自发性脑出血中红细胞对脑含水量的影响及与AQP4表达的关系

发布时间：2018-10-26 15:45

【摘要】：目的：研究自发性脑出血（spontaneous intracerebral hemorrhage, SICH）后红细胞对脑含水量及水通道蛋白-4（aquaporin-4, AQP4）表达的影响；SICH后脑水肿形成过程中，AQP4的表达与脑含水量之间的关系。方法：选用成年雄性SD大鼠130只，随机分为正常组（NG）、假手术组（SO）、全血组（WB）、红细胞组（RBC），其中SO、WB、RBC组按处死时间6h、1d、3d、5d再分为四个亚组，每个亚组10只，其中5只用于脑含水量、RT-PCR及Western Blotting的检测,另外5只用于HE染色及免疫组化检测AQP4。使用立体定向技术向SD大鼠右侧尾状核位置注射自体尾动脉血或自体红细胞建立脑出血动物模型；采用干湿重法测脑含水量；应用免疫组化和Western blotting法检测AQP4蛋白的表达情况，RT-PCR法检测AQP4mRNA的变化情况；结果：脑含水量在RBC组6h、1d、3d及5d的组内比较，RBC-3d组明显高于6h、1d、5d组，各组间比较RBC-6h后脑含水量开始增多（P0.05），在出血后3天到达高峰（P0.01），以后逐渐减少，在出血后5天仍高于正常水平（P0.01）。WB组与RBC组在各时间段的脑含水量变化规律相似，但是均低于RBC组；免疫组化结果WB组及RBC组3d均可见AQP4表达于神经胶质细胞或血管周边的神经胶质细胞足突，胞浆着色，呈棕黄色。Western blotting结果显示RBC组内比较，RBC-3d组的AQP4蛋白量明显高于RBC-6h、1d、5d组，各组间比较RBC-6h后AQP4蛋白量开始增多（P0.05），在出血后3天到达高峰（P0.01），以后逐渐减少，在出血后5天仍高于正常水平（P0.01）；WB组与RBC组在各时间段的AQP4蛋白量变化规律相似，但是均低于RBC组。RT-PCR结果显示RBC组6h、1d、3d及5d的组内比较， RBC-3d组AQP4mRNA明显高于6h、1d、5d组，RBC-6h后血肿周围组织AQP4mRNA表达开始增高（P0.05），在出血后3天到达高峰（P0.01），以后逐渐下降，在出血后5天仍高于正常水平（P0.01），WB组AQP4mRNA表达水平与RBC组规律相似，但均低于RBC组；NG组、SO组的各时间段脑含水量、AQP4蛋白量及AQP4mRNA表达水平无明显差异。结论： 1. AQP4在WB组和RBC组第3天时阳性表达最强，，提示红细胞可刺激AQP4大量表达； 2. WB组和RBC组在第3天时脑含水量最高，提示红细胞在SICH后脑水肿形成中起着重要作用，即红细胞可能是SICH后第3天脑水肿形成的重要物质基础； 3.综合AQP4及脑含水量的结果，AQP4的阳性表达与脑含水量的变化相一致，即WB组和RBC两组在第3天时脑含水量最高，AQP4的阳性表达也最强，说明AQP4的表达与脑含水量的变化趋势一致，提示SICH后第3天所形成的脑水肿可能是通过AQP4的表达上调来实现的。
[Abstract]:Aim: to study the effect of erythrocyte on brain water content and aquaporin-4 (aquaporin-4, AQP4) expression after spontaneous intracerebral hemorrhage (spontaneous intracerebral hemorrhage, SICH) and the relationship between AQP4 expression and brain water content during the formation of cerebral edema after SICH. Methods: 130 adult male SD rats were randomly divided into normal (NG), sham-operation group, (SO), whole blood group, (WB), erythrocyte (RBC), group and SO,WB,RBC group, which were divided into four subgroups according to the time of death for 6 h, 1 d, 3 d and 5 d, respectively. Of the 10 rats in each subgroup, 5 were used for the detection of brain water content, RT-PCR and Western Blotting, and 5 were used for HE staining and immunohistochemical detection of AQP4.. Intracerebral hemorrhage model was established by injecting autologous caudate blood or autologous red blood cells into the right caudate nucleus of SD rats by stereotactic technique. Immunohistochemical and Western blotting methods were used to detect the expression of AQP4 protein, and RT-PCR method was used to detect the changes of AQP4mRNA. Results: the brain water content in the RBC group was significantly higher than that in the RBC-3d group (P 0.05). The brain water content in the RBC-3d group was significantly higher than that in the RBC-3d group at 6 h / 1 d and 5 d after RBC-6h (P0.05), and the brain water content in the RBC-3d group was significantly higher than that in the control group (P < 0.05). The cerebral water content in P0.01). WB group and RBC group was similar to that in RBC group, but lower than that in RBC group, and reached the peak 3 days after bleeding (P0.01), then decreased gradually, and remained higher than that of normal level 5 days after hemorrhage (P0.01). WB group and RBC group in each time period). The results of immunohistochemistry showed that AQP4 was expressed in the foot process of glial cells or peripheral glial cells in WB group and RBC group on the 3rd day, and the cytoplasm was stained. The results of brown. Western blotting showed that the AQP4 expression in RBC group was higher than that in RBC group. The amount of AQP4 protein in RBC-3d group was significantly higher than that in RBC-6h,1d,5d group. Compared with RBC-6h, the amount of AQP4 protein began to increase (P0.05), reached the peak at 3 days after bleeding (P0.01), and then decreased gradually. It was still higher than normal level 5 days after bleeding (P0.01). The changes of AQP4 protein content in WB group and RBC group were similar, but lower than those in RBC group. RT-PCR results showed that AQP4mRNA in RBC-3d group was significantly higher than that in 6 h / 1 day group and 5 d group, compared with 6 h / 1 day group and 5 d group, RT-PCR results showed that AQP4mRNA in RBC-3d group was significantly higher than that in 6 h / 1 day group. After RBC-6h, the expression of AQP4mRNA began to increase (P0.05), reached the peak 3 days after hemorrhage (P0.01), then gradually decreased, still higher than the normal level 5 days after bleeding (P0.01). The expression of AQP4mRNA in WB group was similar to that in RBC group, but lower than that in RBC group. There was no significant difference in brain water content, AQP4 protein content and AQP4mRNA expression in NG group and SO group. Conclusion: 1. The positive expression of AQP4 in WB group and RBC group was the strongest on the 3rd day, suggesting that erythrocytes could stimulate AQP4 expression in large amount; 2. WB and RBC groups had the highest brain water content on the third day, suggesting that red blood cells play an important role in the formation of brain edema after SICH, that is, red blood cells may be an important material basis for the formation of brain edema on the third day after SICH; 3. According to the results of AQP4 and brain water content, the positive expression of AQP4 was consistent with the change of brain water content, that is, WB and RBC groups had the highest brain water content and the strongest positive expression of AQP4 on the third day. The results suggest that the expression of AQP4 is consistent with the change of brain water content, suggesting that the brain edema formed on the 3rd day after SICH may be realized by the up-regulation of AQP4 expression.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34

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