大鼠脉络丛上皮细胞四种酶的超微定位研究
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摘要
目的:位于脑室的脉络丛可以看作是由紧密连接上皮包绕的血管丛,进而形成了血液和脑脊液间的界面。脑脊液分泌是脉络丛上皮的主要特点,是由许多无机离子跨过脉络丛上皮交换的结果,其主要驱动力来自上皮细胞脑脊液面的钠钾泵和碳酸酐酶。本研究将应用4种与细胞功能密切相关的酶(三磷酸腺苷酶—提供能量;酸性磷酸酶—溶酶体的标志酶;)的电镜细胞化学超微定位技术对脉络丛上皮细胞进行研究,以期进一步全面了解脉络丛上皮细胞内的酶定位,并探讨其在脑脊液生成、吸收和循环中的意义,为人类进一步认识脑的解剖和生理提供形态学依据。同时,对于深入研究脑积水、脑膜炎,神经系统变性疾病等疾病的发病机理,具有广阔的应用前景。
方法:选取正常成年雄性SD大鼠80只,体重200-350g,常规饲养,自然光照,随机分为4个组每组20只,用于四种酶的电镜酶组织化学观察。水合氯醛腹腔麻醉后,迅速切取侧脑室、三脑室、四脑室脉络丛。采用柠檬酸铅法做AKPase酶,硝酸铅法做ACPase,Uusitalo-Karnovsky法做5’-NTase,Wachstein-Meisel法做Mg2+-ATPase的组织化学染色。取材后放入相应固定液中短时固定后,放入同种的缓冲液中4℃下过夜,在37℃温箱中孵育,用冷的缓冲液充分洗涤后,再用冷1%四氧化饿后固定1小时,梯度丙酮脱水,包埋剂浸透、包埋和聚合,超薄切片,醋酸双氧铀轻染,JEM-1230透射电镜观察照相。
酶细胞化学对照实验:(1)作用液不加专一性物;(2)作用前用60℃处理30 min;(3)加特异性抑制剂(AKPase用左旋咪唑抑制)。
结果:(1)形态学观察:透射电镜下脉络丛的结构有三种成分,以毛细血管网为中心,周围为结缔组织,表面为脉络丛上皮细胞.上皮细胞呈单层立方形,核位于细胞基底部,呈圆形或椭圆形,胞质内线粒体和小泡状滑面内质网较多,高尔基氏体丰富,它们分布于细胞顶部,游离核糖体丰富,多分散于胞质内,细胞的游离面有密集的微绒毛,上皮细胞基底部质膜有折叠形成许多质膜内褶,胞外有基膜。细胞间有特殊连接构成复合体,毛细血管为有孔型,基质结缔组织由伸长的成纤维细胞分泌的胶原纤维网组成。偶见丛上细胞,这些细胞与脉络丛上皮紧密相关,形态多样。他们位于上皮细胞顶部微绒毛,显示出典型激活巨噬细胞的超微结构特点如小泡和溶酶体。(2)脉络丛上皮酸性磷酸酶(ACPase)的超微分布:当用2%多聚甲醛和2%戊二醛混合固定时,透射电镜下可见ACPase活性出现在上皮细胞的溶酶体;当用2%多聚甲醛和0.5%戊二醛混合固定时,ACPase活性不仅出现在上皮细胞的溶酶体内,还出现在靠近高尔基体成熟面的滑面内质网上。对照组的溶酶体、高尔基体及内质网等细胞器均未显示ACPase活性。各脑室脉络丛上皮酸性磷酸酶的分布未见明显差异。各脑室脉络丛上皮酸性磷酸酶的分布未见明显差异。(3)脉络丛上皮碱性磷酸酶(AKPase)的超微分布: AKPase活性出现在各脑室脉络丛上皮细胞的微绒毛和基底褶的质膜上,上皮连接处、毛细血管内皮细胞、血液、红细胞膜和间质纤维细胞上。AKPase活性还出现在胞质,高尔基体、内质网、线粒体、溶酶体等细胞器的质膜上。三种对照标本均未见阳性反应。各脑室间未见明显差异。(4)镁三磷酸腺苷酶(Mg2+-ATPase)在脉络丛上皮的超微分布:透射电镜下可见Mg2+-ATPase活性出现在脉络丛上皮细胞的微绒毛表面、基底皱褶处、细胞连接处;酶活性还出现在脉络丛的间质和毛细血管内皮细胞上。对照实验未见酶反应产物的沉积。各脑室间未见明显差异。(5) 5’-核苷酸酶(5’-NT ase)在脉络丛上皮的超微分布:透射电镜下可见5’-NTase活性出现在上皮细胞的细胞连接和基底皱褶处,在上皮细胞微绒毛、间质内未见反应产物的沉积。上皮细胞内各种细胞器的质膜亦未见反应产物沉积。各脑室间未见明显差异。
结论:ACPase通过对脑脊液中物质的摄取和细胞代谢产物的储存,在参与脑脊液中物质的清除和维护大脑内环境的平衡中起着重要作用。我们推测脉络丛上皮上AKPase的作用是阻止磷酸酯类入脑和/或参与转磷酸过程,进而参与某些物质的跨膜转运,并可能构成酶屏障。脉络丛上皮缘的ATPase活性可能提供了在细胞质形成的脑脊液进入脑室中所需的能量。位于上皮细胞侧面的细胞连接处5’-NTase,可能有助于细胞连接的形成,构成血脑液屏障的一部分,并可能通过产生腺苷参与细胞间的信息的传导。而位于上皮基底皱褶处的5’-NTase可能与核酸降解代谢和物质运输有密切关系,以维持脑内核酸物质的稳定。
