PAR-1，HIF-1a，MMP-9及TIMP-1在人脑出血灶周表达的实验研究
摘要
目的：近年来，大量动物实验或人外周血中研究表明凝血酶作为神经毒性物质，在高血压脑出血后脑水肿的形成机制中起着重要作用。同时凝血酶通过P38MAPK途径活化血管平滑肌细胞的HIF-1，P38通过使HIF-1磷酸化来调节HIF-1的过量表达，且凝血酶及HIF-1a的激活又启动了另一种脑水肿致病因子MMP-9的表达，共同参与脑水肿的形成。但是，据文献所查，目前少见人类脑组织的PAR-1、HIF-1a、MMP-9、TIMP-1相关临床研究的报道。凝血酶的血管外作用是通过凝血酶受体PAR-1实现的。因此，本组研究了人类高血压脑出血后脑组织PAR-1、HIF-1a和MMP-9、TIMP-1的动态表达，相关性及其与脑水肿的关系。验证PAR-1、HIF-1a、MMP-9、TIMP-1在脑出血后脑水肿形成中的作用，明确人脑出血后凝血酶介导的水肿的具体径路和作用环节。
方法：对2009年1月-2010年7月因高血压脑出血在吉林大学第一医院神经外科，行开颅血肿清除术治疗病人24例，将入路通道范围内紧邻血肿的脑组织做为病例组标本，部分患者皮层“造瘘”起始处即远隔血肿部位脑组织，做为对照组标本，共6例。病例组按发病时间分为<6h、6-24h、24h-3d、>3d组，采用免疫组化法检测24例脑出血灶周组织及6例正常脑组织各时间点PAR-1、HIF-1a和MMP-9、TIMP-1表达情况，RT-PCR法检测HIF-1a和MMP-9，TIMP-1表达情况，Westernblot检测TIMP-1蛋白表达，并通过干湿称重法结合脑水肿进行分析，透视电镜观察血脑屏障动态改变，探讨PAR-1、HIF-1a和MMP-9、TIMP-1在脑水肿发生、发展过程中的可能作用，相关性及临床意义。
结果：
1．脑组织水含量的变化，脑出血后脑水肿程度随出血时间的延长逐渐加重，在出血6h内含水量即开始增高，24h后较明显增高，3d左右达到峰值，之后减轻（P<0.05）。
2．电镜下ICH后6h内，细胞质较少，细胞质内核糖体较多，其它细胞器较少，水肿区神经细胞及星形胶质细胞的胞质和细胞核开始肿胀。6-24h损伤加重，水肿区神经细胞及星形胶质细胞的胞质和核轻度肿胀，神经元有较少量呈暗细胞样改变，还不明显，线粒体轻度肿胀，嵴变短或消失，次级溶酶体增加，核糖体减少，毛细血管周围的星形胶质细胞的足突肿胀，仍然与毛细血管基底膜相连，血管内皮细胞轻度肿胀。24h-3d，脑组织肿胀明显加重，神经元和胶质细胞溶解、线粒体肿胀、变形、固缩，嵴变短或消失，核糖体减少，胞浆空泡化，核染色质边集，细胞器结构紊乱不清、深染，BBB明显破坏，毛细血管无明显的变化。3d后，胞浆内线粒体轻度肿胀，粗面内质网及核糖体等细胞器基本正常，脑组织肿胀稍有好转，胶质细胞开始回缩。
3．在人脑出血血肿灶周中PAR-1、HIF-1a、MMP-9、TIMP-1圴有表达，对照组与出血组相比均有显著性意义（P<0.05），且出血组各组之间比较均有差异（P<0.05）。出血6h后免疫阳性细胞数开始增加，24h-3d组阳性细胞数最多，之后逐渐下降。PAR-1、HIF-1a、MMP-9、TIMP-1与脑水肿成正相关。
4．PAR-1与HIF-1a蛋白表达正相关（r=0.8809，P<0.05），PAR-1与MMP-9蛋白表达正相关（r=0.9080，P<0.05），HIF-1a与MMP-9蛋白表达正相关（r=0.8632，P<0.05）。
结论：
1、人脑出血灶周PAR-1、HIF-1a与MMP-9，TIMP-1的表达明显升高，与脑水肿密切相关。
2、PAR-1、HIF-1a与MMP-9、TIMP-1的异常表达在高血压脑出血后脑水肿中起重要作用。
3、PAR-1、HIF-1a与MMP-9显著相关，以协同或互补的方式共同参与、促进脑水肿的发生。
Experimental study of PAR-1, HIF-1a, MMP-9and TIMP-1in intracerebral hemorrhage in human
Objective:In recent years, a large number of animal experiments or human peripheral blood studies showed that thrombin as neurotoxic substances plays an important role in the formation mechanism of the brain edema of hypertensive cerebral hemorrhage. Thrombin by P38MAPK pathway activated vascular smooth muscle cells of HIF-1, P38to regulate HIF-1over expression, and thrombin, and HIF-la activation by HIF-1phosphorylation and start another brain edema caused cause of the child of MMP-9expression, to participate in the formation of cerebral edema. According to the investigation, the rare human brain tissue PAR-1, HIF-la and MMP-9, TIMP-1in clinical research reports. Extravascular role of thrombin is achieved through the thrombin receptor PAR-1. Therefore, the study of human hypertensive cerebral hemorrhage at different time points after hemorrhage and brain tissue of PAR-1, HIF-la and MMP-9, TIMP-1. Clear specific pathways of the thrombin-mediated edema after intracerebral hemorrhage and the role of link. Validation PAR-1, HIF-la, MMP-9, TIMP-1in the back of the role of intracerebral hemorrhage edema, clearly people after intracerebral hemorrhage of thrombin mediated the specific route and edema action segment.
