section,survival and differentiation of NSC in vivo were investigated by immunohistochemistry and scar tissue were observed by using HE method.Then mRNA of neurotrophins,their receptors and apoptosis genes were tested by using RT-PCR.The SPSS 11.5 statistical analysis software package was used to analyze the data.【Results】1.Neurospheres were cultured in vitro,and the immunohistochemistry against nestin demonstrated that these cells were NSCs.Some appeared NeuN positive staining,while other were GFAP positive staining.All of them have capability of secreting NGF,BDNF,NT-3.2.Behavioral test showed that BBB score in NSC transplanted group was significantly higher than operation group(P＜0.05) 3.Sensory functional test showed that eclipse periods of P1 and N1 in operation group were indifinitly long.In NSCs transplanted group,the eclipse periods of P1 and N1 were significantly shorter than those in sham operation group.4.BDA tracing showed that some brown positive staining fibers were observed to disperse sparsely at caudal side of the injured spinal cord in NSCs transplanted group but not in operation group,5.After transplantation,NSC were survived in host,and about 15%present GFAP positive staining while 10%appeared NeuN positive staining.Other 75%cells, emiting green light,present neither NeuN nor GFAP positive staining,but NGF, BDNF,NT-3 positive.6.After spinal cord transection or transplantation of NSCs,the mRNA expression of various factors in the injured spinal cord and related site showed as follows.①After transection,mRNA of NGF in cerebral cortex decreased at 7th day and there was no obvious change in other factors(P＞0.05).In the rostral side of injured site at different time after transection,mRNA ofNGF(14th day),TrkB(3rd,7th and 14th day),Fas(3rd and 7th day)in operation group were significantly less than the sham operation group(P＜0.05),while PDGF(1st day),TrkC(1st and 7th day),Bcl-2 (1st and 14th day),Caspases-3(1st,7th and 14th day)were more increased(P＜0.05).Other factors,like BDNF,IGF,TGF-B1,FGF,TrkA,BAX and Netrin,had no change(P＞0.05).Comparing with rostral side,the change in caudal side was obvious.The mRNA expression ofCNTF(1st and 3 th day),PDGF(1st,3 th,7th and 14th day),TGF-β1(1st,3 th,7th and 14th day),FGF(1st day),TrkB (1st,7th and 14th day),Bcl-2(1st and 14th day),Fas(1st,3 th and 14th day) and Netrin(7th and 14th day)in operation group were higher than the sham operation group(P＜0.05),but FGF(3rd day)decreased(P＜0.05).There were no differences in expression of NGF,IGF,TrkA,TrkC,Bax.In the muscle dominated by motor neurons,TrkC(3rd day)and Bcl-2(1st day)were significantly increased in operation group(P＜0.05).②After the transplantation of NSCs,the expression of some factors changed in different sites:In cerebral cortex,mRNA of NGF(3rd and 7th day)after transplantation increased much more than the operation group,but without any difference in other factors.At rostral side of injured site in transplanted group,TrkB (3rd,7th and 14th day)and Fas(3rd and 7th day)was significantly increased comparing with the operation group(P＜0.05),while TrkC(1st and 7th day), Bcl-2(1st,3rd,7th and 14th day)and Caspase(1st and 14th day)decreased(P＜0.05).There were no differences in NGF,BDNF,PDGF,IGF,TGF-β1,FGF, TrkA,IGF and Bax.At caudal side after transplantation,mRNA expression of many factors decreased comparing with operation group,including NGF(14th day),CNTF (3rd and 14th day),PDGF(1st and 14th day),IGF(3rd and 14th day),TGFβ1 (1st,7th and 14th day),FGF(1st,7th and 14th day),TrkA(1st,3rd,7th and 14th day),TrkB(1st,3rd,7th and 14th day),TrkC(1st and 3rd day),Bcl-2 (1st,3rd,7th and 14th day),Fas(1st,3rd and 14th day),Bax(3rd and 14th day) Netrin(1st,3rd,7th and 14th day).In musculus soleus after transplantation,mRNA of Bcl-2(7th and 14th day)was increased but TrkA(7th day)and Netrin(7th day) decreased.And TrkC,TrkB,Fas,Bax had no obvious change.【Conclusion】1.NSC derived from the hippocampus of GFP transgenic pregnant mouse could be transplanted to treat spinal cord transection in rats,and could improve hind limb motor function.2.After transplantation,NSC could fill vacancy at injured site,promote the regeneration of motor and sensory fibers and make them pass through scar tissue.3.After transection,the NSC could change the expression of neurotrophins and apoptosis gene so that promote the improvement of motor and sensory funtion.