【作者】 刘宇；

【导师】 杜继曾； 陈学群；

【作者基本信息】 浙江大学 ， 生理学， 2007， 硕士

【摘要】 高原鼠兔（Ochotona curzoniae）、根田鼠（Microtus oeconomus）和高原鼢鼠（Myospalax Baileyi）是青藏高原高寒草甸的优势物种。这些动物在漫长的进化过程中,形成了对高原低氧环境的适应机制。IGF-I（Insulin Like Growth FactorⅠ,类胰岛素生长因子I）作为内分泌和旁分泌/自分泌因子,参与生长发育、细胞分化、损伤和凋亡的保护等一系列生理功能。近年来,证实IGF与低氧关系非常密切,在低氧损伤模型中起保护作用。本论文研究了CoCl₂诱导的低氧和常压低氧（不同氧分压的N₂/O₂混合气模拟低氧）下,高原鼠兔、根田鼠和高原鼢鼠大脑皮层和肝脏组织IGF-I及IGFBP-1表达的变化,并与平原实验小鼠比较,以揭示高原动物IGF-I/IGFBP-1参与低氧损伤的可能机制;并在CoCl₂诱导的低氧和常压低氧下注射NF-κB的抑制剂PDTC（氨基甲酸吡咯铵）,以研究NF-κB参与IGF-I及IGFBP-1表达调节的机制。低氧诱导因子-1α(（HIF 1-α）作为转录因子在细胞低氧信号转导过程中发挥枢纽性的关键作用,所以论文进一步研究了CoCl₂诱导的低氧对高原鼢鼠大脑皮层和肝脏HIF1-α的表达规律和NF-κB参与的可能机制。论文采用Elisa,Western blot和PCR检测大脑皮层和肝脏IGF-I及IGFBP-1的蛋白表达,高原鼢鼠肝脏HIF1-a的表达以及肝脏IGF-I mRNA。实验结果:1.注射CoCl₂（20 mg/kg）6小时后,高原鼢鼠大脑皮层IGF-I的表达无显著变化,而高原鼢鼠肝脏IGF-I/IGFBP-1的表达显著升高;模拟常压低氧（16.0%、10.8%、8.0%O₂）后,小鼠肝脏IGF-I/IGFBP-1的表达显著上升了,在低氧（10.8%、8.0%O₂）下,根田鼠肝脏IGF-I的表达也显著上升,IGFBP-1表达无显著变化,高原鼠兔肝脏IGFBP-1表达显著上升,IGF-I表达无显著变化。2.在CoCl₂和混合气模拟的低氧下,注射NF-κB的抑制剂PDTC后,高原鼢鼠大脑皮层IGF-I的表达显著下降;小鼠和高原鼢鼠肝脏IGF-I/IGFBP-1的表达均显著下降;根田鼠肝脏IGF-I的表达显著下降;高原鼠兔肝脏IGFBP-1的表达显著下降。3.在注射20 mg/kg CoCl₂ 6小时后,高原鼢鼠组肝脏HIF-1α的蛋白表达与对照组相比,有着显著性升高,而高原鼢鼠大脑皮层组织中未检测到HIF-1α的基础表达和低氧应激后表达。4.中华鼢鼠HIF-1αcDNA序列编码区总长度为2457 bp,编码长度为818个氨基酸残基的蛋白序列。中华鼢鼠HIF-1α的蛋白序列与小鼠、大鼠、人类、高原鼢鼠、以色列鼢鼠分别有93%、92%、93%、98%、97%的同源性。以上结果提示:1.高原动物对于高海拔低氧都具有良好的抗损伤和适应能力,这种适应可以部分体现在它们大脑皮层和肝脏组织中IGF-I/IGFBP-1的表达模式中。但不同的高原动物对低氧这一环境的适应也存在着差异性:高原鼠兔和根田鼠分别激活IGFBP-1和IGF-I两种不同的细胞保护因子来适应低氧的环境;高原鼢鼠与高原鼠兔和根田鼠相比,在低氧下反应模式上更与小鼠类似。这些差异可能跟它们各自的生存条件不同有关,另外,也可能是由于这些动物本身的种属差异而造成的。环境和自身的差别导致了同样生活在青藏高原的三种小型哺乳动物产生了不同的低氧适应机制。2.在IGF-I/IGFBP-1表达的调节方面,NF-κB起到了重要的作用:而NF-κB的调节作用在不同动物之间存在差异:NF-κB参与高原动物的IGF-I/IGFBP-1表达调节;NF-κB参与平原小鼠IGF-I表达调节,而部分参与平原小鼠IGFBP-1表达调节。3.HIF-1α作为一种氧敏感的转录调节因子,参与低氧靶基因的表达。但发现在低氧下高原鼠兔肝脏HIF-1α无变化,根田鼠只在严重低氧下HIF-1α表达上升,高原鼢鼠在低氧下肝脏HIF-1α的表达显著上升,明显不同于其他两种高原动物。且NF-κB参与高原鼢鼠肝脏HIF-1α的表达调节。未检测到高原鼢鼠低氧下大脑HIF-1α的变化,注射NF-κB抑制剂后,检测不到HIF-1α的基础表达和低氧下表达。提示不同的转录因子参与高原动物在低氧下的靶基因表达,同时发现不同转录因子之间存在相互作用。4.中华鼢鼠（陕西）属啮齿目,HIF-1α序列高度保守,其与高原鼢鼠（青海）的同源性更高达98%,与以色列鼢鼠同源性高达97%。中华鼢鼠（陕西）和高原鼢鼠（青海）的HIF-1α氨基酸序列在主要功能区相似度为100%,以色列鼢鼠与我国的两种鼢鼠相比在ODD结构域有一个位点不一致,中华鼢鼠和高原鼢鼠的氨基酸为丝氨酸（S）,以色列鼢鼠的为精氨酸（R）。在TAD（N）结构域有4个位点不一致,并多出两个氨基酸。本论文创新点如下:1.首次揭示了不同剂量的CoCl₂模拟低氧,对高原鼢鼠大脑皮层和肝脏组织IGF-I/IGFBP-1蛋白表达的影响;发现高原鼢鼠大脑皮层相对肝脏IGF-I/IGFBP-1受低氧影响较小,对低氧的反应具有组织特异性。2.核因子-κB（NF-κB）参与常压低氧下小鼠肝脏组织IGF-I/IGFBP-1蛋白表达的调节,参常压低氧下根田鼠肝脏组织IGF-I蛋白表达的调节,参与常压低氧下高原鼠兔肝脏组织IGFBP-1蛋白表达的调节,参与CoCl₂模拟低氧下高原鼢鼠肝脏组织IGF-I/IGFBP-1蛋白表达的调节。3.首次揭示了不同强度的CoCl₂模拟低氧对高原鼢鼠的大脑皮层和肝脏组织HIF-1α蛋白表达的影响,核因子-κB（NF-κB）参与肝脏组HIF-1α基础表达和低氧下的表达。4.首次从肝脏组织克隆了中华鼢鼠HIF-1α的cDNA序列。更多还原

