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Influence of Co-injection of Adenoviral-mediated CTLA4Ig Genes on BDNF Gene Transgene Expression in Spinal Cord of Mice with Adenoviral

Author: WangBaoNing
Tutor: HanJiuHui
School: Hebei Medical University
Course: Surgery
Keywords: Adenovirus BDNF gene CTLA4Ig Immunohistochemistry PCR RT-PCR
CLC: R346
Type: Master's thesis
Year: 2010
Downloads: 18
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Abstract

Objective: brain-derived neurotrophic factor (brain-derived neurotrophic factor BDNF) as neurotrophic (neurotrophic factors NTF), the second member of the family, is a major synthesis by the brain tissue, it is possible to maintain a variety of central nervous system neural neuron survival and promote the growth of axons basic protein. BDNF gene in normal rat spinal cord have a small amount of expression, when local tissue cells in the spinal cord injured elevated expression of BDNF, can promote spinal cord neuronal function recovery, but the expression level of instability, of shorter duration. The application of recombinant adenovirus (the Adenovirus ADV) vector into neurotrophic factor gene therapy has become the key topics of the field of neural prosthetics research. Neurotrophic factor into the body tend to see it as a the allogeneic antigen to elicit an immune response, as one of the factors of the application of neurotrophic factor gene therapy. The experiment will carry BDNF gene in ADV (AdBDNF) and without exogenous gene Ad0 or and to carry cytotoxic T lymphocyte-associated antigen 4 immune globulin (Cytotoxic T lymphocyte-associated antigen 4immunoglobulin genes, CTLA4Ig) ADV (AdCTLA4Ig,) Both adenovirus suspension by micro-injection of import rat lumbar enlargement of the spinal cord parenchyma were compared BDNF gene expression in the anterior horn of the spinal cord in order to explore the adenovirus-mediated CTLA4Ig gene BDNF gene expression of common import . 98 Methods: Seven-week-old healthy Wistar rats, weighing 200-250g. The rats were randomly divided into three groups: normal control group (A) normal rats, do not import adenovirus; import AD0 AdBDNF-experimental control group (B group); experimental group (C group) import AdCTLA4Ig AdBDNF. Experimental control group and experimental rats by intraperitoneal injection of ketamine / xylazine anesthesia (75-100mg/kg 5mg/kg), after the success of anesthesia fixed on the stereotaxic instrument. Take about 2cm incision, remove T13 laminectomy exposed spinal cord lumbar enlargement site. Micro-syringe and ST-III-type manual propulsion, the median artery of the right side of the spinal cord after 0.8mm the injections Ad0 (5 × 109pfu/ml) AdBDNF (1 × 109 pfu / ml) suspension Two microliters (group B) or AdBDNF (1 × 109 pfu / ml) AdCTLA4Ig (1 × 109 pfu / ml) 2μl (C group). 45 degree oblique traveling needle tip to the head, oblique piercing depth of 2.5 mm. The injection speed 1μl/min stagnation needle 5min after injection the slow needle injection. Full stop bleeding, wash the wound the local spraying antibiotics to prevent infection, wound closure. B, C rats transfected virus 2 days, 4 days, 7 days, 15 days, 30 days, 45 days, 60 days drawn specimens of the rat spinal cord. Three groups of rats spinal cord thickness of 40 consecutive frozen cross sections through the line immunohistochemical staining, counting the three groups at different time points of BDNF in the anterior horn motor neurons staining positive cell counts and BDNF immunoreactive products integrated optical density (IOD) value determined using immunofluorescence staining continued AdCTLA4Ig expression time and polymerase chain reaction (PCR) monitoring B, C two sets of the survival time of the virus injected into the spinal cord, and reverse transcription polymerase chain reaction (RT-PCR) assay group BDNF gene expression in rat spinal cord. Results: 1 BDNF, of CTLA4Ig transgene expression 1.1 CTLA4Ig transgene expression: experimental group after two days can be seen immunoreactive cells, sustainable over time to 45 days after surgery in 60 days after no immunoreactive cells. 