| Apoptosis is one of the patterns of cell death during cerebral ischemia. As an antiapoptotic gene, it has been proved in many studies that bcl-2 gene ( B-cell lymphoma/leukemia-2 gene) can protect neural cells from ischemic brain injury. Bax gene (bcl-2 associated x gene) has been proved to be a proapoptotic gene, colaberating with bcl-2 to adjust apoptosis. With the developments of molecular biology and genetic engineering, gene therapy may provide a new strategy for clinical therapy of cerebral infarction. Up to now virus vectors are utilized in most studies of gene therapy, but it is still difficult to be applied in the patients because it is not safe enough. In our experiment before we have injected plasmid-mediated bcl-2 cDNA directly into cerebral tissues near the penumbra and showed that pLXSN-bcl-2 cDNA can inhibit apoptosis of neural cells safely and effectively during focal cerebral ischemia in rats. Nevertheless this transgen-ic method of direct injection in local brain is too traumatic and limited to be applicable to clinic. In order to discover a more convenient, simple, safe and effective therapeutic method, we use plasmid pLXSN as a vector to transfer human bcl-2 cDNA and inject it intraventricularly into the rats of middle cerebral artery occlusion model, observing the dynamic changes of the infarct volume, gene expression of bcl-2 and bax and neuronal apoptosis at different time point after cerebral ischemia and reperfusion to research the neuroprotection of pLXSN mediated bcl-2 gene during focal cerebral ischemia and provide an experimental proof for clinical treatment of cerebral infarction.MethodpLXSN and pLXSN-bcl-2 were identified, extracted and amplified in normal molecular biological method. All the 180 male rats were made into animal models of middle cerebral artery occlusion by Zea-longa thread occlusion method and were randomly divided into three groups: normal saline(NS) control group (n =60) , plasmid control group(n =60) , bcl-2 group(n =60). Separate in-traventricular administration of NS, pLXSN and pLXSN-bcl-2 20ui were progressed immediately after MCAO in stereotactic method. TTC staining, immuno-histochemistry and in site terminal labeling (TUNEL) method were used respectively to determine the infarct volume, expression of bcl-2/bax and the apoptosis of neuron after cerebral ischemia for 2h and reperfusion for 3h, 6h, 24h, 48h, 72h.Results(1) The infarct volume at different time point after cerebral ischemia and reperfusion was detected by TTC staining, and the images of each specimen were collected and analysed by microimage analyse system. Compared with NS control group, plasmid control group, the infarct volume of bcl-2 group was significantly decreased at each time point observed (P < 0. 05) and the infarct volume of each group enlarged with the time of ischemia and reperfusion delayed before 24 h, got a peak at 24 h point and decreased later.(2) The positive area percents of bcl-2 and bax protein expression were determined by immunohistochemistry, and the images of each specimen were collected and analysed by microimage analyse system.(1)The expression of bcl-2 protein: Compared with NS control group and plasmid control group, the bcl-2 protein expression of bcl-2 group at the same time point was significantly increased (p <0.05). Bcl-2 protein expression of NS control group and plasmid control group increased slowly after 3h, reached to a peak at 48h, and decreased soon. In bcl-2 group, it increased slowly in 6h,then rapidly after 6h, reached to a peak at 24h, and decreased subsequently.(2)Protein expression of bax. No significant differences were determined a-mong the three groups after ischemia and reperfusion for 3h (p >0. 05). It was less in bcl-2 group than plasmid control group at 6h point (p < 0. 05) , though there was no significant difference between SN contral group and plasmid control group (P >0.05). At the subsequent 24h to 72h time point, it decreased significantly in bcl-2 group contrast with the two control groups at the sa...
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