Objectives: The choroid plexuses (CPs), located within the brain ventricles, can be viewed as a tight epithelium enclosing a vascularized stroma, and as such form an interface between the blood and the cerebrospinal fluid (CSF). CSF secretion is obviously a key feature of the CPs. This secretion is the result of complex inorganic ion exchanges across the tight epithelium of the CP, mainly driven by the activities of the Na+- K+-ATPase located at the CSF-facing membrane of the epithelial cells, and of the carbonic anhydrase. By electron microscopic cytochemical techniques, we will focus on four enzymes in the choroid plexus epithelium of SD rats which are closely concerned with cell function (ATPase—providing energy, ACPase—the sign enzyme of lysosome),hoping to more realize the subcellular localization in choroid plexus epithelium.We will also discuss its significance in the secretion, absorbtion and circulation of CSF. It will offer morphological evidence for us to more realize the anatmization and physiology of the brain. At the same time, It will have wide application foreground in the pathogenesis of hydrocephalous, meningitis, neurodegeneration diseases and so on.
Methods: 80 normal adult SD rats of both sexes are selected, weighted 200-350 gram, routine breeding, natruly illumination, randomely grouping into four groups for the four enzymes. After intraperitoneal hydrochloride aldehyde anesthesia,the choriod plexus of lateral, third, fourth ventricle are rapidly removed and immersed in the corresponding fixative for 1 hour for AKPase,5’-NTase and Mg2+-ATPase,and for 3 hour for ACPase cytochemistry. For AKPase, the lead citrate method is selected; For ACPase, the lead nitrate method is selected; For 5’-NTase,the Uusitalo-Karnovsky method is selected; For Mg2+-ATPase, the Wachstein-Meisel method is selected. Following fixation, the choriod plexus was washed 2 hour or overnight in a cold 0.1M cacodylate buffer pH 7.2 with 8% sucrose. The washed tissues were immensed in the incubation media at 37℃. The choriod plexus was rainsed again in the buffer solution, postfixed in 1% osmium tetroxide for 60min at 0-4℃,dehydrated in a graded series of acetone and then in propylene oxide and Epon mixture for endosmosis, and then embedded in Epon 812. After ultrathin sectioning with a LKB Ultrotome, the specimen were usually stained with uranyl acetate for 5 min, and observed at an JEM-1230 transmission electron microscope.
Cytochemistry control experiment:(1)absence of the substrate in the incubation medium;(2)60℃for 30 min before incubation;(3)in the presence of specific inhibitor for incubation(levomisole for AKPase).
