烟草烟雾吸入对大鼠脑组织E-selectin和TNF-α的影响
|
摘要
前言
缺血性脑血管病是常见病、多发病，其致死率与致残率均很高，是导致患者致残和认知功能障碍的主要原因，是威胁人们健康和导致生活质量下降的主要疾病之一。吸烟是缺血性脑血管病的危险因素，近年来的研究提示，粘附分子及细胞因子在缺血性脑血管病的形成中起重要作用，吸烟可引起血管内皮损伤及动脉粥样硬化，其病理变化与粘附分子及细胞因子有密切关系。
粘附分子是由细胞产生，能介导细胞与细胞、细胞与细胞外基质间相互接触和结合的一类分子。其中与动脉硬化及脑血管病发生研究较多的为细胞间粘附分子(ICAM-1)、血管细胞间粘附分子-1(VCAM-1)、E-选择素(E-selectin)、P-选择素(P-selectin)等。E-selectin能介导多形核白细胞与内皮细胞的起始粘附。缺血性脑血管病的形成与白细胞、血小板及血管内皮细胞表面的粘附分子有密切关系，即粘附分子促进白细胞、血小板聚集于血管内皮细胞，引起血流障碍，使缺血性脑损伤加重。
在缺血性脑血管病的形成过程中，肿瘤坏死因子(TNF-α)是造成损伤的重要因素。TNF-α在缺血性脑血管病中的作用日益受到众多学者的重视，TNF-α能够刺激血管内皮细胞表达粘附分子，介导白细胞与血管内皮细胞的粘附。
本实验通过大鼠吸烟模型，应用免疫组织化学方法研究吸烟对动物脑组织中E-selectin和TNF-α的表达影响，探讨E-selectin及TNF-α在吸烟引起脑血管病方面的可能机制，为缺血性脑血管病的预防和治疗提供理论依据。
材料和方法
二．实验材料：健康wistar大鼠60只，由中国医科大学实验动
物中心提供，实验用烟为市售某牌香烟，自制 80 x 60 x 58cm’的烟
箱，E－selectin、TNF－a单克隆抗体及 SABC试剂盒均购自武汉
博士德生物工程有限公司。
2．实验方法：健康 SIStal大鼠 60只，随机分成六组／组：正
常对照组中组：非吸烟对照组人组：短期吸烟组中组：长期小量
吸烟组在组：长期大量吸烟组J组：戒烟组。长期大量组、短期
吸烟组、戒烟组每次吸烟10支，长期小量吸烟组每次吸烟4支，每
组每天吸烟两次，每次30分钟，两次间隔4小时。长期组吸烟三
个月，短期组吸烟一个月，戒烟组吸烟一个月，戒烟一个月。各组
实验动物在各时间点，经10％水含氯醛麻醉，以150毫升肝素化
生理盐水和4％多聚甲醛250Inl进行灌流固定，将固定后的标本，
常规进行水洗，材料脱水、浸蜡、包埋，在石蜡切片机上切取冠状脑
片，连续切片 10张，厚度约 5 pm，分别行 HE染色，E＊elechn及
’YNF－a免疫组化染色。免疫组化染色采用SABC法，DAB显色，
光学显微镜下观察各组免疫组化切片阳性反应物沉积情况，在切
片白皮质处观测5个不重复高倍视野，分别计数E1elechn各组
阳性血管内皮细胞数及TNF－a阳性细胞数，实验数据以均数。
标准差表示，用SPSS统计软件进行方差分析，应用q检验，做组间
两两分析比较，P＜0．05有统计学意义。
实验结果
二．E＊electin免疫组化结果：E＊electin阳性细胞呈棕色，主
要表达在血管内皮细胞。A组及B组平均每5个视野血管内皮细
胞无表达或偶有l一二个血管内皮细胞表达，A为两组比较无差异
河＞0．05人吸烟各组表达均升高，分别与A在组比较有明显差
·2·
异（P＜0．05）＊组较C、D上组表达减少（P＜0．05）。
2．’INF－a免疫组化结果：TNF－a阳性细胞呈棕色，阳性细
胞为神经元、脑胶质细胞和血管内皮细胞，A组及B组平均每5
个视野有少量阳性细胞表达。A在两组比较无差异（P＞o．仍人
吸烟各组表达均升高，分另与A在组比较有明显差异（P＜o．05＊
F组较C、D＊组表达减少（P＜0．05）。
讨 论
吸烟是促进脑梗死形成的重要危险因素，吸烟引起缺血性脑
血管病的确切机制不清楚，大量研究表明粘附分子及炎性细胞因
子介导的炎性反应在脑梗死形成中起着重要作用，研究吸烟后E
＊elechn和’YNF－。的变化对探讨吸烟引起脑梗死发病机制有
重要意义。
香烟燃烧的烟雾可分为气体和微粒两部分，超过4000多种有
害物质已被证实，其中主要的有毒复合物包括一氧化碳（CO人尼
古丁和焦油。CO进入人体后大部分与红细胞内血红蛋白结合，
形成碳氧血红蛋白（COHB人引起缺氧和血管损伤。此外，尼古丁
和焦油等有害物质可直接损害血管内皮细胞，导致血管内皮功能
失调。目前，普遍认为，血管内皮细胞不仅是血管腔内的一层保护
细胞，而且有抗凝、抗血栓形成及纤溶等广泛生理功能，并且广泛
参与了许多疾病的病理过程，内皮细胞损伤是动脉粥样硬化及脑
梗死的早期病变和基本动因。本实验发现E－selechn主要表达
在活化的血管内皮细胞上。各个吸烟组脑组织中均有 E－selectin
表达，与对照组比较有显著升高，戒烟后E－selechn表达与各吸
烟组比较表达下降。结果提示。吸烟可使脑组织E－selechn升
高。吸烟对E－selectin的影响以及发生的机制可能与香烟的毒
性成份造成血管内皮细
The ischemic cerebrovascular disease ( ICVD) has been commonly seen in the disorders of nervous system in our country and in the world. The incidence and the mortality of the disease have been keeping high. ICVD usually leaves the serious with various dysfunctions. It is the main cause of disability and recognizing. Smoking as a risk factor for ICVD. In recent years, Adhesion molecules and cytokines have played an important role in ICVD. Smoking may damage vascular epithelium cell and cause atherosclerosis ( As). The pathophysiological processes have connection with adhesion molecules and cytokines.
