脑出血大鼠脑内细胞外基质相关基因表达的研究
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摘要
背景和目的：脑出血(intracerebral hemorrhage, ICH)属于脑血管病中的危重类型,在中国的发病率明显高于西方国家。尽管ICH急性期治疗策略已经越来越规范化,但仍局限于血肿清除以及对症支持治疗,针对脑出血造成的神经功能缺失目前尚无有效的治疗方法,致使其病死率及致残率近十年来仍居高不下。已知的脑出血后损伤机制表明细胞外基质(extracellular matrix, ECM)的组成、结构和分布均发生了变化,血脑屏障(blood brain barrier, BBB)破坏、脑水肿的形成等都与ECM密切相关。既往研究证实ECM参与胚胎及各种组织、器官的形成、发育、修复和再生。ECM的组成成分与神经系统的多种生理病理过程密切相关。此外,与ECM功能相关的蛋白如基质金属蛋白酶家族(matrix metalloproteinases, MMPs),整合素家族(integrins),选择素家族(selectins)以及透明质酸酶等均可作用于ECM,参与炎症反应、肿瘤转移、血管发生及创伤愈合等过程。因此,本研究拟从脑出血后ECM相关基因表达变化的角度,探讨脑出血后组织和神经功能恢复的作用机制,将为基础和临床探索脑出血治疗策略提供新的线索和思路。
方法：本研究分为2个部分。第一部分：通过立体定位向大鼠脑内注射Ⅶ型胶原酶复制脑出血模型,假手术组注入等体积生理盐水。采用基因芯片法检测脑出血后ECM相关基因表达变化。第二部分：根据第一部分实验结果,选择胶原(collagen)Ⅰ、collagen Ⅲ、collagen Ⅳ,MMP-2、MMP-9、膜型(memberane-type, MT)1-MMP以及integrin αvβ3、integrin α5β1进行蛋白表达验证。大鼠分假手术组及脑出血组,处理同第一部分。采用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)-右旋糖酐(dextran)灌注结合激光共聚焦显微镜观察损伤区微血管构筑及血流灌注情况；免疫组化方法检测脑出血损伤区增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)+/血管性血友病因子(von Willebrand factorv, WF)+标记的新生血管密度变化及collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、MMP-9、MT1-MMP以及integrin αvβ3、integrin α5β1的时空分布；Western blot技术检测脑出血后collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、MMP-9、MT1-MMP以及integrin β3、integrin a5的蛋白表达。
结果：基因芯片结果显示：脑出血后ECM相关基因如细胞分化抗原(cluster of differentiation, CD)44、骨桥蛋白(oesteopontin, OPN/SPP-1)、钙粘蛋白(cadherin)-1、弹性蛋白微原纤维界面因子(elastin microfibril interfacer, EMILIN)-1、胞间粘附因子(intercellular adhesion molecule, ICAM)-1、转化生长因子(transforming growth factor, TGF)-β以及大部分的ECM胶原成分、MMPs及integrins成员等于不同时间点均有不同趋势的变化。FITC-dextran灌注结果显示：脑出血后4天,损伤处周围血流灌注较假手术组明显减少,脑出血后7天到14天损伤脑组织周围微血管密度较4天时逐渐增大,血流灌注增多,21天到达高峰。免疫组化结果显示：脑出血后4天血肿周围即可见至JPCNA+/vWF+标记的细胞核,且持续增高至14天。在血肿周围可见大量collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、MMP-9、 MT1-MMP以及integrin αvβ3、integrin α5β1阳性血管。Western blot结果显示：脑出血后4天collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、 MMP-9、MT1-MMP以及integrin β3、integrin a5蛋白表达较假手术组明显增高。Collagen Ⅰ、collagen Ⅲ蛋白表达增高至14天,持续到21天；collagenⅣ蛋白表达从14天开始有所降低；MMP-9蛋白表达7天时即开始有所减少,MMP-2、MT1-MMP、integrin β3、integrin α5蛋白表达随着时间延长逐渐增高至14天,21天时开始降低。
结论：1.脑出血后,ECM相关基因表达改变,可能参与脑内炎症反应、组织修复过程；2.脑出血后,collagens、MMPs和integrins表达持续增高,可能改变脑微血管与周围ECM的相互作用,影响血管新生,修复损伤组织。
Background and purpose:Intracerebral hemorrhage (ICH) is a critical subtype of cerebrovascular disease, with higher morbility in China than western countries. In spite of the medical strategy for the acute phase of ICH is becaming more and more standardized, it still be limited to evacuation of the hematoma and supporting therapy. At present, there is no effective therapeutic method for patients to improve neurological deficits caused by ICH. So the fatality rate and disability rate still remain high in the last ten years. The foregone injured mechanism of ICH demonstrated that the composition, structures and distribution of extracellular matrix (ECM) all have changed. Destruction of blood brain barrier (BBB), formation of brain edema, and etc are closely relevant to ECM. The former research confirmed that ECM participate in formation, development, restoration and regeneration of embryo and all kinds of tissues and organs. Moity of ECM were relevated to physiological and pathological process of nervous system. In addition, matrix metalloproteinases (MMPs), integrins, selectins, hyaluronidase and the other proteins associated with function of ECM, participating in inflammatory reaction, neoplasm metastasis, vasculogenesis, wound healing and the other process. Accordingly, the purpose of the present study is to clarify the mechanism of neurological function recovery following ICH by investigating genes associated ECM, and to provide new clues and ideas for exploring the clinical and basic treatment of ICH.