Rsults: (1) Morphological Observation: There are three components in TEM: the capicillary core, the epithelium cells surrounding the connective tissue. The choroid plexus epithelium is cuboid monolayer, with its round or oval nucleus at the basal part. Lots of mitochondria, smooth endoplasmic reticulum, Golgi complex are at the top part of the epithelial cells. The free ribosomes and some lysosomes are diffusely through the cell cytoplasm. The apical surface is extended as numerous digiform microvilli, and on the basal surface of the cell, the plasmalemma tends to be extensively infolded. The lateral cell membranes of adjoining cells are tortuous, interdigitating, and possess an apical tight junction. The capillaries of the choroid plexus stroma are of the fenestrated type with very thin endothelial walls. The stromal connective tissue is composed of a loose network of collagen fibers, secreted by occasional elongated fibroblasts. Polymorphic epiplexus cells lie on the ventricular surface of the epithelial cells, in close association with the microvilli border. (2) The enzyme cytochemistry observation of Acid phosphatase on the Rat choroid plexus: In the transmission electron microscope, we observed that electron dense reaction products are only at the sites of lysosomes with prefixication in 2% paraformaldehyde and 2% glutaraldehyde ,we also qbserved positive reaction for this enzyme in the endocytoplasmic reticulum near the trans face of Golgi cisternae with prefixication in 2% paraformaldehyde and 0.5% glutaraldehyde. There are no reaction products in the control group and no difference between the three ventricles. (3) Ultrascytochemical observation of Alkaline phoshatase on the Rat choroid plexus epithelium: In the transmission electron microscope, we observed that electron dense reaction products are at the sites of the plasma membrane of mocrovilli and the basal infoldings, at the junctions between epithelial cells, in the firocytes of the stroma and the endothelial cells of the capillary wall. We also observed positive reaction for this enzyme in the cytoplasm, the plasma membrane of various cell organelles such as lysosome, Golgi omplexs, mitochondria etc. We see no difference between the three ventricles. (4) Ultracytochemical observation of Mg2+-ATPase on the Rat choroid plexus epithelium: In the transmission electron microscope, we observed that electron dense reaction products are at the sites of the plasma membrane of microvilli and the basal infoldings, at the junctions between epithelial cells. Mg2+-ATPase reaction products are also found at the stroma and the endothelium cells of the capillary vessels. There are no reaction products in the control group, and no differences are seen between the three ventricles. (5) Ultracytochemical observation of 5’-NTase on the Rat choroid plexus epithelium: In the transmission electron microscope, we observed that electron dense reaction products are at the junctions between epithelial cells. There are no reaction products neither at the sites of the plasma membrane of microvilli and the basal infoldings, nor at the stroma and the endothelium cells of the capillary vessels. There are no reaction products in the control group, and no differences are seen between the three ventricles.
Conclusion: We discussed the subcellular localization of ACPase and speculated that the role of acid phosphatase in the choroid plexus is probably in the uptake of materials from the CSF or the storage of the waste products of cell metabolism. ACPase may play a great role in maintain internal environment homeostasis. We speculated that the role of Alkaline phosphatase in the choroid plexus is probably in preventing the entry of phosphate esters into brain tissue and/or participating in transphosphorylation process, and then participating in the active transport of some substances. It can also be considered as part of the enzyme barrier. The magnesium-activated ATPase at the borders of the choroid plexus epithelial cells might be interpreted in terms of its role in providing the energy needed for the passage of CSF formed in the cell cytoplasm to the ventricle of the brain. And the 5’-NTase between the epithelial cells may help to form the cell junction, be considered as part of the blood-cerebrospinal fluid barrier, and is likely to participate in signal transferation by producing adenosine. The 5’-NTase at the basal infoldings may be concerned with the metabolization of nucleic acid and matter transportation, and may play a great role in maintain nucleic acid homeostasis of the brain.
