摘要
目的探讨内质网应激在慢性间歇低氧幼鼠脑损伤中的作用。方法取无特定病原体（SPF）级健康雄性SD幼鼠48只，采用数字表法随机分为4组：间歇低氧2、4周组，对照2、4周组。标本经苏木精-伊红染色，用原位末端标记技术（TUNEL）观察幼鼠海马、皮层神经元病理学改变及凋亡变化；反转录PCR法检测幼鼠海马、皮层的葡萄糖调节蛋白78（GRP78）mRNA及皮层的半胱氨酸天冬氨酸蛋白酶12（Caspase-12）mRNA基因表达；Western印迹法检测幼鼠海马、皮层GRP78mRNA蛋白表达；免疫组织化学法检测幼鼠皮层Caspase-12蛋白表达。单因素方差分析4组数据。结果各间歇低氧组海马及前额叶皮层神经元均有明显凋亡，均明显高于相应对照组，尤以间歇低氧4周组最为明显（海马8．78％±0．71％比3．26％±0．45％，皮层6．02％±0．32％比2．91％±0．29％，均P〈0．01）；间歇低氧2、4周组幼鼠海马和前额叶皮层GRP78mRNA表达均高于对照组（海马0．424±0．033比0．326±0．013、0．444±0．028比0．310±0．015，皮层0．514±0．038比0．430±0．017、0．524±0．038比0．439±0．033），蛋白表达也均高于对照组（海马0．221±0．032比0．178±0．014、0．241±0．019比0．170±0．013，皮层0．307±0．012比0．226±0．022、0．311±0．023比0．225±0．025，均P〈0．05）；前额皮层Caspase-12mRNA（0．396±0．004比0．323±0．014、0．417±0．011比0．313±0．011）和蛋白的表达（0．334±0．035比0．197±0．023、0．368±0．079比0．215±0．024）也均高于相应对照组（均p〈0．05）；两对照组之间差异均无统计学意义（均P〉0．05）。结论慢性间歇低氧可上调学习记忆相关脑区GRP78mRNA和蛋白的表达，启动内质网应激，从而诱导Caspase-12介导的细胞凋亡，可能在慢性间歇低氧所导致的脑损伤中起重要作用。
Objective To explore the role of endoplasmic reticulum stress in brain injury following chronic intermittent hypoxia （CIH） in weanling rats. Methods A total of 48 male healthy Sprague-Dawley rats （ 3 - 4-week-old, 80 - 100 g） were randomly divided into 4 groups ： 2-week-CIH （2IH） group, 4-week- CIH （4IH） group, 2-week-control （2C） group and 4-week-control （4C） group. The morphologic changes were observed by hematoxylin-eosin （HE） staining and cell apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Then hippocampus and prefrontal cortices were collected for transcription and expression analysis of glucose regulated protein 78 （GRP78） by reverse transcription （RT） -PCR and Western blotting respectively. And the expressions of Caspase-12 mRNA and Caspase-12 protein in prefrontal cortexes were analyzed by RT-PCR and immunohistochemistry. Results The neuronal apoptosis in hippocampus and prefrontal cortices in CIH exposed groups were more pronouncedthan those of the control groups （ all P 〈 0. 01 ）, especially in the 4IH group （ hippocampus ： 8.78% ± 0. 71% vs 3.26% ±0. 45% , cortices： 6. 02% ± 0. 32% vs 2. 91% ±0. 29% ）. The expression levels of GRP78 mRNA （ hippocampus ： 0. 424 ± 0. 033 vs 0. 326 ± 0. 013 and 0. 444 ± 0. 028 vs 0. 310 ± 0. 015, cortices ： 0. 514 ± 0. 038 vs 0. 430 ± 0. 017 and 0. 524± 0. 038 vs 0. 439 ± 0. 033 ） and GRP78 protein in hippocampus and prefrontal cortices （hippocampus： 0. 221 ±0. 032 vs 0. 178 ±0. 014 and 0. 241±0. 019 vs 0. 170 ±0. 013, cortices：0. 307 ±0. 012 vs 0. 226 ±0. 022 and 0. 311±0. 023 vs 0. 225 ±0. 025）, and the expression levels of Caspase-12 mRNA （0. 396 ± 0. 004 vs 0. 323 ± 0. 014, 0. 417 ± 0. 011 vs 0. 313 ± 0. 011 ） and Caspase-12 protein （0. 334 ± 0. 035 vs 0. 197 ± 0. 023, 0. 368 ± 0. 079 vs 0. 215 ± 0. 024） in prefrontal cortexes in the IH groups all were more than those in the 2C and 4C groups （ all P 〈 0. 05 ）. Conclusions Chronic intermittent hypoxia can up-regulate the GRP78 transcription and expression in brain regions associated with learning and memory. This may induce the endoplasmic reticulum stress and activate the Caspase-12 mediated apoptosis signaling pathway. In the end, neuronal apoptosis occurs. All these factors may play an important role in the impairment of learning memory during the exposure of growing rats to chronic intermittent hypoxia.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2012年第24期1706-1710,共5页
National Medical Journal of China
基金
浙江省自然科学基金（Y2110277）
浙江省科技厅科研项目（2008C33011）