1.2 BDNF turn gene expression: the ventral horn of the spinal cord of rats in the control group (A), movement of God moving element and spinal dorsal horn neurons and glial cells shows a small amount of BDNF positive cells, subcellular localization of the positive products were mainly distributed in the cytoplasm. Experimental control group and experimental group positive cells present in the injected side of the anterior horn of the spinal cord motor neurons and surrounding glial cells. Experimental control group (B group) import Ad0 AdBDNF after BDNF number of positive cells and immunoreactive product integrated optical density (IOD) significantly increased over time and peaked 7 days after 30 days expression level is close to the normal control group experimental group (C group) import CTLA4Ig AdBDNF BDNF positive cells and immune-positive products integrated optical density (IOD) significantly increased after 7 days and the same to the peaked and the expression level is higher than the normal group (group A) and experimental group (B) peak lasted significantly longer in expression in 60 days with the normal group (A) and the experimental group (B) no significant difference. Statistical analysis showed that: three groups of BDNF positive number of stained cells immunoreactive products integrated optical density (IOD) in 4 days, 7 days, 15 days, 30 days, 45 days there are statistically significant differences (P lt; 0.05 ), BDNF positive number of stained cells and immunoreactive products product spectral density (IOD) value in 2 days and 60 days between the three groups was no statistically significant difference (P gt; 0.05). 2 PCR detection: 2.1 adenosis venom detection of PCR: the adenovirus expression level with decreasing concentration gradually decreases. The minimum specific bands after the extraction of the virus solution 10-fold dilution series of DNA by the PCR reaction can be detected adenovirus dilution 10-4, AdlacZ (1 × 109pfu/ml) and AD0 (5 x 109 pfu / ml) The lowest dilution of specific bands consistent. 2.2 spinal cord specimens adenovirus PCR detected: adenoviral DNA levels were detected by PCR method, the survival time in the experimental control group, the experimental group of the spinal cord specimens. Adenovirus DNA is gradually decreased with time, the experimental control group (B) group to 45 days, while the experimental group to # 60 were detected in less than adenovirus expression. 3 RT-PCR detection: of 3.1 BDNFmRNA expression in rat spinal cord tissue: from the mRNA level by RT-PCR were used to detect expression of BDNF in the control group, the experimental control, experimental group: 2 days after the transfection of three groups could be detected in the BDNFmRNA peak upregulated expression in rat spinal cord tissue, experimental control group and experimental group were after 7 days, the experimental group and the experimental control group high expression level can be sustained for 30 days and 15 days, the control group disappears completely to the 45th day . Experimental group to 60 days completely disappear. Statistical analysis showed that during peak periods amount of BDNFmRNA expression between the two groups were significantly different (P lt; 0.05) group at different time points compare BDNFmRNA the expression of a significant difference (P lt; 0.05). 3.2 CTLA4IgmRNA in rat spinal cord tissues: from the mRNA level by RT-PCR method were used to detect expression the CTLA4IgmRNA in the experimental group. Transfected 2 days after the experimental group can be detected CTLA4IgmRNA expressed in the rat spinal cord tissues, increase peak after 4-7 days, high expression levels can be continued until two weeks to 45 days completely disappeared. CONCLUSION: In the normal Wistar rat spinal cord, the expression of a small amount of BDNF staining positive cells and a trace amount of BDNFmRNA. AdV mediated CTLA4Ig gene injected into the spinal cord can be effectively suppressed Partly T lymphocyte infiltration, thereby reducing the local inflammatory reaction mediated by AdV inhibit the body's rejection of viral vectors experimental group significantly prolonged common import BDNF gene expression of time in the spinal cord, and enhanced its import BDNF gene expression intensity.

Influence of Co-injection of Adenoviral-mediated CTLA4Ig Genes on BDNF Gene Transgene Expression in Spinal Cord of Mice with Adenoviral

Abstract

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