Methods:From January2009to July2010in JiLin university for high blood pressure cerebral hemorrhage, the first hospital neurosurgery, could work clear hematoma surgical treatment patients24cases, the road will be within the scope of the hematoma channel next to the brain as the case group specimens, some patients cortex "colostomy " starting place namely hematoma parts over brain tissue, as the control group specimens, a total of6cases. According to the time of the case group is divided into<6h,6to24h,24h-3d,>3d group, using immunohistochemical method to detect24cases focal cerebral hemorrhage week6cases organization and normal brain tissue each time point PAR-1, HIF-la and MMP-9, TIMP-1expression, RT-PCR method to detect HIF-1a and MMP-9, TIMP-1expression, the Western blot test TIMP-1the protein expression, and through the dry wet weighing method combined with brain edema carries on the analysis, the electron microscope blood brain barrier dynamic change, discusses PAR-1、HIF-la and MMP-9、TIMP-1in the brain edema occurrence, development process in the possible role, and the correlation and clinical significance.
Results:
1. The water content of the brain changes, cerebral hemorrhage afterbrain edema degree with bleeding the extension of time progressive, in bleeding within6h water content, it started to increase,24h after a significantly higher,3d peak around, and then reduce (P<0.05).
2. ICH under electron microscopy after6h inside, less cytoplasm, cytoplasm ribosomes is more, organelles other less. Edema area nerve cells and stellate cell cytoplasm and the nucleus began swelling.6to24h damage increase, edema area nerve cells and stellate cell cytoplasm and nuclear mild swelling, neurons have less show dark cell samples change, still not obvious, the mitochondria swelling of the mild, crest a shorter or disappear, secondary lysosomes increase, ribosomes reduce, capillary surrounding astrocytes foot processes swelling, still and capillary basement membrane is linked together, endothelial cells mild swelling.24h-3d and brain swelling obviously increase, neurons and glia dissolution, mitochondria swell, deformation, solid shrinkage, crest a shorter or disappear, ribosomes reduce, the empty bubble cytoplasm, nuclear chromatin the edge set, organelles structure disorder is not clear, deep dye, BBB obvious damage. Capillary no significant change.3d, the cytoplasm mitochondria swelling of the mild, rough endoplasmic reticulum and ribosomes organelles such as the basic normal. Swelling of the brain is somewhat better, glial cells start to shrink.
3. In one week in focal cerebral hemorrhage hematoma PAR-1, HIF-la, MMP-9, TIMP-1a rate of expression, the control group and bleeding group significantly by sexual meaning (P<0.05), and bleeding between groups are comparison of difference (P<0.05). Bleeding after6h immune cells began to increase the number of positive,24h-3d group is the maximum number of positive cells, and then gradually decreases. PAR-1, HIF-la, MMP-9, TIMP-1a positive correlation with cerebral edema.