方法:选取正常成年雄性SD大鼠80只,体重200-350g,常规饲养,自然光照,随机分为4个组每组20只,用于四种酶的电镜酶组织化学观察。水合氯醛腹腔麻醉后,迅速切取侧脑室、三脑室、四脑室脉络丛。采用柠檬酸铅法做AKPase酶,硝酸铅法做ACPase,Uusitalo-Karnovsky法做5’-NTase,Wachstein-Meisel法做Mg2+-ATPase的组织化学染色。取材后放入相应固定液中短时固定后,放入同种的缓冲液中4℃下过夜,在37℃温箱中孵育,用冷的缓冲液充分洗涤后,再用冷1%四氧化饿后固定1小时,梯度丙酮脱水,包埋剂浸透、包埋和聚合,超薄切片,醋酸双氧铀轻染,JEM-1230透射电镜观察照相。
酶细胞化学对照实验:(1)作用液不加专一性物;(2)作用前用60℃处理30 min;(3)加特异性抑制剂(AKPase用左旋咪唑抑制)。
结果:(1)形态学观察:透射电镜下脉络丛的结构有三种成分,以毛细血管网为中心,周围为结缔组织,表面为脉络丛上皮细胞.上皮细胞呈单层立方形,核位于细胞基底部,呈圆形或椭圆形,胞质内线粒体和小泡状滑面内质网较多,高尔基氏体丰富,它们分布于细胞顶部,游离核糖体丰富,多分散于胞质内,细胞的游离面有密集的微绒毛,上皮细胞基底部质膜有折叠形成许多质膜内褶,胞外有基膜。细胞间有特殊连接构成复合体,毛细血管为有孔型,基质结缔组织由伸长的成纤维细胞分泌的胶原纤维网组成。偶见丛上细胞,这些细胞与脉络丛上皮紧密相关,形态多样。他们位于上皮细胞顶部微绒毛,显示出典型激活巨噬细胞的超微结构特点如小泡和溶酶体。(2)脉络丛上皮酸性磷酸酶(ACPase)的超微分布:当用2%多聚甲醛和2%戊二醛混合固定时,透射电镜下可见ACPase活性出现在上皮细胞的溶酶体;当用2%多聚甲醛和0.5%戊二醛混合固定时,ACPase活性不仅出现在上皮细胞的溶酶体内,还出现在靠近高尔基体成熟面的滑面内质网上。对照组的溶酶体、高尔基体及内质网等细胞器均未显示ACPase活性。各脑室脉络丛上皮酸性磷酸酶的分布未见明显差异。各脑室脉络丛上皮酸性磷酸酶的分布未见明显差异。(3)脉络丛上皮碱性磷酸酶(AKPase)的超微分布: AKPase活性出现在各脑室脉络丛上皮细胞的微绒毛和基底褶的质膜上,上皮连接处、毛细血管内皮细胞、血液、红细胞膜和间质纤维细胞上。AKPase活性还出现在胞质,高尔基体、内质网、线粒体、溶酶体等细胞器的质膜上。三种对照标本均未见阳性反应。各脑室间未见明显差异。(4)镁三磷酸腺苷酶(Mg2+-ATPase)在脉络丛上皮的超微分布:透射电镜下可见Mg2+-ATPase活性出现在脉络丛上皮细胞的微绒毛表面、基底皱褶处、细胞连接处;酶活性还出现在脉络丛的间质和毛细血管内皮细胞上。对照实验未见酶反应产物的沉积。各脑室间未见明显差异。(5) 5’-核苷酸酶(5’-NT ase)在脉络丛上皮的超微分布:透射电镜下可见5’-NTase活性出现在上皮细胞的细胞连接和基底皱褶处,在上皮细胞微绒毛、间质内未见反应产物的沉积。上皮细胞内各种细胞器的质膜亦未见反应产物沉积。各脑室间未见明显差异。
结论:ACPase通过对脑脊液中物质的摄取和细胞代谢产物的储存,在参与脑脊液中物质的清除和维护大脑内环境的平衡中起着重要作用。我们推测脉络丛上皮上AKPase的作用是阻止磷酸酯类入脑和/或参与转磷酸过程,进而参与某些物质的跨膜转运,并可能构成酶屏障。脉络丛上皮缘的ATPase活性可能提供了在细胞质形成的脑脊液进入脑室中所需的能量。位于上皮细胞侧面的细胞连接处5’-NTase,可能有助于细胞连接的形成,构成血脑液屏障的一部分,并可能通过产生腺苷参与细胞间的信息的传导。而位于上皮基底皱褶处的5’-NTase可能与核酸降解代谢和物质运输有密切关系,以维持脑内核酸物质的稳定。
Objectives: The choroid plexuses (CPs), located within the brain ventricles, can be viewed as a tight epithelium enclosing a vascularized stroma, and as such form an interface between the blood and the cerebrospinal fluid (CSF). CSF secretion is obviously a key feature of the CPs. This secretion is the result of complex inorganic ion exchanges across the tight epithelium of the CP, mainly driven by the activities of the Na+- K+-ATPase located at the CSF-facing membrane of the epithelial cells, and of the carbonic anhydrase. By electron microscopic cytochemical techniques, we will focus on four enzymes in the choroid plexus epithelium of SD rats which are closely concerned with cell function (ATPase—providing energy, ACPase—the sign enzyme of lysosome),hoping to more realize the subcellular localization in choroid plexus epithelium.We will also discuss its significance in the secretion, absorbtion and circulation of CSF. It will offer morphological evidence for us to more realize the anatmization and physiology of the brain. At the same time, It will have wide application foreground in the pathogenesis of hydrocephalous, meningitis, neurodegeneration diseases and so on.
