【作者】 张严峻；

【导师】 魏尔清；

【作者基本信息】 浙江大学 ， 药理学， 2006， 博士

【摘要】 半胱氨酰白三烯（cysteinyl leukotrienes,CysLTs,包括LTC₄,LTD₄,LTE₄）是花生四烯酸5-脂氧酶（5-lipoxygenase,5-LOX）的代谢产物。CysLTs是很强的炎症介质,参与多种炎症反应过程,如介导呼吸道平滑肌痉挛、微血管渗漏等病理反应。在中枢神经系统内,半胱氨酰白三烯参与许多疾病的发生、发展,如脑缺血、脑外伤、脑出血、脑肿瘤、脑脊髓炎、多发性硬化、癫痫以及衰老等。在脑外伤、脑肿瘤、脑缺血等病理状态下,脑内CysLTs产生增加,并参与血脑屏障破坏、脑水肿的发生。 CysLTs通过其受体发挥生理作用。目前,已经发现和克隆了两种半胱氨酰白三烯受体（cysteinyl leukotrienes receptors）,即CysLT₁和CysLT₂受体,它们均为经典的7次跨膜G蛋白偶联受体。CysLT₁受体有多种拮抗剂,如zafirlukast、montelukast、tomelukast、pobilukast、pranlukast（ONO-1078）和MK-571,它们均能拮抗CysLTs引起的人支气管收缩。目前,已有部分拮抗剂在临床用于支气管哮喘的治疗。但是,CysLT₂受体只有非选择性拮抗剂Bay u9773、FPL55712。 CysLT₁受体分布在脾脏、肺、胎盘、小肠、表皮和支气管平滑肌等组织,以及单核淋巴细胞、巨噬细胞、肥大细胞、嗜酸性粒细胞、CD34⁺细胞、中性粒更多还原

