| Objective: Injuries of peripheral nerves have long been a common clinical problem. 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. Successful gene transfer in peripheral and central nervous system is dependent on efficient methods of introducing and expressing a particular transgene in the appropriate population of neural cells effectively. 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. This technique is used in human.Most humans are infected with wild-type adenovirus in the first few years of life. So, in considering the clinical application of gene therapy with adenovirus vectors, it is very important to analyze and control the immune response to the vector in animals previously sensitized with the virus. In this study, in order to improve the express of adenoviral, recombinant replication defective adenoviral vectors encoding LacZ gene (AdLacZ) jointly with adenoviral vector without encoding any exogenous gene (Ad0) or with CTLA4Ig gene (AdCTLA4Ig) was transferred to lumbar enlargement through micro-injector in rats, in order to investigate the effect of immune tolerant induced by CTLA4Ig and its mechanism.Methods: Rats were primed by subcutaneous injection of AdLacZ into the dorsal thorax 7 days before injection of virus into the Spinal Cord. 120 Wister female rats aged 7 weeks were randomly divided into 2 groups: group A, the AdlacZ+Ad0 transfer group and group B, the AdlacZ+AdCTLA4Ig transfer group. After anesthetized , the animal were fixed on the stereotaxic instrument. About 3cm length of posterior median incision was made, T13 vertebral plate was removed and the spinal cord was exposed. At the site 1.0mm right to the posterior median artery of spinal cord, 1.0μl (1×109pfu/ml )AdlacZ and 1.0μl (5×109pfu/ml) Ad0 in group A, or 1.0μl (1×109pfu/ml) AdlacZ 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 5 different time points, 3.5ml blood was taken from rat heart of two groups. Lumbar enlargement of spinal cord was taken out at 5 different time points post injection, cut into 50μm thick successive cross section and stained with X-gal solution. Then positive sections were counted. The peak time and persisting period of gene expression in two groups were studied. The effect of AdCTLA4Ig on LacZ gene expression was observed. The titer of serum neutralizing antibody was assayed. By polymerase chain rection (PCR)to monitor the adenovirus that disappearance of time in blood after adenovirus injection in the spinal.Results :1 X-gal expression in spinal cord: Ventral horn motor neurons and glial cells in spinal cord was targeted by the LacZ gene and CTLA4Ig gene transfer .The scope of transgene was limited in 0.5cm at each side of the injective spot. Persisting period of the positive sections was 30 days in the group A; but 60 days in the group B. The positive sections were counted and showed that the peak time of gene expression were 9～15days, there was significant difference ofβ-gal expression in two groups. (P<0.05). And can improve the expression of time of AdvLacZ.2 The detection of serum anti-adenoviral neutralizing antibodies : The low serum anti-adenoviral neutralizing antibodies titers ( 1 : 4 ) was detected from 15days post transfer in A groups, and remained until day 60. In the B groups, the low serum anti-adenoviral neutralizing antibodie titers(1:4)was detected from 30days post transfer and maintaining day 60 post transfer. The titer of serum anti-adenoviral neutral- lizing antibodies in A group are higher than that in B group , and there there was significant difference in statistically. The standard serum of anti-adenoviral neutralizing antibodies was≥1:128.3 Using PCR method to detect the level of adenovirus expression in group A and group B, the expression of adenovirus gradually decresed. Adenovirus does not be detected the expression of adenovirus until 30 day in group A. And in group B the expression of adenovirus is detected at the 60 day. Conclusion:In the sensitized rats'body, CTLA4Ig can effectually inhibit cytotoxicity T- lymphocyte cellular immune generation and activation, and also can inhibit local inflammatory reaction resulted from AdV. The transgene achieved long-term expression through restrained the immune reaction. The expression of other co-injection of adenoviral-mdiated transgene in spinal cord will be influenced significantly. In adddition, CTLA4Ig can obviously inhibit the humoral immunoresponse. It can lengthen the adenovirus to preserve the time obviously in vivo.
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