The Effect Of Adenovirus-mediated BDNF Gene On The Expression Of BDNF Gene In Spinal Cord

Objective:With the development of transportation, industry and agriculture,peripheral nerve injury become a common disabling disease on clinic.Sophisticated microsurgery skills have made neurorrhaphy precise,however,the functional recovery of injured nerve is unsatisfactory.Researches of delivery neurotrophic factors encoded by adenoviral vector to spinal cord for protecting the original neurons of injured nerves and promoting peripheral regeneration have become more popular.The process of the nerve regenerating is a very complex prosess of biochemistry and cytology.BDNF is the second member of the neurotrophic factor(NTF),in the brian and spinal.BDNF can maintenance many kinds of nerve cells in central nervous system,promote nerve cells axon growth.But BDNF expression is a small quantity in the normal central nervous system.BDNF expression obviously promote after injure the peripheral nerve,but the expression is not stable,the expression time is short.Researches of delivery neurotrophic factors encoded by adenoviral vector for protecting the original neurons of injured nerves and promoting peripheral regeneration have become more popular.Successful gene transfer in peripheral and central nervous system is characteristic,high performance and safe.But there have been disadvantages of low level and short period of transgene expression in vector infected site with the present used method of delivery of vectors to nervous system.Immune response to adenoviral vectors have severely affected the ability of these vectors to induce long-term gene expression and even caused side effects.There were three group in this study:normal control group(A group), experimental control group(B group),experimental group(C group).Recombinant replication- defective adenoviral vectors encoding BDNF gene(AdBDNF)(C group) or adenoviral vector without encoding any exogenous gene(Ad0)(B gruop) was transferred to lumbar enlargement through micro-injector in spinal cord of Wistar rats.Compare the BDNF expression of A,B and Cgroup.By polymerase chain reaction(PCR) to monitor the exist time after adenovius with the minimum dilution.By reverse transcriptase polymerse chain reaction (RT-PCR) to investigate encoding BDNF gene expression in the rat spinal cord.By above of the methods to research the Influencing Factor of Adenoviral-mediated BDNF Genes Expression in Spinal Cord.Methods:180 Wister female rats aged 7 weeks were randomly divided into 3 groups:group A,the AdBDNF+Ad0 transfer group and group B,the AdBDNF+AdCTLA4Ig transfer group.After anesthetized by ketamine,the animal were fixed on the stereotaxic instrument.About 2cm length of posterior median incision was made,T13 vertebral plate was removed and the spinal cord was exposed.At the site 0.8mm right to the posterior median artery of spinal cord,1.0μl (1×109pfu/ml )AdBDNF and 1.0μl(5×109pfu/ml) Ad0 in group A,or 1.0μl(1×109pfu/ml) AdBDNF and 1.0μl(5×109pfu/ml) AdCTLA4Ig in group B was injected with micro-injector under a propeller by the same person.The needle tip is 45°headward and downward at the depth of 2.5mm.The injecting speed is 1μl /min.Needles were stuck 5min and withdrew slowly after the injection.The wound was rinsed,stopped bleeding thoroughly, sprinked antibiotics to and then closed.At 7 different time points within 5 weeks post injection of the B and C group,in any time of A group,lumbar enlargement of spinal cord was taken Out,cut into 40μm thick successive cross section to stain the BDNF and count positive cells.The peak time and persisting period of gene expression in three groups were studied.The titer of serum neutralizing antibody was assayed.By polymerase chain reaction(PCR) to monitor the apperarance of time after adenovirus injection in the spinal cord.By reverse transcriptase polymerase chain reaction(RT-PCR) to test BDNF gene expression in the spinal cord.Results:1 BDNF expression in spinal cord:Ventral horn motor neurons and glial cells in spinal cord was targeted by the BDNF gene and CTLA4Ig gene transfer.The scope of transgene was limited in 0.5cm at each side of the injective spot..The expression in the injective side was much more fierce than that of the opposite side.Persisting period of the positive sections was 3 weeks in the group A;but 5 weeks in the group B.The positive sections were counted and showed that the peak time of gene expression were 2～8days,there was no significant.1 BDNF expression in spinal cord:Ventral horn motor neurons and glial cells in spinal cord was targeted by the BDNF gene transfer.The scope of transgene was limited in 0.5cm at each side of the injective spot.The expression in the injective side was much more fierce than that of the opposite side. Persisting period of the positive cells was 5 weeks in the group B and group C.The positive cells were counted and showed that the peak time of gene expression were 7days after injecting in the group B and group C.The postive cells in group B was more than group A in any time,and the postive cells in group C was more than group B.Analyzed by statistics and showed that there were significant difference of the postive in the theree group.2 PCR and RT-PCR detection2.1 Adenovirus in spinal cord PCR detection:Using PCR method to detect the level of adenovirus expression in group and the experimental group,the expression of Adenovirus gradually decresed,Adenovirus dosen't be detected the expression of adenovirus until 10 week.2.2 BDNF geng expression in spinal cord RT-PCR detectionAfter operation we can detect BDNF mRNA expression.BDNF gene expression in group B is longer than in group A.Statistical analysis showed that there was no significant difference of BDNF expression between the two groups during peak periods(P＞0.05).Statistical analysis showed that there are significant difference during different time between the two groups.Conclusion:The postive cells of the BDNF stain in the normal rat exist in the Ventral horn motor neurons cells in spinal cord.Spinal cord can be injured mechanically (micro-injector stimulation) and chemically(partial inflammation and organism immunoreactions caused by adenovirus in rat's spinal cord) by the injection of non-mediated ectogenic gene adenovirus Ad0,which can increase the expression of BDNF in spinal cord.BDNF can be more expressed in spinal cord by the injection of adenovirus-mediated ectogenic BDNF gene than AD0.This result showed BDNF had the ability of aggregation.The expression of BDNF in spinal cord can last for three weeks by the injection of AdBDNF which was inspected by the RT-PCR of BDNFmRNAd.