The Effect Of SA Liposome Mediated Human Interleukin-10 Gene Transfection On The Focal Cerebral Ischemia-reperfusion Injury In Rats

Part 1:The effect of human interleukin IL-10 gene transfection on the focal cerebral ischemia-reperfusion injury in ratsObjective: to detect the effect of SA liposome mediated human IL-10 gene transfer on the focal cerebral ischemia-reperfusion injury in rats and to observe the protective effect.Methods: We used the Longa method to establish the MCAO model. The SA liposome/hIL-10 mixture was injected into the lateral ventricle after the ischemia but before the reperfusion. Then we detected the hIL-10 content both in brain tissue and in serum on the 24 h (1 d), 72 h (3 d), 168 h (7 d) after ischemia-referfusion procedure. Also we use RT-PCR to detect the hIL-10 mRNA in the cortex and hippocampus. The damages to hippocampus neurons in CA1 subfield, brain infarct volume and behavior scores were also determined in order to evaluated the protective effect.Results:(1) hIL-10mRNA can be detected in the hIL-10 gene transfection group both in cortex and in hippocampus. But cannot be detected in the other three group. The IL-10 concentration of brain tissue and serum on the 1st day, 3rd day and 7th day after gene transfer were much higher respectively than those in normal group, ischemia-reperfusion group and in empty plasmid transfer group. But the absolute amount of hIL-10 in brain tissue is significantly higher than that in serum.(2) The hippocampal neuron in the CA1 subfield was lost or degenerated in the ischemia-reperfusion sham group and in the empty sham group, In the hIL-10 gene treated group, damage to hippocampal neurons was lesser.(3) The brain infarction detected by the TTC staining was markedly reduced by the gene transfer of hIL-10. The calculated infart volume of the control group was 107.2± 6.1mm~3 ,which was significantly larger than that of the hIL-10 transfection group(85.1±4.3 mm~3).(4) The bevior scores of the hIL-10 transfection group(1.7±0.4) was much lower than that of control group(2.3±0.5).Conclusion:(1) The SA liposome can effectively mediated the hIL-10 transfected in the rat brain tissue following the ischemia-reperfusion procedure. Also it can effectively express the hIL-10 protein.(2): The hIL-10 transfection can produce the protective effect on the rat ischemia-reperfusion brain injury.Part 2:The effect of human IL-10 gene transfection on the mRNA and protein expression of IL-1βand TNF-αof the brain tissue following focal the cerebral ischemia-reperfusion in ratsObjective: to observe the effect of human IL-10 gene therapy on the mRNA and protein expression of IL-1βand TNF-αof the brain tissue following the cerebral ischemia-reperfusion in rats, and to reveal the mechanism of its neuroprotective effect.Methods: We use realtime PCR to detect the mRNA of IL-1βand TNF-αin each group, and use ELISA to detect the protein of IL-1βand TNF-αin each group.Results: (1): The expression of mRNA of IL-1βand TNF-αin brain tisse in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.(2): The content of IL-1βand TNF-αin brain tissue in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.(3): The content of IL-1βand TNF-αin serum in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.Conclusion: The hIL-10 plays neuropective effect maybe through inhibiting increase of the mRNA and protein expression of IL-1βand TNF-αinducing by the cerebral ischemia-reperfusion in rats.Part 3:The effect of human IL-10 gene transfection on the SOD, GSH-Px, MDA and NO of the brain tissue following the focal cerebral ischemia-reperfusion in ratsObjective: to observe The effect of human IL-10 gene transfection on the SOD, GSH-Px, MDA and NO of the brain tissue in the cerebral ischemia-reperfusion rats and to reveal the mechanism of neuroprotective effect of hIL-10.Methods: We use the assay pyrogallol autoxidation to determining SOD activity in each group. And we use spectrophotometer to determine the activity of GSH-Px in each group.Results: (1): The content of SOD and GSH-Px of brain tisse in the ischemia control group and in empty plasmid group are much lower than those in normal control group. The hIL-10 gene transfection can inhibit this decrease.(2): The content of MDA of brain tissue in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.(3): The content of NO of brain tissue in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.Conclusion: The hIL-10 plays neuropective effect maybe through modulating the imbalance of oxidant and antioxidant inducing by the cerebral ischemia-reperfusion.Part 4:The effect of human IL-10 gene transfection on the expression of NHE-1 mRNA of the brain tissue following focal cerebral ischemia-reperfusion in ratsObjective: to observe the effect of human IL-10 gene transfection on the mRNA of NHE-1 of the brain tissue in the cerebral ischemia-reperfusion rats, and to reveal the mechanism of neuroprotective effect of hIL-10.Methods: We use realtime PCR to detect the mRNA of NHE-1 and NF-κB in each group.Results: (1): The expression of mRNA of NHE-1 of brain tisse in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.(2): The content of NF-κB of brain tissue in the ischemia control group and in empty plasmid group are much higher than those in normal control group. The hIL-10 gene transfection can inhibit this increase.Conclusion: The hIL-10 plays neuropective effect maybe through inhibiting the increase of NHE-1 mRNA and NF-κB mRNA expression inducing by the cerebral ischemia-reperfusion.