| Objective: With the development of society, all kinds of trauma has occurred more and more nervous system damage associated with the number rapidly increased. but because of the complexity of the nervous system and special repair nervous system damage repair is still a very difficult problem. With the development of molecular biology, for the repair of injured nervous system using gene therapy approach, exogenous neurotrophic factor gene by vector-oriented nerve stump into the use of receptor cells and efficient secretion of the long-term neurotrophic factor, promoting the growth of nerve axons, thus promoting nerve injury repair, in order to obtain good results. Although the use of transgenic technology for nerve repair provides a very good direction, but because of exogenous receptor gene and vector role of immune rejection, making a long-term efficiency of exogenous gene expression can not be achieved, which also limits the the effect of nerve repairing. In response to this bottleneck, the present study, to import with a cytotoxic T lymphocyte-associated antigen-4 Ig (Cytotoxic T lymphocyte- associated antigen 4 immunoglobulin, CTLA4Ig) gene adenovirus AdV (Ad CTLA4Ig) and carrying LacZ gene AdV (AdLacZ ) micro-syringe into the common rat spinal cord lumbar enlargement division, as the experimental group, will not carry the exogenous gene Ad0 and carrying LacZ gene AdV (AdLacZ) through the micro-syringe into the common rat spinal cord lumbar enlargement division, as the control group, two groups of by comparing theβ-gal expression changes in the spinal cord and by polymerase chain reaction (PCR) to monitor the spinal cord after injection of adenovirus amount of change and disappearance time and the use of reverse transcription-polymerase chain reaction (PT-PCR) to detect CTLA4Ig gene and LacZ gene expression in the rat spinal cord to explore the CTLA4Ig induce local immune tolerance to the AdLacZ the role of 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. 54 Wister female rats aged 7 weeks were randomly divided into 4 groups: group A, C, the AdlacZ+Ad0 transfer group and group B,D, 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,C, or 1.0μl (1×109pfu/ml) AdlacZ and 1.0μl (5×109pfu/ml) AdCTLA4Ig in group B,D 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. group C and group D after 30 days from the first operation after the median artery in the left side of the spinal cord 1mm injections AdlacZ (1×109pfu/ml) and Ad0 (5×109pfu/ml) of 1μl (group C) or AdLacZ (1×109 pfu / ml), and AdCTLA4Ig (5×109 pfu / ml) of 1μl (group D). The remaining operations the same way. The experimental group and control group transfected with adenovirus to take after the 9 time points near and far side of rat spinal cord injection sites 5mm segment of the lumbar enlargement, injection nodding side of the spinal segment to 5mm thick frozen cross-sections 50μm continuum, the experimental group and the control group, X-gal staining of the sections counted, and by polymerase chain reaction (PCR) to monitor the spinal cord after injection of adenovirus amount of change and disappearance time, using reverse transcription-polymerase chain reaction (PT-PCR) Detection of CTLA4Ig gene and LacZ gene expression in rat spinal cord. Results:1 X-gal, CTLA4Ig transgene expression: LacZ gene and CTLA4Ig gene transfection of the spinal cord anterior horn motor cells of bilateral and surrounding glial cells. Transgenic expression of the upper and lower limit the scope of the 0.5cm section of the injection site and expressed from time to time the peak of transgenic expression of the number of positive frozen cross-sections≤120 Pian. Slice the control group were observed under experimental X-gal staining positive expression of time is about 15 days; while the experimental group of X-gal staining positive expression of time is about 60 days. Count of the experimental group of spinal cord sections showed positiveβ-gal expression in the spinal cord lumbar enlargement of the peak are in four days and 9 days, 15 days after theβ-gal in the spinal cord lumbar enlargement of X-gal staining positive for expression of the gradual decrease in the 60 Days can still see the low-expression. Statistical analysis showed that between the two groups during the height of the number of positive biopsy were significantly different (P <0.05). And AdLacZ gene expression in the spinal cord significantly longer time.2 Ad. PCR tests: the expression of adenovirus with the decrease of the concentration gradually decreased. Solution using 10-fold serial dilutions of the virus extracted DNA by PCR reaction can detect adenovirus-specific bands of the lowest dilution of 104. AdlacZ (1×109pfu/ml) and Ad0 (5×109 pfu / ml) specific bands of the lowest dilution line.3 PCR detection of adenovirus samples of spinal cord: The PCR method from the adenovirus DNA levels were detected in the experimental control group, the expression of the experimental group, adenovirus of the DNA volume is decreasing with time, the experimental control group to 39 days detected the expression of adenovirus in the experimental group 60 days can still detect the expression of adenovirus .4 RT-PCR results of the experiment4.1 The expression ofβ-gal RNA in the rat lumbar spinal cord enlargement at local Using RT-PCR method from the mRNA levels were detectedβ-gal in the experimental control group, the expression of the experimental group: 2 days after transfection of the two groups can be detected inβ-galmRNA lumbar enlargement in the rat spinal cord tissue of the control group Service was not detected in the 30-day goal ofβ-galm bands, the experimental group at 60 days, one can still see the expression ofβ-galm target band, but in order to significantly darken the brightness can be seen in the initial and secondary adenovirus-Import Import Adenovirus 30-dayβ-galmRNA is still a low level of expression.4.2 CTLA4Ig mRNA in the rat spinal cord lumbar enlargement of local expression of the organization (see figure 20) using RT-PCR method to detect the CTLA4IgmRNA from the mRNA level of expression in the experimental group. Transfected 2 days after the experimental group could be detected CTLA4IgmRNA lumbar enlargement in the rat spinal cord tissues, in the first 60 days of adenovirus into the second import of adenovirus at 30 days of the objectives can be seen CTLA4IgmRNA bands can be seen most light and for the first time in four days and 9 days into office, the second into the first nine days of adenovirus initial import of adenovirus compared to the first 9 days out of dark.Conclusion: In the pre-sensitized rats, the AdV-mediated CTLA4Ig gene was injected into the spinal cord lumbar enlargement Department can effectively inhibit local T lymphocyte infiltration, thereby reducing by AdV-mediated local inflammatory response, inhibit the body of the virus vector rejection, significantly increased the expression of LacZ gene in the spinal cord the time, and can significantly enhance its co-import intensity of LacZ gene expression. And AdCTLA4Ig on humoral immune response significantly inhibited, could significantly prolong adenovirus in vivo lifetime.
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