| Objective: BDNF (Brain Derived Neurotrophic Factor) is the second member of the neurotrophic factor (NTF), in the brain and spinal. BDNF is a major synthesis by brain tissue to able to maintain a variety of the central nervous system, neuronal survival and promote the growth of nerve axons basic protein. But BDNF expression is a small quantity in the normal central nervous system. When the injured spinal cord tissue following a partial increase in expression of BDNF can promote functional recovery of spinal cord neurons, but the expression of instability and shorter processing time. Researches of delivery neurotrophic factors encoded by adenoviral vector for protecting the original neurons of injured nerves and promoting peripheral regeneration with the specificity and efficiency have become the forefront of research areas of nerve repair. In this study, recombinant replication defective adenoviral vectors encoding BDNF gene (AdBDNF) jointly with adenoviral vector without encoding any exogenous gene (Ad0) or with CTLA4Ig gene (AdCTLA4Ig) was transferred to the spinal cord parenchyma of rat lumbar enlargement, in order to Compared two groups of BDNF gene expression in the spinal cord anterior horn of the situation, in order to study of adenovirus-mediated CTLA4Ig gene into the BDNF gene co-expression.Methods: 98 Wistar rats aged 7-week-old, weighing 200-250g. The rats were randomly divided into three groups: The normal control group (A group) Adenovirus does not import; experimental control group (B group) Ad0 + AdBDNF transfer group; experimental group (C group):AdCTLA4Ig + AdBDNF transfer group. Group B and Group C rats fixed on the stereotaxic instrument after intraperitoneal injection of ketamine / Sierra hydrochloride anesthesia (75-100mg/kg +5 mg / kg). 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 (1.0×109pfu/ml) AdBDNF and 1.0μl (5×109pfu/ml) Ad0 in group B, or 1.0μl (1.0×109pfu/ml) AdBDNF and 1.0μl (1.0×109pfu/ml) AdCTLA4Ig in group C was injected with micro-injector under a propeller by the same person. The needle tip is 45°head ward 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 A,B and C group, lumbar enlargement of spinal cord was taken 0ut ,cut into 40μm thick successive cross section to stain by Immunohistochemistry the BDNF and count positive cells and Determination immunoreactive products were integrated optical density (IOD ). Immunofluorescence staining AdCTLA4Ig continued expression of time and using PCR to monitor B, C groups exist time of the virus, and RT-PCR to detect the three groups BDNF gene in the rat expressed in the spinal cord situation.Results:1 BDNF and CTLA4Ig expression1.1 CTLA4Ig expression in spinal cord: the experimental group after two days it can be seen positive cells, 45 days after operation for sustainable over time after 60 days in non-immune cells were positive.1.2 BDNF expression in spinal cord: group A, rat spinal cord ventral horn motor God moving yuan and spinal cord dorsal horn sensory neurons and some glial cells can be seen a small amount of BDNF-positive cells, the positive product subcellular localization mainly distributed in the cytoplasm. Group B and group C was more positive cells in the presence of bilateral anterior horn of spinal cord motor neurons and surrounding glial cells. Group B the BDNF-positive staining cells, and immunoreactive products in the value of integrated optical density (IOD) significantly increased and peaked at 7 days after operation, over time, the expression of 30 days is close to normal control group, Group C the BDNF-positive staining cells, and immunoreactive products in the value of integrated optical density (IOD) increased significantly and similarly in the peak after 7 days and the expression was higher than the normal group (A group) and experimental group (B group), but the peak was significantly prolonged the duration of expression at 60 days with the normal group (A group) and experimental group (B group) no significant difference. Statistical analysis showed that three groups of BDNF-positive staining cells, and immunoreactive products in the value of integrated optical density (IOD) in 4 days, 7 days, 15 days, 30 days 45 days were significantly statistical difference (P <0.05), BDNF-positive staining cells and immune-IR spectral density plot (IOD) in 2 days and 60 days among the three groups had no significant statistical difference (P> 0.05).2 PCR and RT-PCR Detection:2.1 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, the group B to 45 days and group C to 60 days were not detect the expression of adenovirus.3 RT-PCR detection3.1 BDNFmRNA expressions in the rat spinal cord: The RT-PCR method from the mRNA levels were detected BDNF group A group B and group C: 2 days after transfection of the three groups can be detected the BDNFmRNA in spinal cord. Group B and Group C expression of the peak are 7 days after operation. Experimental group and the high expression levels of experimental control group can continue to 30 days and 15 days. Statistical analysis shows that between the two groups during peak periods BDNFmRNA expression were significantly difference (P <0.05), group at different time points compared BDNFmRNA expression were significantly difference (P <0.05).3.2 CTLA4IgmRNA expression in the rat spinal cord tissue: The RT-PCR method from the mRNA levels was detected CTLA4IgmRNA expression in the experimental group. 2 days after transfection the experimental group could be detected CTLA4IgmRNA the expression in the rat spinal cord tissue to express the peak increase after 4-7 days, high expression can be continued until 2 weeks, to the first 45 days to disappear.Conclusion: In normal Wistar rat spinal cord, there are a small number of BDNF positive cells and expression of trace amounts of BDNFmRNA. The experimental group will AdV-mediated CTLA4Ig gene was injected can effectively inhibit local T lymphocyte infiltration, thereby reducing by AdV-mediated local inflammatory response, inhibit the body's rejection of the viral vector and significantly extend the common import BDNF gene in spinal cord expression of time, and enhance its co-import intensity of BDNF gene expression.
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