【Abstract】 Ochotona curzoniae,Microtus oeconomus and Myospalax Baileyi are native mammals resident at Qinghai-Tibetan plateau,and they have well acclimatized to the environmental hypoxia,but the adaptive principle has not been entirely explored.IGF-I （Insulin-like Growth FactorⅠ）,an endocrinal and paracrinal/autocrinal factor,is linked to hypoxia closely.Recently more and more papers showed that IGF may play protective roles in hypoxic model.This study reports the effects of hypoxia on IGF-I/IGFBP-1 expressions in both the prefrontal cortex and the liver of Ochotona curzoniae,Microtus oeconomus,Myospalax haileyi and mice,and the potential involvement of nuclear factor kappa-B（NF-κB）in mediating the effects of normobaric hypoxia.IGF-1 work as a key regulator of cellular apoptosis and play an important role in preventing tissue damage.In the plateau mammal study,hypoxia was simulated by two methods:the cobalt chloride（CoCl₂）treatment and the normobaric chamber. PDTC,an inhibitor of NF-κB,was injected just before the hypoxia exposure in the normobaric hypoxia study.IGF-I/IGFBP-1 protein was determined by Elisa.Results:1.Six hours after the injection of CoCl₂,IGF-I in the prefrontal cortex of M.Baileyi （20,40 mg/kg）dose not change evidently;IGF-I/IGFBP-1 in the liver of M.Baileyi （20,40 mg/kg）significantly increased.During normobaric hypoxia（10.8%O₂ or 8.0%O₂）,IGF-I expressions in the liver of M.oeconomus and the IGFBP-1 expressions in the liver of O.curzoniae has been greatly enhanced;16.0%,10.8% O₂ or 8.0%O₂ hypoxia remarkably enhanced IGF-I/IGFBP-1 expressions in the liver of mice.Under the hypoxic induction（10.8%and 8,0%O₂）,hepatic IGF-I of Microtus oeconomus significantly enhanced,but hepatic IGFBP-1 did not show such change.Hepatic IGF-I and IGFBP-1 of Ochotona curzoniae showed reverse changes of hepatic IGF-I and IGFBP-1 from Microtus oeconomus.2.IGF-I expressions of the PDTC treatment during hypoxia in prefrontal cortex of M. Baileyi reduced remarkedly;PDTC treatment during hypoxia significantly reversed the hypoxia-enhanced IGF-I expressions in the liver of mice,M.Baileyi and M. oeconomus,PDTC treatment during hypoxia significantly reversed the