【作者】 李丹；

【导师】 李昱；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2017， 硕士

【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(P<0.01),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(p<0.05),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。更多还原

【Abstract】 Objectives: Long time cerebral hypoperfusion can lead to excessive oxidative stress in many organs,which plays an important role in the process of neuronal damage and cognitive dysfunction.Heme oxygenase(HO)is the key enzyme and the rate-limiting enzyme in the decomposition of heme metabolism,and it is also an important endogenous antioxidant system,which has remarkable anti-oxidative stress ability.In the present study,the old SD rats with chronic cerebral hypoperfusion models were set up,and the expression of HO-1 and HO-2,which were key elements of HO family were detected,and their roles and significance were explored in the liver tissues.Methods: The old SD rat models with cerebral hypoperfusion were established by permanent bilateral common carotid artery occlusion(2VO).The cortical blood flow was detected.Hematoxylin eosin staining was used for observing the morphological changes of the rats’ liver cells.Serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured in the blood samples collected from inferior vena cava? The activity of HO in the liver tissues was detected by ELISA.The location and expression of HO-1 and HO-2 was determined by immunohistochemical(IHC)staining.Finally,the expression of HO-1 and HO-2 at mRNA and protein levels were detected by RT-PCR and Western blot,respectively.Results: Compared with the sham-operated group,on the 7th d after 2VO operation,the area ratio of cortical blood flow was significantly decreased,and it reached the lowest value;at 14 d,21 dafter 2 VO,the area ratio of cortical cerebral blood flow was gradually increased,and at 28 d,it recovered to the normal level.The HE staining results showed that compared with sham operated group,on the first day after 2VO,the liver cells had hydropic degeneration due to ischemia,manifested with increased cell volume,transparent cytoplasm;and on the seventh day after 2VO,the hydropic degeneration was the most significant;after 14,21,28 days,liver cells vacuslation has improved,but has not yet recovered to the normal level.Liver function test results showed thatALT and AST were significantly increased 7 d after 2VO,and the difference was statistically significant(P<0.01),but they were still in normal range,there was no clinical significance;and with the extension of time,the concentration of ALT and AST were gradually decreased,28 d after operation,they were both recovered to normal level.IHC,RT-PCR and Western blot has showed that,compared with the sham operated group,the expression of HO-1 was rapidly increased on the first day after 2VO,and on the seventh day,it reached a peak(p<0.01),which was consistent with the changes of HO activity in the livers.Nevertheless,the changes of HO-2 in the liver tissues were not significant.Conclusion: Chronic cerebral hypoperfusion can lead to the slight damage of the morphological structure of liver cells,but the functions of liver is in normal?status,all of which is tightly related with the dynamic changes of HO-1 and heme oxygenase activity,and they suggest that HO-1 may play a protective role in the liver tissue of chronic cerebral hypoperfusion.更多还原