Adhesion molecules are caused by cell. Intercellular adhesion molecule - 1 ( ICAM - 1 ) , vascular cell adhesion molecule - 1 ( VCAM - 1). E - selectin, P - selectin have been studied commonly in As and ICVD. E - selectin may mediate the first adhesion of polymorphic neutrophic and endothelia cell. Adhesion molecules have played an important role in ICVD. They promote leukocytes and platelets gathering the endothelial and cause the damage of cerebrovascular. TNF - a is the important factor in the development of adhesion molecules, mediate the adhesion of leukocytes and endothelial cell.
In this study, we observed the irnmunoactivities expression of E-selectin and TNF - a in rat brain. Using the rat smoking models
and immunohistochemical staining. We try to explore the possible mechanisms of E-selectin and TNF - a in ICVD.
Matherial and Method
Matherial - Healthy Wistar rats were supplied by Department of Experiment Animals of China Medical University. Cigarettes were obtained from market. Single clone antibody of E-selectin and TNF - a SABC test box were purchased from Wuhan Boster Biological Technology CO. Ltd.
Method - Healthy Wistar rats were random divided into six groups of ten animals for each one. (A) the normal control group. ( B) the none - smoking control group. ( C ) short - time smoking group. ( D ) long - time and small - dose smoking group. ( E) long - time and big
- dose smoking group. ( F) giving - up smoking group. Ten cigarettes for (C) group, (E) group and (F) group each time. Four - cigarettes for ( D) group each time. Smoking twice one day for each group, and last 30 minutes. Four hours for interval. Smoking three months for (D) group and (E) group, and one month for (C) group. Smoking one month and giving - up one month for ( F) group. Rats were transcardially perfused with heparinized saline and buffered formalin, then brains were removed and fixed with buffered formalin. 5
- jxm - thick sections were cut. Immunohistochemical staining and HE staining for E-selectin and TNF - a. Immunostaining was evaluated by light microscopy. The numbers of E-selectin positive vascular endothelial cells were counted in nonoverlapping fields under the light microscope at 400 X magnification. One section from each animals, 5 nonover lapping microscopic fields were collected. With the above methods. TNF - a count was also collected. The results were
expressed as mean ± standard error. Spss 10.0 was performed to analyse the results of E-selectin and TNF - a. P <0.05 was regard as statistically significant.