hypoxia-enhanced IGFBP-1 expressions in the liver of mice,M.Baileyi and O. curzoniae;3.Six hours after the injection of CoCl₂,HIF-1αin the prefrontal cortex and liver of M. Baileyi（20 mg/kg）increased significantly compared to the control group.We did not detect any expression of HIF-1,however,in the cortex of M.Baileyi.4.The coding region of M.cansus HIF-1αcDNA was 2457 bp,encoding 818 amino acids.The amino acid sequence of M.cansus HIF-1αhas 93%,92%,93%,98% identities of those from mice,rat,human and M.Baileyi.Conclusions:1.Ochotona curzoniae,Microtus oeconomus and Myospalax Baileyi are well acclimatized to the environmental hypoxia.Ochotona curzoniae,Microtus oeconomus and Myospalax Baileyi may have different signaling pathways to prevent the hypoxic damage.These differences might be caused by their living environments or the differences of their species.2.NF-κB may play important,but different roles in the regulation of IGF-I/IGFBP-1 expressions of different animals.The IGF-I/IGFBP-1 expressions of Ochotona curzoniae,Microtus oeconomus and Myospalax Bailevi linked to NF-κB.NF-κB also partly participates in the regulation of IGFBP-1 expression of mice.3.As an oxygen-sensitive transcription factor,NIF-1αis important for the response of ischemia-hypoxia.Although some transcription factors,like AP-1,Egr-1,may mediate some special gene’s expressions under hypoxia,HIF-1αplays an indispensable role in the oxygen balance.Expression of HIF-1αof Ochotona curzoniae did not show changes under hypoxia,expression of HIF-1αof Microtus oeconomus enhanced only in the circumstance of dramatic hypoxia,while expression of hepatic HIF-1αof Myospalax Baileyi enhanced significantly during hypoxia.Moreover,NF-κB may mediate the hepatic expression of HIF-1αof Myospalax Baileyi.After injection of the antagonist of NF-κB,we did not detect the basic expression and the expression of HIF-1αunder hypoxia. 4.The homology of M.cansus HIF-1αcDNA sequence shows 98%identity to M. Baileyi and 97%identity to M.judaei.The amino acids alignment exhibits 100% identity in all key domain between M.Baileyi and M.cansus which lived in Western China,but there are four different amino acids in TAD（N）domain of M. judaei which lived in Israel,The amino acids in ODD domain of M.baileyi and M. cansus is S.but R instead of S in M.judaei.更多还原