Methods:This study was divided into two parts.(1) Models of ICH were administered by injecting type Ⅶ collagenase into brain of the rats. Sham controls received the same volume of0.9%sterile saline. Gene chip technology was used to detect genes correlated with ECM following ICH.(2) According to the results in the first part, collagen Ⅰ, collagen Ⅲ, collagenⅣ, MMP-2, MMP-9, MT1-MMP, integrin αvβ3and integrin α5β1were selected for further validation. The rats were divided into sham-operated group and model group, and given the same treatment with the rats in the first part. Confocal laser scanning microscopy was used to observed angioarchitecture and blood perfusion of damage zone after fluorescein isothiocyanate (FITC)-dextran injection. Immunohistochemisty were used to identify proliferating cell nuclear antigen (PCNA)+/von Willebrand factor+(vWF) nuclei, and the spatial and temporal distribution of collagen Ⅰ, collagen Ⅲ, collagen Ⅳ, MMP-2, MMP-9, MT1-MMP, integrin αvβ3and integrin α5β1. The expression level of protein collagen Ⅰ, collagenⅢ, collagenⅣ, MMP-2, MMP-9, MT1-MMP, integrin β3and integrin α5were evaluated by Western blot.
Results:Results of gene chip detection showed that ECM-related genes such as cluster of differentiation (CD)44, oesteopontin (OPN/SPP-1), cadherin-1, elastin microfibril interfacer (EMILIN)-1, intercellular adhesion molecule (ICAM)-1, transforming growth factor (TGF)-β as well as most members of collagens, MMPs, intergrins all have different tendency at different time points after ICH induction, and it suggests that ECM may be revelant to repairing of neural injury following ICH. The result after FITC-dextran perfusion showed that the blood perfusion around the hematoma at4days after ICH decrease obviously than that of sham operation, The density of microvessel around the injured brain tissue continuously increasd from7days to14days after ICH and reach peak value at21days. Immunohistochemisty displayed that PCNA+nuclei in vWF+dilated vessels could be observed around the hematoma at4days, and peaked at14days after ICH induction. Numerous collagen Ⅰ, collagen Ⅲ, collagen Ⅳ, MMP-2, MMP-9, MT1-MMP, integrin αvβ3and integrin α5β1positive blood vessel could be detected around the hematoma. Results of Western blot suggested that the expression of protein collagen Ⅰ, collagen Ⅲ, collagen Ⅳ, MMP-2, MMP-9, MT1-MMP, integrin β3and integrin a5all increased obviously at4days after ICH than that in the sham operation group. Expression of collagen Ⅰ and collagen Ⅲ arrived climax at14days and lasted to21days, while expression of collagen Ⅳ started to reduce from14days. Protein level of MMP-9decreased from at7days, while the expression of MMP-2, MT1-MMP, integrin β3and integrin α5gradually increased untill14days and decreased at21days.