4. PAR-1and HIF-la protein expression are related (r=0.8809, P<0.05), PAR-1and MMP-9protein expression are related (r=0.9080, P<0.05), HIF-la and MMP-9protein expression are related (r=0.8632, P<0.05).
Conclusion:
1. Surrounding cerebral hemorrhage, the expression of PAR-1, HIF-la, MMP-9and TIMP-1increased significantly, and brain edema closely related.
2. The abnormal expression of PAR-1, HIF-la and MMP-9, TIMP-1plays an important role in early brain edema.
3. PAR-1, HIF-1a and MMP-9significantly related to collaborative or complementary approach to the common participation, promote the occurrence of cerebral edema.
方法：对2009年1月-2010年7月因高血压脑出血在吉林大学第一医院神经外科，行开颅血肿清除术治疗病人24例，将入路通道范围内紧邻血肿的脑组织做为病例组标本，部分患者皮层“造瘘”起始处即远隔血肿部位脑组织，做为对照组标本，共6例。病例组按发病时间分为<6h、6-24h、24h-3d、>3d组，采用免疫组化法检测24例脑出血灶周组织及6例正常脑组织各时间点PAR-1、HIF-1a和MMP-9、TIMP-1表达情况，RT-PCR法检测HIF-1a和MMP-9，TIMP-1表达情况，Westernblot检测TIMP-1蛋白表达，并通过干湿称重法结合脑水肿进行分析，透视电镜观察血脑屏障动态改变，探讨PAR-1、HIF-1a和MMP-9、TIMP-1在脑水肿发生、发展过程中的可能作用，相关性及临床意义。
结果：
1．脑组织水含量的变化，脑出血后脑水肿程度随出血时间的延长逐渐加重，在出血6h内含水量即开始增高，24h后较明显增高，3d左右达到峰值，之后减轻（P<0.05）。
2．电镜下ICH后6h内，细胞质较少，细胞质内核糖体较多，其它细胞器较少，水肿区神经细胞及星形胶质细胞的胞质和细胞核开始肿胀。6-24h损伤加重，水肿区神经细胞及星形胶质细胞的胞质和核轻度肿胀，神经元有较少量呈暗细胞样改变，还不明显，线粒体轻度肿胀，嵴变短或消失，次级溶酶体增加，核糖体减少，毛细血管周围的星形胶质细胞的足突肿胀，仍然与毛细血管基底膜相连，血管内皮细胞轻度肿胀。24h-3d，脑组织肿胀明显加重，神经元和胶质细胞溶解、线粒体肿胀、变形、固缩，嵴变短或消失，核糖体减少，胞浆空泡化，核染色质边集，细胞器结构紊乱不清、深染，BBB明显破坏，毛细血管无明显的变化。3d后，胞浆内线粒体轻度肿胀，粗面内质网及核糖体等细胞器基本正常，脑组织肿胀稍有好转，胶质细胞开始回缩。
3．在人脑出血血肿灶周中PAR-1、HIF-1a、MMP-9、TIMP-1圴有表达，对照组与出血组相比均有显著性意义（P<0.05），且出血组各组之间比较均有差异（P<0.05）。出血6h后免疫阳性细胞数开始增加，24h-3d组阳性细胞数最多，之后逐渐下降。PAR-1、HIF-1a、MMP-9、TIMP-1与脑水肿成正相关。
4．PAR-1与HIF-1a蛋白表达正相关（r=0.8809，P<0.05），PAR-1与MMP-9蛋白表达正相关（r=0.9080，P<0.05），HIF-1a与MMP-9蛋白表达正相关（r=0.8632，P<0.05）。
结论：
1、人脑出血灶周PAR-1、HIF-1a与MMP-9，TIMP-1的表达明显升高，与脑水肿密切相关。
2、PAR-1、HIF-1a与MMP-9、TIMP-1的异常表达在高血压脑出血后脑水肿中起重要作用。
3、PAR-1、HIF-1a与MMP-9显著相关，以协同或互补的方式共同参与、促进脑水肿的发生。
Experimental study of PAR-1, HIF-1a, MMP-9and TIMP-1in intracerebral hemorrhage in human
Objective:In recent years, a large number of animal experiments or human peripheral blood studies showed that thrombin as neurotoxic substances plays an important role in the formation mechanism of the brain edema of hypertensive cerebral hemorrhage. Thrombin by P38MAPK pathway activated vascular smooth muscle cells of HIF-1, P38to regulate HIF-1over expression, and thrombin, and HIF-la activation by HIF-1phosphorylation and start another brain edema caused cause of the child of MMP-9expression, to participate in the formation of cerebral edema. According to the investigation, the rare human brain tissue PAR-1, HIF-la and MMP-9, TIMP-1in clinical research reports. Extravascular role of thrombin is achieved through the thrombin receptor PAR-1. Therefore, the study of human hypertensive cerebral hemorrhage at different time points after hemorrhage and brain tissue of PAR-1, HIF-la and MMP-9, TIMP-1. Clear specific pathways of the thrombin-mediated edema after intracerebral hemorrhage and the role of link. Validation PAR-1, HIF-la, MMP-9, TIMP-1in the back of the role of intracerebral hemorrhage edema, clearly people after intracerebral hemorrhage of thrombin mediated the specific route and edema action segment.
