两种脑出血动物模型的比较以及AQP4表达调控机制的研究

【作者】 张波；

【导师】 孙善全；

【作者基本信息】 重庆医科大学 ， 人体解剖与组织胚胎学， 2012， 硕士

【摘要】 目的：对胶原酶和自体血注入法建立的大鼠脑出血动物模型进行比较，为脑出血发病机制和／或干预性治疗的研究提供可靠的动物模型。方法：健康成年SD大鼠300只，雄性，体重200-250g，按照随机数字表分为脑出血模型组和假手术组，将胶原酶和自体血注入到尾状核建立脑出血模型，假手术组除不注入药物外其他操作与脑出血组相同。分别在术后6h、l2h、24h、48h、72h和7d时运用磁共振成像（mageticresonanceimaging，MRI）、干湿重法、伊文思蓝(EvansBlue，EB)测定法、免疫荧光和Western-blot分别比较两种脑出血动物模型的血肿体积、脑含水量（BWC），血脑屏障(BBB)通透性和AQP4的表达变化。结果：胶原酶模型组的血肿体积在术后24h达到最大，并持续至72h，自体血模型组的血肿体积在术后12h达到最大，72h已接近正常，假手术组无脑出血。胶原酶模型组的BWC在术后24h达到峰值，并持续至72h，7d时仍高于自体血模型组，自体血模型组的BWC在术后24h达到峰值，48h逐渐下降，至7d已恢复正常，假手术组无明显水肿，两组模型在48h及其以后各时间点，BWC差异具有统计学意义（P＜0.05）。胶原酶模型组的EB含量在术后12h达到高峰，并持续至48h，7d时仍高于自体血模型组，自体血模型组的EB含量在术后12h达到高峰，但在24h逐渐下降，至72h已恢复正常，假手术组无明显变化，两组模型在24h及其以后各时间点，EB含量差异具有统计学意义（P＜0.05）。胶原酶模型组在出血12h以后在大脑皮质、软脑膜、室管膜和血肿周围组织AQP4阳性细胞表达逐渐减弱，仅在血肿周围可见阳性细胞表达，且细胞轮廓不清楚，而自体血模型组出血24h以后在大脑皮质颗粒细胞层可见AQP4阳性细胞大量表达，细胞轮廓清楚，假手术组无明显变化。Western-blot结果显示，两组脑出血模型在6h及以后时间点，AQP4的表达量差异具有统计学意义（P＜0.05）。结论：两组脑出血模型之间存在差异，自体血脑出血模型组与人类自发性脑出血病理生理过程更为接近，是更为接近临床脑出血的一种动物模型。目的：研究精氨酸加压素1a受体（V1aR）和水通道蛋白4(aquaporin4，AQP4)在脑出血大鼠脑组织中的表达变化，并初步探讨两者在出血性脑水肿形成过程中的作用。方法：健康成年SD大鼠120只，雄性，体重200-250g，随机分为脑出血组和假手术组，将自体血注入大鼠尾状核建立脑出血模型，假手术组除不注入自体血外，其余操作与脑出血组相同。每组大鼠分别于术后6h、l2h、24h和48h运用干湿重法、免疫荧光和Western-blot检测脑含水量（BWC）、V1aR和AQP4蛋白的表达。结果：脑出血组的BWC随着出血时间的延长而逐渐加重，至24-48h时最为明显，显著高于假手术组（P＜0.05）。脑出血组的V1aR和AQP4共表达于软脑膜、脉络丛和血肿周围等处，尤其是24-48h，两者在上述区域的表达明显增多，假手术组可见V1aR和AQP4在脉络丛有少量的表达，而在脑室周围未见表达。在6h及其以后各时间点，V1aR和AQP4的表达量显著升高（P＜0.05）。Western-blot结果显示，在6h及其以后各时间点，V1aR和AQP4的表达量显著高于假手术组（P＜0.05）。结论：脑出血后AQP4的异常升高是引起脑水肿的主要原因之一，而AQP4的升高主要是由于V1aR的调控所导致的。更多还原

【Abstract】 Objective:The aim of this study was to compare collagenase injection induced intracerebral hemorrhage animal model with that induced by autologous blood injection, in order to provide reference for study of pathologic mechanism and/or intervening therapy of intracerebral hemorrhage.Methods:300healthy male adult SD rats (200-250g) were randomly divided into ICH group and the sham operation group. ICH models were established by infusing Collagenase and autologous blood into right caudate nucleus in rats. The operation procedure of the sham group was as same as the ICH group but without infusing drug. The hematoma volume were measured by magnetic resonance imaging (MRI).The changes of brain water content (BWC) were measured by wet and dry weight methods. EB was used to measure the integrity of BBB. The expression of AQP4were detected by immunofluorescence and Western-blot after6h、12h、24h、48h,72h and7d.Results:The hematoma volume in collagenase group reached its peak at24h and continues at72h, in autologous blood group reached its peak at12h and already close to normal at72h, sham group no bleeding in the brain. BWC was reached its peak at24h and continues at72h in collagenase group, whereas on7d is still higher than autologous blood group, in autologous blood group, BWC was reached its peak at24h and gradually decreased at48h and return to normal at7d, sham group without obvious brain edema, At48h and after each time point in two groups, BWC has a significant different (P<0.05).EB contents was reached its peak at12h and continues at48h in collagenase group, whereas on7d is still higher than autologous blood group, in autologous blood group, EB contents was reached its peak at12h and gradually decreased at24h and return to normal at48h, At24h and after each time point in two groups, EB contents has a significant different (P<0.05).A number of swelling AQP4positive cells appeared in the granulose cell layer of rat cerebral cortex in autologous blood group at24h, but none of it was observed in collagenase group at12h. The contents of AQP4had a significant different (P<0.05) in the two groups at6h and after each time point by Western-blot.Conclusions:There was different in the two ICH groups, autologous blood group is relatively close to clinical ICH conditions, Objective:To investigate the changes of arginine-vasopressin Via receptor (V1aR) and aquaporin4(AQP4) expression, to explore the mechanism of brain edema caused by ICH.Methods:120healthy male adult SD rats (200-250g) were randomly divided into two groups, the ICH group and the sham group. ICH models were established by infusing autologous blood into right caudate nucleus in rats. The operation procedure of the sham group was as same as the ICH group but without infusing autologous blood. The changes of brain water content (BWC) were measured by wet and dry weight methods. The expression of V1aR and AQP4were detected by immunofluorescence and Western-blot after6h、12h、24h and48h.Results:BWC was reached its peak during the24-48h in ICH group, significant higher than sham group (P<0.05), V1aR and AQP4co-expression increased at leptomeningeal、chorioid plexus and the tissue surrounding the hematoma at24h in ICH group, whereas in sham group, V1aR and AQP4co-expression small amount of at chorioid plexus, none of it is observed in the surrounding of ventricle.The contents of ViaR and AQP4has a significant higher than sham group (P<0.05) at6h and after each time point by Western-blot.Conclusions:The increased expression of AQP4mediated by V1aR played an important role in the brain edema after ICH.更多还原