Result
The immunohistochemistry stain of E-selectin
The positive cells of E-selectin showed brown and located only in the endothelial cell. There wasn' t expression in the endothelial cells for group A and group B. There was a significant increment expression in the endothelial cells following smoking, the expression in group E was much stronger than those in the rest groups, group C and group D came in second, and that in the giving - up one located in third position.
The immunohistochemistry stain of TNF - a
The positive cells of TNF - a showed brown and located neuro-cyte^neurogliocyte and vascular endothelial cell. There are a few expression for group A and group B. The expression in group C, D, E were stronger than group A, B. The expression in group F was lower than group C, group D and group E.
Discussion
Study has already demonstrated that th
缺血性脑血管病是常见病、多发病，其致死率与致残率均很高，是导致患者致残和认知功能障碍的主要原因，是威胁人们健康和导致生活质量下降的主要疾病之一。吸烟是缺血性脑血管病的危险因素，近年来的研究提示，粘附分子及细胞因子在缺血性脑血管病的形成中起重要作用，吸烟可引起血管内皮损伤及动脉粥样硬化，其病理变化与粘附分子及细胞因子有密切关系。
粘附分子是由细胞产生，能介导细胞与细胞、细胞与细胞外基质间相互接触和结合的一类分子。其中与动脉硬化及脑血管病发生研究较多的为细胞间粘附分子(ICAM-1)、血管细胞间粘附分子-1(VCAM-1)、E-选择素(E-selectin)、P-选择素(P-selectin)等。E-selectin能介导多形核白细胞与内皮细胞的起始粘附。缺血性脑血管病的形成与白细胞、血小板及血管内皮细胞表面的粘附分子有密切关系，即粘附分子促进白细胞、血小板聚集于血管内皮细胞，引起血流障碍，使缺血性脑损伤加重。
在缺血性脑血管病的形成过程中，肿瘤坏死因子(TNF-α)是造成损伤的重要因素。TNF-α在缺血性脑血管病中的作用日益受到众多学者的重视，TNF-α能够刺激血管内皮细胞表达粘附分子，介导白细胞与血管内皮细胞的粘附。
本实验通过大鼠吸烟模型，应用免疫组织化学方法研究吸烟对动物脑组织中E-selectin和TNF-α的表达影响，探讨E-selectin及TNF-α在吸烟引起脑血管病方面的可能机制，为缺血性脑血管病的预防和治疗提供理论依据。
材料和方法
二．实验材料：健康wistar大鼠60只，由中国医科大学实验动
物中心提供，实验用烟为市售某牌香烟，自制 80 x 60 x 58cm’的烟
箱，E－selectin、TNF－a单克隆抗体及 SABC试剂盒均购自武汉
博士德生物工程有限公司。
2．实验方法：健康 SIStal大鼠 60只，随机分成六组／组：正
常对照组中组：非吸烟对照组人组：短期吸烟组中组：长期小量
吸烟组在组：长期大量吸烟组J组：戒烟组。长期大量组、短期
吸烟组、戒烟组每次吸烟10支，长期小量吸烟组每次吸烟4支，每
组每天吸烟两次，每次30分钟，两次间隔4小时。长期组吸烟三
个月，短期组吸烟一个月，戒烟组吸烟一个月，戒烟一个月。各组
实验动物在各时间点，经10％水含氯醛麻醉，以150毫升肝素化
生理盐水和4％多聚甲醛250Inl进行灌流固定，将固定后的标本，
常规进行水洗，材料脱水、浸蜡、包埋，在石蜡切片机上切取冠状脑
片，连续切片 10张，厚度约 5 pm，分别行 HE染色，E＊elechn及
’YNF－a免疫组化染色。免疫组化染色采用SABC法，DAB显色，
光学显微镜下观察各组免疫组化切片阳性反应物沉积情况，在切
片白皮质处观测5个不重复高倍视野，分别计数E1elechn各组
阳性血管内皮细胞数及TNF－a阳性细胞数，实验数据以均数。
标准差表示，用SPSS统计软件进行方差分析，应用q检验，做组间
