Influence Of Co-injection Of Adenoviral-mediated CTLA4Ig Genes On Other Transgene Expression In Spinal Cord

Objective: Researches of delivery neurotrophic factors encoded by adenoviral vector (Adv) 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. In this study, mice were injected with the mixture of Adv containing LacZ gene (AdLacZ) and with no transgene (Ad0) or with CTLA4Ig gene (AdCTLA4Ig) in lumbar enlargement through micro-injector, in order to investigate the effect of immune tolerant induced by CTLA4Ig and its mechanism. Methods: 160 Wister female rats aged 7 weeks were randomly divided into 2 groups: in the control group (group A) co-injected with AdlacZ and Ad0; in experimental group( group B) co-injected with AdlacZ and AdCTLA4Ig. After general anesthetized with katamine and xylazine , the animal were position in the stereotaxic instrument.. In group A, a total of 2.0μl of the mixture of AdlacZ 1.0μl (1×109pfu/ml ) and Ad0 1.0μl (5×109pfu/ml) was injected into the site of 0.8mm beside the posterior median artery of spinal cord using micro-injector. AdlacZ 1.0μl (1×109pfu/ml) and AdCTLA4Ig 1.0μl (5×109pfu/ml) in group B was injected by the same way. The needle tip was 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 closed. Mice were killed at 10 different timepionts after the initial injection into spinal cord within 6 weeks. 2ml blood was collected from each mouse and the serum was used for the detection of neutualizing antibody. Frozen sections (50μm) of the injection site were stained by X-gal solution to detectβ-galactosidase. 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 immunohistochemical staining of lymphocyte subgroup of spinal cord sections was performed; the titer of serum neutralizing antibody was assayed. Results1 Effect of co-administration of AdCTLA on transgene In group A, a small amount ofβ-galactosidase was detected only in the nucleus of ventral horn neurons and glial cells at the inject site on day1. Thereafter, positive cells were increased and gradually appeared in the opposite side, peaked on day2 to 8.β-galactosidase expression persisted in the spinal cord around the needle trace for 2 weeks. In group B, cells ofβ-galactosidase and CTLA4Ig expression were detected at the injection site on day1 in spinal cord ventral horn neurons and glial cells. Cells ofβ-galactosidase and CTLA4Ig expression were increased and gradually appeared in the opposite side. The expression in the injective side was much stronger than that of the opposite side. Positive cell increased gradually until day8,β-galactosidase and CTLA4Ig expression decreased and disppared at 4 weeks. The scope of transgene was limited in 0.6cm at each side of the injective spot. The counting of positive sections of X-gal staining peaked from 2d to 8d post injection, but there was no significant difference ofβ-gal expression in two groups(P>0.05). 2 Effect of co-administration of AdCTLA on cellular immune responeIn group A co-injected with AdlacZ and Ad0, CD68 leukocytes were detected in spinal cord at the injection site on day1, and CD8 T-lymphocytes were detected on day2. A small number CD4 T-lymphocyte were detected on day3. CD68 leukocytes disappeared mostly within 6 days. CD8 T- lymphocyte infiltration was mainly detected and only few CD4 T-lymphocyte infiltration was detected from day8. CD8 T-lymphocyts increased until week2, and then decreased gradually. CD8 T-lymphocyts infiltration is more severe than that in group B. Some leukocytes infiltration was still obvious until week6. In group B, CD8 T-lymphocyts infiltration was similar to that in group A on day6. After day9, the degree of cell infiltration was supperssed. CD8 T-lymphocyte infiltration was mainly detected, and couldn't be detected at week4. The degree of CD4 lymphocyte infiltration was not obviously increased in the two groups at all timepoints.3 Effect of co-administration of adCTLA on production serum anti- adenoviral neutralizing antibodiesThe serum anti-adenoviral neutralizing antibodies was detect from day3 post transfer in two groups, and increased similarly within 3 weeks post transfect. The serum anti-adenoviral neutra- lizing antibody was maintaining the high level until week4. The titer of serum anti-adenoviral neutrallizing antibodies in group A was lower than that in group B, but there was no significant differences statistically.Conclusion: Local injection of AdCTLA4Ig into spinal cord supressed cellular immune response effectivelly, and prolonged the duration of transgene expression of the co-injected adenoviral-mediated gene. But the degree of transgene expression of the co-injected gene wasn't affected. Injection of AdCTLA4Ig didn't obviously reduce the humoral immune response to the co-injected adenovirus.