【Abstract】 Cysteinyl leukotrienes （CysLTs, including LTC₄, LTD₄ and LTE₄） are 5-lipoxygenase （5-LOX） metabolites of arachidonic acid. As potent inflammatory mediators, CysLTs can induce hyperresponsiveness and inflammation in asthmatic airways, reduce blood flow in coronary, femoral and carotid vessels, induce plasma extravasation and tissue edema;finally result in circulatory and respiratory dysfunctions in the peripheral organs. In the central nervous system, CysLTs are also involved in pathogenesis of various diseases, such as cerebral ischemia, trauma, hemorrhage, tumor, encephalomyelitis, multiple sclerosis, epileptic seizures and aging. Production of CysLTs is increased in the ischemic brain, and the increased CysLTs are correlated to blood-brain barrier （BBB） dysfunction and brain edema.The actions of CysLTs are mediated via activating their receptors. Two CysLT receptors have been identified and cloned, i.e. CysLT₁ and CysLT₂ receptors. Both of them are classical Gq protein-coupled seven transmembrane receptors. A lot of CysLTi receptor selective antagonists have been discovered, such as zafirlukast, montelukast, tomelukast, pobilukast, pranlukast （ONO-1078）, and MK-571. They can antagonize the constriction of bronchi induced by CysLTs. Some of these antagonists are now clinically used as anti-inflammatory and anti-asthma drugs. However, only non-selective antagonists, Bay u9773 and FPL 55712, are available for CysLT₂ receptor. As the distribution of these receptors, CysLTi receptor is highly expressed in spleen, lung, placenta, small intestine, as well as in lots of cells, including bronchial smooth muscle cells, monocytes, macrophages, mast cells, eosinophils, CD34⁺hemopoietic progenitor cells, neutrophils, human umbilical vein endothelial cells （HUVECs） and fibroblasts. On the other hand, the expression pattern of human CysLT2 receptor is substantially different from that of CysLTi receptor. It is also highly expressed in spleen and peripheral blood leukocytes, as well as in the heart and adrenal glands. In the central nervous system, CysLT2 receptor expression is widespread, including most regions of the brain and spinal cord. Similar expression patterns of CysLT2 receptor were seen in mouse tissue to that in human.Although CysLTi and CysLT2 receptors are widely studied, their physiological and pathological implications are still not clarified fully, especially in following aspects. One aspect is that the expression of CysLTi and CysLT2 receptors in the brain and lung is unclear in the developing process of individuals. The second aspect is that the expression and distribution of CysLTi and CysLT2 receptors in the brain and the changes after injuries （such as ischemic injury） are unknown, although CysLTs can induce disruption of BBB and brain edema and CysLTi receptor antagonists or 5-LOX inhibitors can protect from ischemic brain injury. The third aspect is that no direct evidence indicates the involvement of CysLTi and CysLT2 receptors in eosinophilic inflammation of airways in asthmatic animals, although CysLTs induce such an inflammation in airways is well known.Therefore, to determine whether CysLTi and CysLT2 receptors are expressed in different physiological and pathological conditions of the lung and brain, we observed the following changes in the present study. One is the expression property of CysLTi and CysLT2 receptors in mouse development. The second is the expression of CysLTi and CysLT2 receptors in the brain after focal cerebral ischemia, the relation between receptor expression and acute ischemic neuronal injury, and the localization and modulating role of CysLTi receptor in the ischemic brain. The third is the expression of CysLTi and CysLT2 receptors in the lung after eosinophilic inflammation, and the modulating effect of CysLTi receptor antagonist in asthmatic mice.We hope to clarify the expressions of CysLTi and CysLT2 receptors in lung and brain development, cerebral ischemia and bronchial asthma. These findings willcontribute to well understand the mechanisms of the actions CysLTi and CysLT2 receptors on physiological and pathological processes.Part 1 The expressions of cysteinyl leukotriene receptors in mousebrain and lung at various developmental stagesAim: To determine the mRNA expressions of cysteinyl leukotriene receptors （CysLTi and CysLT2 receptors） in the brain and lung in various stages of the development in mice. Methods: The brain and lung tissues were obtained from mice at end stage of embryos, 1, 3, 7, 10, 30 days after birth, 2 months （adult） and 2 years （elder） after birth. The mRNA expressions of CysLTi and CysLT2 receptors were analyzed by quantitative RT-PCR. Results: In the brain, CysLTi receptor mRNA expression was relatively higher 1 day before and 1 day after birth;became lower 3 days after birth, reached the minimal level 7 days and maintained until 10 days after birth;then increased 30 days after birth and maintained until adult;reduced to the minimal level again in elder mice. However, CysLT2 receptor mRNA expression was gradually reduced from 1 day to 7 days after birth;but reached a peak at 10 days after birth;reached the minimal level in elder mice. In the lung, CysLTi receptor mRNA expression was lower at the end stage of embryo;remained at a relatively higher levels within 7 days after birth;reduced 10 days after birth;reached a peak 30 days after birth;then gradually reduced and reached the minimal level in elder mice. While CysLT2 receptor mRNA expression reached a peak 10 days after birth and the minimal level in elder mice. Conclusion: The expressions of CysLTi and CysLT2 receptor mRNAs largely varied between 1 week to 1 month after birth;their expressions were at minimal levels in elder mice.Part 2 The expressions of cysteinyl leukotriene receptors in mouse brain after focal cerebral ischemia.Aim: To determine whether cysteinyl leukotriene receptors （CysLTi and CysLT2 receptors） are involved in acute neuronal injury after focal cerebral ischemia in mice, and to confirm CysLTi receptor localization. Methods: After permanent focal cerebral ischemia was induced by middle cerebral artery occlusion （MCAO）, neurological deficits and neuron loss was determined at various time points within 48 h. The mRNA expressions of CysLTi and CysLT2 receptors in the brain were analyzed by RT-PCR, CysLTi receptor localization was detected by double immunofluorescence. Results: Neurological deficits and neuron loss were found 6, 24 and 48 h after MCAO. The mRNA expressions of both CysLTi and CysLT2 receptors were up-regulated in the ischemic hemisphere 1, 24 and 48 h after MCAO with peaks at 24 h. CysLTi receptor was selectively localized in neurons 24 h after MCAO. Conclusion: CysLTi and CysLT2 receptors are involved in acute neuronal injury after focal cerebral ischemia. CysLTi receptor is localized in neurons after ischemia.Part 3 The expressions of cysteinyl leukotriene receptors in the lungs of asthmatic mice and the modulation by CysLTireceptor antagonist montelukastAim: To determine the expressions of cysteinyl leukotriene receptors （CysLTi and CysLT2 receptors） in airway eosinophilic inflammation of ovalbumin （OVA）-induced asthmatic mice and the modulation by montelukast, a CysLTi receptor antagonist. Methods: Asthma model was induced by challenge with aerosol OVA in C57BL/6 mice that were chronically exposed to OVA. The eosinophils in bronchoalveolar lavage fluid （BALF） and lung tissues were counted, interleukin-5 （IL-5） level in BALF was measured, and CysLTi and CysLT2 receptor mRNA expressions were detected by semi-quantitative RT-PCR. Results: After asthmatic reactions were induced by OVA challenge in the sensitized mice, CysLTi receptor mRNA expression was increased by 32.4% but CysLT2 receptor mRNA was decreased by 68.0%. OVA challenge also increased eosinophils in lung tissue and BALF, and elevated the level of IL-5 in BALF. Montelukast （6 mg/kg, once per dayfor 20 d） significantly suppressed the increased eosinophils in lung tissue and BAL fluid, and increased IL-5 level in BAL fluid in OVA challenged mice. Montelukast also inhibited the increased CysLTi receptor mRNA expression by 70.9%, but did not affect the reduced CysLT2 receptor mRNA expression after OVA challenge. Conclusion: CysLT receptors are modulated immunologically, and montelukast inhibits up-regulation of CysLTi receptor and airway eosinophilic inflammation in asthmatic mice.CONCLUSION1. The expressions of CysLTi and CysLT2 receptors were related to growth in mouse brain and lung. The mRNA expressions are different between CysLT i and CysLT2 in brain and lung.2. CysLTi and CysLT2 receptors are involved in acute neuronal injury after focal cerebral ischemia. CysLTi receptor is localized in neurons 24 h after ischemia.3. CysLT receptors are modulated immunologically, and montelukast inhibits up-regulation of CysLTi receptor and airway eosinophilic inflammation in asthmatic mice.更多还原