两两分析比较，P＜0．05有统计学意义。
实验结果
二．E＊electin免疫组化结果：E＊electin阳性细胞呈棕色，主
要表达在血管内皮细胞。A组及B组平均每5个视野血管内皮细
胞无表达或偶有l一二个血管内皮细胞表达，A为两组比较无差异
河＞0．05人吸烟各组表达均升高，分别与A在组比较有明显差
·2·
异（P＜0．05）＊组较C、D上组表达减少（P＜0．05）。
2．’INF－a免疫组化结果：TNF－a阳性细胞呈棕色，阳性细
胞为神经元、脑胶质细胞和血管内皮细胞，A组及B组平均每5
个视野有少量阳性细胞表达。A在两组比较无差异（P＞o．仍人
吸烟各组表达均升高，分另与A在组比较有明显差异（P＜o．05＊
F组较C、D＊组表达减少（P＜0．05）。
讨 论
吸烟是促进脑梗死形成的重要危险因素，吸烟引起缺血性脑
血管病的确切机制不清楚，大量研究表明粘附分子及炎性细胞因
子介导的炎性反应在脑梗死形成中起着重要作用，研究吸烟后E
＊elechn和’YNF－。的变化对探讨吸烟引起脑梗死发病机制有
重要意义。
香烟燃烧的烟雾可分为气体和微粒两部分，超过4000多种有
害物质已被证实，其中主要的有毒复合物包括一氧化碳（CO人尼
古丁和焦油。CO进入人体后大部分与红细胞内血红蛋白结合，
形成碳氧血红蛋白（COHB人引起缺氧和血管损伤。此外，尼古丁
和焦油等有害物质可直接损害血管内皮细胞，导致血管内皮功能
失调。目前，普遍认为，血管内皮细胞不仅是血管腔内的一层保护
细胞，而且有抗凝、抗血栓形成及纤溶等广泛生理功能，并且广泛
参与了许多疾病的病理过程，内皮细胞损伤是动脉粥样硬化及脑
梗死的早期病变和基本动因。本实验发现E－selechn主要表达
在活化的血管内皮细胞上。各个吸烟组脑组织中均有 E－selectin
表达，与对照组比较有显著升高，戒烟后E－selechn表达与各吸
烟组比较表达下降。结果提示。吸烟可使脑组织E－selechn升
高。吸烟对E－selectin的影响以及发生的机制可能与香烟的毒
性成份造成血管内皮细
The ischemic cerebrovascular disease ( ICVD) has been commonly seen in the disorders of nervous system in our country and in the world. The incidence and the mortality of the disease have been keeping high. ICVD usually leaves the serious with various dysfunctions. It is the main cause of disability and recognizing. Smoking as a risk factor for ICVD. In recent years, Adhesion molecules and cytokines have played an important role in ICVD. Smoking may damage vascular epithelium cell and cause atherosclerosis ( As). The pathophysiological processes have connection with adhesion molecules and cytokines.
Adhesion molecules are caused by cell. Intercellular adhesion molecule - 1 ( ICAM - 1 ) , vascular cell adhesion molecule - 1 ( VCAM - 1). E - selectin, P - selectin have been studied commonly in As and ICVD. E - selectin may mediate the first adhesion of polymorphic neutrophic and endothelia cell. Adhesion molecules have played an important role in ICVD. They promote leukocytes and platelets gathering the endothelial and cause the damage of cerebrovascular. TNF - a is the important factor in the development of adhesion molecules, mediate the adhesion of leukocytes and endothelial cell.