Conclusions:1. Expression of genes correlated with ECM changed after ICH, the alteration may be relevant to inflammatory reaction and tissue repair process in the brain.2. Collagens, MMPs and integrins contiued to increase following ICH, and they may modify interaction between cerebral microvessel and peripheral ECM, thus to affect angiogenesis and restore injured brain tissue.
方法：本研究分为2个部分。第一部分：通过立体定位向大鼠脑内注射Ⅶ型胶原酶复制脑出血模型,假手术组注入等体积生理盐水。采用基因芯片法检测脑出血后ECM相关基因表达变化。第二部分：根据第一部分实验结果,选择胶原(collagen)Ⅰ、collagen Ⅲ、collagen Ⅳ,MMP-2、MMP-9、膜型(memberane-type, MT)1-MMP以及integrin αvβ3、integrin α5β1进行蛋白表达验证。大鼠分假手术组及脑出血组,处理同第一部分。采用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)-右旋糖酐(dextran)灌注结合激光共聚焦显微镜观察损伤区微血管构筑及血流灌注情况；免疫组化方法检测脑出血损伤区增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)+/血管性血友病因子(von Willebrand factorv, WF)+标记的新生血管密度变化及collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、MMP-9、MT1-MMP以及integrin αvβ3、integrin α5β1的时空分布；Western blot技术检测脑出血后collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、MMP-9、MT1-MMP以及integrin β3、integrin a5的蛋白表达。
结果：基因芯片结果显示：脑出血后ECM相关基因如细胞分化抗原(cluster of differentiation, CD)44、骨桥蛋白(oesteopontin, OPN/SPP-1)、钙粘蛋白(cadherin)-1、弹性蛋白微原纤维界面因子(elastin microfibril interfacer, EMILIN)-1、胞间粘附因子(intercellular adhesion molecule, ICAM)-1、转化生长因子(transforming growth factor, TGF)-β以及大部分的ECM胶原成分、MMPs及integrins成员等于不同时间点均有不同趋势的变化。FITC-dextran灌注结果显示：脑出血后4天,损伤处周围血流灌注较假手术组明显减少,脑出血后7天到14天损伤脑组织周围微血管密度较4天时逐渐增大,血流灌注增多,21天到达高峰。免疫组化结果显示：脑出血后4天血肿周围即可见至JPCNA+/vWF+标记的细胞核,且持续增高至14天。在血肿周围可见大量collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、MMP-9、 MT1-MMP以及integrin αvβ3、integrin α5β1阳性血管。Western blot结果显示：脑出血后4天collagen Ⅰ、collagen Ⅲ、collagen Ⅳ, MMP-2、 MMP-9、MT1-MMP以及integrin β3、integrin a5蛋白表达较假手术组明显增高。Collagen Ⅰ、collagen Ⅲ蛋白表达增高至14天,持续到21天；collagenⅣ蛋白表达从14天开始有所降低；MMP-9蛋白表达7天时即开始有所减少,MMP-2、MT1-MMP、integrin β3、integrin α5蛋白表达随着时间延长逐渐增高至14天,21天时开始降低。
结论：1.脑出血后,ECM相关基因表达改变,可能参与脑内炎症反应、组织修复过程；2.脑出血后,collagens、MMPs和integrins表达持续增高,可能改变脑微血管与周围ECM的相互作用,影响血管新生,修复损伤组织。
Background and purpose:Intracerebral hemorrhage (ICH) is a critical subtype of cerebrovascular disease, with higher morbility in China than western countries. In spite of the medical strategy for the acute phase of ICH is becaming more and more standardized, it still be limited to evacuation of the hematoma and supporting therapy. At present, there is no effective therapeutic method for patients to improve neurological deficits caused by ICH. So the fatality rate and disability rate still remain high in the last ten years. The foregone injured mechanism of ICH demonstrated that the composition, structures and distribution of extracellular matrix (ECM) all have changed. Destruction of blood brain barrier (BBB), formation of brain edema, and etc are closely relevant to ECM. The former research confirmed that ECM participate in formation, development, restoration and regeneration of embryo and all kinds of tissues and organs. Moity of ECM were relevated to physiological and pathological process of nervous system. In addition, matrix metalloproteinases (MMPs), integrins, selectins, hyaluronidase and the other proteins associated with function of ECM, participating in inflammatory reaction, neoplasm metastasis, vasculogenesis, wound healing and the other process. Accordingly, the purpose of the present study is to clarify the mechanism of neurological function recovery following ICH by investigating genes associated ECM, and to provide new clues and ideas for exploring the clinical and basic treatment of ICH.
