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Influencing Factor of Adenoviral-mediated LacZ Genes Expression in Spinal Cord

Author: LiNing
Tutor: HanJiuHui
School: Hebei Medical University
Course: Surgery
Keywords: Adenoviral vector LacZ gene CTLA4Ig Spinal cord Transfection efficiency PCR Flow cytometry Immune response
CLC: R651.3
Type: Master's thesis
Year: 2008
Downloads: 22
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Abstract

Objective:With the development of transportation and industry and agriculture, peripheral nerve injury become a common disabling disease on clinic, and the treatment of peripheral nerve injury has been a difficult clinical problem. The research of gene therapy has become the forefront and hot spots in the field of peripheral nerve injury; adenovirus vector (Adenovirus, AdV)-mediated gene transfection has been shown many advantages, which AdV become the one of most charismatic carrier in the study of nerve regeneration, nerve tracer neuromuscular disease research and gene therapy experiment. Some exogenous gene was transferred into the nervous system with adenoviral vector, adenoviral-mediated exogenous genes expression in the nervous system play a role on promoting the regeneration of peripheral nerve, however the most important issue is adenoviral-mediated exogenous genes expression in Spinal Cord and peripheral nerve cells. In this study, 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. By polymerase chain reaction (PCR) to monitor the loss of time after adenovirus injection in the spinal cord and the specificity of the adenovirus with the minimum dilution. By reverse transcriptase polymerase chain reaction (RT-PCR) compare encoding CTLA4Ig to LacZ gene expression in the rat spinal cord. Using flow cytometry in order to investigate the effect of immune tolerance induced by CTLA4Ig and its mechanism and influencing factor of adenoviral-mediated LacZ gene expression in spinal cord.Methods: 200 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 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)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 5 min and withdrew slowly after the injection. The wound was rinsed, stopped bleeding thoroughly, sprinked antibiotics to and then closed. At 10 different time points within 10 weeks post injection, 2ml blood was taken from caudal vein of two groups. Lumbar enlargement of spinal cord was taken out at 10 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. By polymerase chain reaction(PCR)to monitor the disappearan- ce of time after adenovirus injection in the spinal cord and the specificity of the sensitivity of adenovirus.By reverse transcriptase polymerase chain reaction(RT-PCR) to test and compare encoding CTLA4Ig to LacZ gene expression in the spi- nalcord. Using flow cytometry to detect blood T lymphocyte’s change.Results1 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 0.6cm at each side of the injective spot. The expression in the injective side was much fiercer than that of the opposite side. Persisting period of the positive sections was 2weeks in the group A, but 4weeks 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 difference ofβ-gal expression in two groups (P>0.05). 2 PCR detection2.1 Adenovirus PCR detection: Adenoviral expression decrease with the lowering of the concentration.The virus after the series of diluting could be detected by PCR reaction,the specificity of the adenovirus with the minimum dilution is 10-4. The specificity of AdlacZ (1×109pfu/ml) and Ad0 (5×109 pfu/ml) with the minimum dilution are the same concentration.2.2 Adenovirus in spinal cord PCR detection: Using PCR method to detect the level of adenovirus expression in the control group and the experimental group, the expression of adenovirus gradually decresed, Adenovirus dosen’t be detected the expression of adenovirus until 10th week.3 RT-PCR detection After operation we can detect CTLA4IgmRNA andβ-gal mRNA expression.β-gal expression in Group B is longer than that in group A. Statistical analysis showed that there was no significant difference ofβ-gal expression in two groups during peak periods(P>0.05). Statistical analysis showed that during different time in the two groups there are significant difference(P<0.05).4 Flow cytometry test: Infiltrating leukocytes were detectable within 2 days of injection and reached a maximum by 9 days. Thereafter the number of infiltrating cells decreased, but a small number persisted in Spinal Cord until day60. In 2 day after injection, the percentage of CD8 T-lymphocyte was detected increase whereas the percentage of CD4 T-lymphocyte presented in the Infiltrating population did not significantly increase until day 60, peaking on day 15. Experimental group CD4 and CD8 T-lymphocyte radio were less than the control group, statistical analysis showed that during peak periods the two groups of cells have no significant difference(P>0.05). Statistical analysis showed that during different time the two groups of cells have significant difference (P<0.05).Conclusion: 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 not be influenced. CTLA4Ig can’t obviously inhibit humoral immunoresponse. Thus we can conclude that time, immune response and the concentration of adenovirus are influencing factor of adenoviral-mediated LacZ gene expression in Spinal Cord.

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