In this study, we observed the irnmunoactivities expression of E-selectin and TNF - a in rat brain. Using the rat smoking models
and immunohistochemical staining. We try to explore the possible mechanisms of E-selectin and TNF - a in ICVD.
Matherial and Method
Matherial - Healthy Wistar rats were supplied by Department of Experiment Animals of China Medical University. Cigarettes were obtained from market. Single clone antibody of E-selectin and TNF - a SABC test box were purchased from Wuhan Boster Biological Technology CO. Ltd.
Method - Healthy Wistar rats were random divided into six groups of ten animals for each one. (A) the normal control group. ( B) the none - smoking control group. ( C ) short - time smoking group. ( D ) long - time and small - dose smoking group. ( E) long - time and big
- dose smoking group. ( F) giving - up smoking group. Ten cigarettes for (C) group, (E) group and (F) group each time. Four - cigarettes for ( D) group each time. Smoking twice one day for each group, and last 30 minutes. Four hours for interval. Smoking three months for (D) group and (E) group, and one month for (C) group. Smoking one month and giving - up one month for ( F) group. Rats were transcardially perfused with heparinized saline and buffered formalin, then brains were removed and fixed with buffered formalin. 5
- jxm - thick sections were cut. Immunohistochemical staining and HE staining for E-selectin and TNF - a. Immunostaining was evaluated by light microscopy. The numbers of E-selectin positive vascular endothelial cells were counted in nonoverlapping fields under the light microscope at 400 X magnification. One section from each animals, 5 nonover lapping microscopic fields were collected. With the above methods. TNF - a count was also collected. The results were
expressed as mean ± standard error. Spss 10.0 was performed to analyse the results of E-selectin and TNF - a. P <0.05 was regard as statistically significant.
Result
The immunohistochemistry stain of E-selectin
The positive cells of E-selectin showed brown and located only in the endothelial cell. There wasn' t expression in the endothelial cells for group A and group B. There was a significant increment expression in the endothelial cells following smoking, the expression in group E was much stronger than those in the rest groups, group C and group D came in second, and that in the giving - up one located in third position.
The immunohistochemistry stain of TNF - a
The positive cells of TNF - a showed brown and located neuro-cyte^neurogliocyte and vascular endothelial cell. There are a few expression for group A and group B. The expression in group C, D, E were stronger than group A, B. The expression in group F was lower than group C, group D and group E.
Discussion
Study has already demonstrated that th
引文
1. AropovSE, Simonian NA, Gorigorian CTS. Dynamics of poly morphonuclear leukocyte accumulation in acute cerebral imfarction and their correlation with brain tissue damage [J]. stroke, 1996,27:1739-1743
2. Hynes RO, Lander AD. Contact and adhesive specificities in the associations, migration and targeting of cells and axons. Cell, 1992, 68(2):303-322
3. Piecrmann P, Michel O, Alberecht M, et al. Cerebral endothelial cells are a major source for soluble intercelluer adhesion molecule-1 in the human central nervous system. Neuroscience., 1995, 186(1): 61-64
4. Chopp M, Li Y, Jidng N, et, al. Antibodies against adhesion molecules reduce apoptosis after transient middle cerebral artery occlusion in rat brain [J], J cereb Flow Metab, 1996,16:578-584
5. Hating HP, Borg EL, Sturnshita N, et, al. E-selectin appears in nonischemic tissue during experiment focal cerebral ischemica. Stoke, 1996,27:1386
