| Objective:In the experiment, the vitro recombination technology was used to prepare pcDNA3.1-HIF-1αplasmid. We injected HIF-1αplasmid into the middle cerebral artery (MCA) occlusion reperfusion of rats through external-internal carotid artery. After that, specificity immunohistochemistry was applied to stain for subtypes of glial cells of cerebral tissue in 6h, 12h, 24h, 72h and 7d, respectively. We then observed the changes in each cell group. In this process, double staining immunofluoresence technique was used to stain for HIF-1α,VEGF, and subtypes of glial cells, respectively, and we examined the co-expression of the positive cells under a confocal laser scanning microscope. Based on these experiment results, we finally came to the conclusions and the regulation mechanism of HIF-1αto cell reparation after rats ischemia-reperfusion injury.Methods:1 The cDNA of HIF-1αobtained in vitro was cloned into pcDNA3.1 the eukaryotic expression vectors after sequencing and restriction enzyme digestion was employed to identify the recombinants;2 The modeling for the MCA occlusion reperfusion of rats consulted the reports of Zea Longa, etc and made some improvements. Rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate and were conducted median neck incision. We separated the muscle tissue to have the neck vessels exposed and inserted filament into the internal carotid through the external carotid artery. The filament end which was about 1.8 cm away from the bifurcation of vessels was regarded as the filament infarction MCA. After 2 hours, we removed the filament and injected HIF-1αplasmid. For other time points at 6h,12h,24h,72h and 7d, we carried out the same operations and injected PBS into their control groups;3 The rats of experimental groups and control groups of corresponding time points were decapitated after brain death. We made 2mm thick serial sections from posterior optic chiasm among which the first one was stained by TTC and the rest ones were embedded in paraffin and made into 5μm thick serial paraffin sections. The paraffin sections were then stained by HE. And specificity immunohistochemistry was applied to stain for subtypes of glial cells. Then, at high magnification, we counted the number of subtypes of glial cells in each group at different time points;4 HIF-1α,VEGF, and subtypes of glial cells, were stained respectively, by paraffin sections using double staining immunofluoresence technique. We examined the overlapping of the positive cells under a confocal laser scanning microscope and analyzed the problem that whether there was coexpression of the subtypes of glial cells and HIF-1αas well as VEGF;5 Statistical analysis: Use SPSS15.0 statistical software to conduct statistical analysis of the experimental data. T test was used to compare measurement data between groups. P <0.05 indicated that there were statistically significant differences;Results:1 The sequencing results of HIF-1αcDNA amplified by RT-PCR were exactly the same with the Genebank records. We obtained pcDNA3.1-HIF-1αfor experimental use in the following operations;2 The modeling of MCA occlusion reperfusion of rats was finished. There showed no significant differences in rats death rate between groups whose external carotid artery injected with HIF-1αplasmid and those with PBS. However, the nerve function score of 7-day survival rats were actually obviously lighter than that of the control groups injected with PBS. The staining results by TTC could be observed from 72h groups that the area of cerebral infarction of the treatment groups was smaller than that of the control groups;3 The effect of HIF-1αto the subtypes of glial cells of rats with ischemia-reperfusion injury: After the carotid artery injection of HIF-1αplasmid, the means of astrocytes (GFAP-positive cells) number were respectively 12.20±2.864, 18.80±4.207, 30.00±4.301, 38.40±2.881 and 43.20±6.978, and the means of GFAP-positive cells number of the PBS injection groups were respectively 15.80±4.494, 18.60±1.673, 23.00±4.583, 31.60±3.361 and 29.00±6.000. Using T test to compare the means of GFAP-positive cell number of the two groups at the same time points, we observed that, after 24h of cerebral infarction treatment, there were significant differences in the numbers of positive cells between the groups injected with HIF-1αand the control groups (P<0.05); After the carotid artery injection of HIF-1αplasmid, the means of microglia (Isolectin-B4 positive cell) number were respectively 8.400±2.074, 14.40±4.393, 20.80±2.280, 17.00±3.162 and 11.60±2.408, and the means of positive cell number of the PBS control groups were respectively 8.400±2.302, 15.40±2.402, 25.80±4.147, 37.80±6.181 and 24.80±4.325. Using T test to compare the means of positive cell number of the two groups at the same time points, we observed that after 24h of cerebral infarction treatment, there were differences between the two groups and they were of statistical significance (P<0.05). The means of oligodendrocyte (CNPase positive cell) number of HIF-1αinjection groups were respectively 17.00±2.916, 11.40±2.302, 10.00±3.526, 16.40±2.302 and 19.20±2.388, and the means of positive cell number of PBS control groups were respectively 16.60±2.408, 10.60±2.074, 7.200±1.924, 4.000±1.581 and 13.20±2.388. Using T test to compare the means of positive cell number of the two groups at the same time points, we observed that after 72h of cerebral infarction treatment, the numbers of positive cells of the two groups were of statistical significance;4 Coexpression of HIF-1α, VEGF and subtypes of glial cells: We used double staining immunofluoresence technique to stain for the paraffin sections. Under a confocal laser scanning microscope, we oberserved the coexpression phenomenon of a large number GFAP positive cells together with HIF-1αand VEGF protein, whereas we did not find significant overlapping of a large number of Isolectin-B4 positive cells and CNPase positive cells with HIF-1αand VEGF protein.Conclusions:1 The pcDNA3.1-HIF-1αplasmid injected through external-internal carotid artery could effectively reduce the infarction size in the rat cerebral tissue.2 The injection of recombination pcDNA3.1-HIF-1αplasmid after rat ischemia-reperfusion injury.could increase the number of astrocytes and oligodendrocytes, and at the same time, could effectively inhibit microglia cell proliferation to protect the neurons.3 HIF-1αprotein in subtypes of glial cells was mainly expressed in the form of astrocytes.
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