HO-1和HO-2在老年SD大鼠慢性脑血流低灌注状态下肝脏中的动态变化及意义

发布时间：2018-08-26 17:58

【摘要】：目的:长期的脑血流灌注不足,会导致体内多脏器内过度的氧化应激的产生。血红素加氧酶(heme oxygenase,HO)是血红素代谢分解过程中的关键酶和限速酶,也是体内重要的内源性抗氧化体系,具有显著的抗氧化应激能力。本课题通过建立老年SD大鼠脑血流低灌注模型,研究HO家族中重要成员HO-1和HO-2在肝脏中的表达,并探讨其在脑血流灌注不足引起的损伤时对肝脏的作用及意义。方法:采用持久性双侧颈总动脉结扎法(2VO)致老年SD大鼠脑血流灌注不足,首先检测脑皮质血流的变化情况;采用苏木素-伊红染色观察大鼠肝细胞形态学改变;采集下腔静脉的静脉血检测血清中丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的活性;采用ELISA法检测肝脏组织中HO的活性变化;采用免疫组织化学法检测肝脏组织中HO-1和HO-2的表达情况;并分别通过RT-PCR和Western blot法检测肝脏组织中HO-1和HO-2在mRNA和蛋白水平的表达。结果:术后第7天,大鼠皮质血流的面积比值明显降低,达到最低值,而术后14、21、28天,大鼠皮质脑血流的面积比值逐渐升高,到28天基本恢复的正常水平。HE染色结果表明:与假手术组比,在术后第1天,肝细胞因缺血出现水样变性,即细胞体积明显增大,胞质变得透明,第7天其水样变性最为显著;在术后14、21、28天,肝细胞的水样变性有所改善,但仍未恢复到正常水平。肝功能检测结果显示:ALT和AST在2VO术后7 d明显增高,差异有统计学意义(P0.01),但增高范围仍属于正常,无临床意义;随着时间的延长,ALT和AST的含量逐渐降低,到28 d,数值基本恢复至假手术组的水平。免疫组化、RT-PCR和Western blot显示:与假手术组比较,HO-1在术后1d即有增加,并在第7d达到高峰(p0.05),这样的变化与HO酶活性变化一致;而HO-2在慢性脑血流灌注不足的肝脏中却没有显著变化。结论:慢性脑血流灌注不足时,可导致肝细胞的形态结构有轻微的损伤,但肝功能基本正常,这与HO-1和血红素加氧酶活性的动态增加密切相关,这提示HO-1在慢性脑血流灌注不足的肝脏中起着保护作用。
[Abstract]:Objective: chronic cerebral blood perfusion deficiency may lead to excessive oxidative stress in multiple organs in vivo. Heme oxygenase (heme oxygenase,HO) is a key enzyme and rate-limiting enzyme in heme catabolism and an important endogenous antioxidant system in vivo. The purpose of this study was to study the expression of HO-1 and HO-2 in the liver of aged SD rats by establishing a model of cerebral blood flow hypoperfusion, and to explore the role and significance of HO-1 and HO-2 in liver injury caused by cerebral perfusion insufficiency. Methods: chronic bilateral common carotid artery ligation (2VO) was used to induce cerebral blood perfusion insufficiency in aged SD rats. The changes of cerebral cortical blood flow were detected by hematoxylin and eosin staining, and the morphological changes of hepatic cells were observed by hematoxylin eosin staining. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected by collecting venous blood from inferior vena cava, and the changes of HO activity in liver tissue were detected by ELISA method. The expression of HO-1 and HO-2 in liver tissue was detected by immunohistochemical method, and the expression of HO-1 and HO-2 in liver tissue was detected by RT-PCR and Western blot methods respectively. Results: on the 7th day after operation, the area ratio of cortical blood flow decreased significantly to the lowest value in rats, while the area ratio of cerebral blood flow increased gradually on day 14, 21 and 28 after operation. The results of HE staining showed that, compared with the sham operation group, on the first day after operation, the hepatocytes showed water-like degeneration due to ischemia, that is, the cell volume was obviously increased, the cytoplasm became transparent, and the water-like degeneration was the most obvious on the 7th day. On the 14th day, 21 ~ (th) day after operation, the degeneration of hepatocytes was improved, but it did not return to normal level. The results of liver function test showed that the levels of alt and AST increased significantly 7 days after 2VO, the difference was statistically significant (P0.01), but the range of increase was normal and had no clinical significance, and the contents of alt and AST decreased gradually with the prolongation of time. By 28 days, the value basically returned to the level of the sham operation group. Immunohistochemistry RT-PCR and Western blot showed that compared with sham-operated group, the expression of ro-HO-1 increased on the 1st day after operation and reached its peak on the 7th day (p0.05), which was consistent with the change of HO enzyme activity, but HO-2 did not change significantly in the liver of chronic cerebral blood perfusion deficiency. Conclusion: chronic cerebral blood perfusion deficiency can cause slight damage to the morphology and structure of hepatocytes, but the liver function is basically normal, which is closely related to the dynamic increase of HO-1 and heme oxygenase activities. This suggests that HO-1 plays a protective role in the liver with chronic cerebral blood perfusion deficiency.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

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