6．蔡宏道(主编)，现代环境卫生学，第一版，北京人民卫生出版社，1995，989
7. Davis TW, Lack effect of aspirin on cigarette smoke-induced increase in circulating endothelial cells. Heamostasis, 1987, 17(1-2): 66-69
8. Lin SJ, Risk factors, endothelial cell tumover and lipid transport in atherogensis. Neuroscience, 1996,58 (5):309-316
9. Iimmerman M, The effect of nicotine on aortic endothelium: A quantitative ultrastructural study, Atherosclerosis, 1987,63 (1):
33-41
10. Villablanca AC, Nicotine stimulates DNA synthesis and preliferation in vascular endothelial cells in vivio. J Appl physiol, 1998, 64(6): 2089-2098
11. Moyer CF, Sajuthi, et al. Synthetis of IL-1. alpha and IL-1 beta by arterial cells in atherosclerosis. Am J Pathol, 1991, 138 (4): 951-960
12. Palmobo JD, Biaclaburn GL, Forese RA, Endothelia cell factors and response to injury. Surg Gynecol obstet, 1991. 173:505-518
13. O Brien KD, Mcdoncld To, chait A et al. Neovaseular expression of E-selectin intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte contact. Circulation, 1996, 93: 672-682
14. Zhang RL, Chopp M, Jiang N, et al. Anti-interrecellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the wister rat. Stroke, 1995, 26:1438-1443
15. Lin T, Clark R, Yong PR, Tumor necrosis factor in ischemic neurons [J], stroke, 1994.25:1481-1488
16．吴苏宁、张晓霞，急性脑梗死患者血清肿瘤坏死因子及循环内皮细胞含量变化的对比研究[J]，中风与神经疾病杂志2000年17(3)：149-151
17. Pavel V, Petrik, MD, Hugh A et al. Cigarette smoking accelerate carotid artey intimal hyperplasia in a dose-dependent manner.. stroke, 1995: 26: 1409-1414
2. Hynes RO, Lander AD. Contact and adhesive specificities in the associations, migration and targeting of cells and axons. Cell, 1992, 68(2):303-322
3. Piecrmann P, Michel O, Alberecht M, et al. Cerebral endothelial cells are a major source for soluble intercelluer adhesion molecule-1 in the human central nervous system. Neuroscience., 1995, 186(1): 61-64
4. Chopp M, Li Y, Jidng N, et, al. Antibodies against adhesion molecules reduce apoptosis after transient middle cerebral artery occlusion in rat brain [J], J cereb Flow Metab, 1996,16:578-584
5. Hating HP, Borg EL, Sturnshita N, et, al. E-selectin appears in nonischemic tissue during experiment focal cerebral ischemica. Stoke, 1996,27:1386
6．蔡宏道(主编)，现代环境卫生学，第一版，北京人民卫生出版社，1995，989
7. Davis TW, Lack effect of aspirin on cigarette smoke-induced increase in circulating endothelial cells. Heamostasis, 1987, 17(1-2): 66-69
8. Lin SJ, Risk factors, endothelial cell tumover and lipid transport in atherogensis. Neuroscience, 1996,58 (5):309-316
9. Iimmerman M, The effect of nicotine on aortic endothelium: A quantitative ultrastructural study, Atherosclerosis, 1987,63 (1):
33-41
10. Villablanca AC, Nicotine stimulates DNA synthesis and preliferation in vascular endothelial cells in vivio. J Appl physiol, 1998, 64(6): 2089-2098
11. Moyer CF, Sajuthi, et al. Synthetis of IL-1. alpha and IL-1 beta by arterial cells in atherosclerosis. Am J Pathol, 1991, 138 (4): 951-960
12. Palmobo JD, Biaclaburn GL, Forese RA, Endothelia cell factors and response to injury. Surg Gynecol obstet, 1991. 173:505-518
13. O Brien KD, Mcdoncld To, chait A et al. Neovaseular expression of E-selectin intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte contact. Circulation, 1996, 93: 672-682
14. Zhang RL, Chopp M, Jiang N, et al. Anti-interrecellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the wister rat. Stroke, 1995, 26:1438-1443
15. Lin T, Clark R, Yong PR, Tumor necrosis factor in ischemic neurons [J], stroke, 1994.25:1481-1488
16．吴苏宁、张晓霞，急性脑梗死患者血清肿瘤坏死因子及循环内皮细胞含量变化的对比研究[J]，中风与神经疾病杂志2000年17(3)：149-151
17. Pavel V, Petrik, MD, Hugh A et al. Cigarette smoking accelerate carotid artey intimal hyperplasia in a dose-dependent manner.. stroke, 1995: 26: 1409-1414