Methods:From January2009to July2010in JiLin university for high blood pressure cerebral hemorrhage, the first hospital neurosurgery, could work clear hematoma surgical treatment patients24cases, the road will be within the scope of the hematoma channel next to the brain as the case group specimens, some patients cortex "colostomy " starting place namely hematoma parts over brain tissue, as the control group specimens, a total of6cases. According to the time of the case group is divided into<6h,6to24h,24h-3d,>3d group, using immunohistochemical method to detect24cases focal cerebral hemorrhage week6cases organization and normal brain tissue each time point PAR-1, HIF-la and MMP-9, TIMP-1expression, RT-PCR method to detect HIF-1a and MMP-9, TIMP-1expression, the Western blot test TIMP-1the protein expression, and through the dry wet weighing method combined with brain edema carries on the analysis, the electron microscope blood brain barrier dynamic change, discusses PAR-1、HIF-la and MMP-9、TIMP-1in the brain edema occurrence, development process in the possible role, and the correlation and clinical significance.
Results:
1. The water content of the brain changes, cerebral hemorrhage afterbrain edema degree with bleeding the extension of time progressive, in bleeding within6h water content, it started to increase,24h after a significantly higher,3d peak around, and then reduce (P<0.05).
2. ICH under electron microscopy after6h inside, less cytoplasm, cytoplasm ribosomes is more, organelles other less. Edema area nerve cells and stellate cell cytoplasm and the nucleus began swelling.6to24h damage increase, edema area nerve cells and stellate cell cytoplasm and nuclear mild swelling, neurons have less show dark cell samples change, still not obvious, the mitochondria swelling of the mild, crest a shorter or disappear, secondary lysosomes increase, ribosomes reduce, capillary surrounding astrocytes foot processes swelling, still and capillary basement membrane is linked together, endothelial cells mild swelling.24h-3d and brain swelling obviously increase, neurons and glia dissolution, mitochondria swell, deformation, solid shrinkage, crest a shorter or disappear, ribosomes reduce, the empty bubble cytoplasm, nuclear chromatin the edge set, organelles structure disorder is not clear, deep dye, BBB obvious damage. Capillary no significant change.3d, the cytoplasm mitochondria swelling of the mild, rough endoplasmic reticulum and ribosomes organelles such as the basic normal. Swelling of the brain is somewhat better, glial cells start to shrink.
3. In one week in focal cerebral hemorrhage hematoma PAR-1, HIF-la, MMP-9, TIMP-1a rate of expression, the control group and bleeding group significantly by sexual meaning (P<0.05), and bleeding between groups are comparison of difference (P<0.05). Bleeding after6h immune cells began to increase the number of positive,24h-3d group is the maximum number of positive cells, and then gradually decreases. PAR-1, HIF-la, MMP-9, TIMP-1a positive correlation with cerebral edema.
4. PAR-1and HIF-la protein expression are related (r=0.8809, P<0.05), PAR-1and MMP-9protein expression are related (r=0.9080, P<0.05), HIF-la and MMP-9protein expression are related (r=0.8632, P<0.05).
Conclusion:
1. Surrounding cerebral hemorrhage, the expression of PAR-1, HIF-la, MMP-9and TIMP-1increased significantly, and brain edema closely related.
2. The abnormal expression of PAR-1, HIF-la and MMP-9, TIMP-1plays an important role in early brain edema.
3. PAR-1, HIF-1a and MMP-9significantly related to collaborative or complementary approach to the common participation, promote the occurrence of cerebral edema.
引文
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