Methods:This study was divided into two parts.(1) Models of ICH were administered by injecting type Ⅶ collagenase into brain of the rats. Sham controls received the same volume of0.9%sterile saline. Gene chip technology was used to detect genes correlated with ECM following ICH.(2) According to the results in the first part, collagen Ⅰ, collagen Ⅲ, collagenⅣ, MMP-2, MMP-9, MT1-MMP, integrin αvβ3and integrin α5β1were selected for further validation. The rats were divided into sham-operated group and model group, and given the same treatment with the rats in the first part. Confocal laser scanning microscopy was used to observed angioarchitecture and blood perfusion of damage zone after fluorescein isothiocyanate (FITC)-dextran injection. Immunohistochemisty were used to identify proliferating cell nuclear antigen (PCNA)+/von Willebrand factor+(vWF) nuclei, and the spatial and temporal distribution of collagen Ⅰ, collagen Ⅲ, collagen Ⅳ, MMP-2, MMP-9, MT1-MMP, integrin αvβ3and integrin α5β1. The expression level of protein collagen Ⅰ, collagenⅢ, collagenⅣ, MMP-2, MMP-9, MT1-MMP, integrin β3and integrin α5were evaluated by Western blot.
Results:Results of gene chip detection showed that ECM-related genes such as cluster of differentiation (CD)44, oesteopontin (OPN/SPP-1), cadherin-1, elastin microfibril interfacer (EMILIN)-1, intercellular adhesion molecule (ICAM)-1, transforming growth factor (TGF)-β as well as most members of collagens, MMPs, intergrins all have different tendency at different time points after ICH induction, and it suggests that ECM may be revelant to repairing of neural injury following ICH. The result after FITC-dextran perfusion showed that the blood perfusion around the hematoma at4days after ICH decrease obviously than that of sham operation, The density of microvessel around the injured brain tissue continuously increasd from7days to14days after ICH and reach peak value at21days. Immunohistochemisty displayed that PCNA+nuclei in vWF+dilated vessels could be observed around the hematoma at4days, and peaked at14days after ICH induction. Numerous collagen Ⅰ, collagen Ⅲ, collagen Ⅳ, MMP-2, MMP-9, MT1-MMP, integrin αvβ3and integrin α5β1positive blood vessel could be detected around the hematoma. Results of Western blot suggested that the expression of protein collagen Ⅰ, collagen Ⅲ, collagen Ⅳ, MMP-2, MMP-9, MT1-MMP, integrin β3and integrin a5all increased obviously at4days after ICH than that in the sham operation group. Expression of collagen Ⅰ and collagen Ⅲ arrived climax at14days and lasted to21days, while expression of collagen Ⅳ started to reduce from14days. Protein level of MMP-9decreased from at7days, while the expression of MMP-2, MT1-MMP, integrin β3and integrin α5gradually increased untill14days and decreased at21days.
Conclusions:1. Expression of genes correlated with ECM changed after ICH, the alteration may be relevant to inflammatory reaction and tissue repair process in the brain.2. Collagens, MMPs and integrins contiued to increase following ICH, and they may modify interaction between cerebral microvessel and peripheral ECM, thus to affect angiogenesis and restore injured brain tissue.
引文
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