Methods: 80 normal adult SD rats of both sexes are selected, weighted 200-350 gram, routine breeding, natruly illumination, randomely grouping into four groups for the four enzymes. After intraperitoneal hydrochloride aldehyde anesthesia,the choriod plexus of lateral, third, fourth ventricle are rapidly removed and immersed in the corresponding fixative for 1 hour for AKPase,5’-NTase and Mg2+-ATPase,and for 3 hour for ACPase cytochemistry. For AKPase, the lead citrate method is selected; For ACPase, the lead nitrate method is selected; For 5’-NTase,the Uusitalo-Karnovsky method is selected; For Mg2+-ATPase, the Wachstein-Meisel method is selected. Following fixation, the choriod plexus was washed 2 hour or overnight in a cold 0.1M cacodylate buffer pH 7.2 with 8% sucrose. The washed tissues were immensed in the incubation media at 37℃. The choriod plexus was rainsed again in the buffer solution, postfixed in 1% osmium tetroxide for 60min at 0-4℃,dehydrated in a graded series of acetone and then in propylene oxide and Epon mixture for endosmosis, and then embedded in Epon 812. After ultrathin sectioning with a LKB Ultrotome, the specimen were usually stained with uranyl acetate for 5 min, and observed at an JEM-1230 transmission electron microscope.
Cytochemistry control experiment:(1)absence of the substrate in the incubation medium;(2)60℃for 30 min before incubation;(3)in the presence of specific inhibitor for incubation(levomisole for AKPase).
Rsults: (1) Morphological Observation: There are three components in TEM: the capicillary core, the epithelium cells surrounding the connective tissue. The choroid plexus epithelium is cuboid monolayer, with its round or oval nucleus at the basal part. Lots of mitochondria, smooth endoplasmic reticulum, Golgi complex are at the top part of the epithelial cells. The free ribosomes and some lysosomes are diffusely through the cell cytoplasm. The apical surface is extended as numerous digiform microvilli, and on the basal surface of the cell, the plasmalemma tends to be extensively infolded. The lateral cell membranes of adjoining cells are tortuous, interdigitating, and possess an apical tight junction. The capillaries of the choroid plexus stroma are of the fenestrated type with very thin endothelial walls. The stromal connective tissue is composed of a loose network of collagen fibers, secreted by occasional elongated fibroblasts. Polymorphic epiplexus cells lie on the ventricular surface of the epithelial cells, in close association with the microvilli border. (2) The enzyme cytochemistry observation of Acid phosphatase on the Rat choroid plexus: In the transmission electron microscope, we observed that electron dense reaction products are only at the sites of lysosomes with prefixication in 2% paraformaldehyde and 2% glutaraldehyde ,we also qbserved positive reaction for this enzyme in the endocytoplasmic reticulum near the trans face of Golgi cisternae with prefixication in 2% paraformaldehyde and 0.5% glutaraldehyde. There are no reaction products in the control group and no difference between the three ventricles. (3) Ultrascytochemical observation of Alkaline phoshatase on the Rat choroid plexus epithelium: In the transmission electron microscope, we observed that electron dense reaction products are at the sites of the plasma membrane of mocrovilli and the basal infoldings, at the junctions between epithelial cells, in the firocytes of the stroma and the endothelial cells of the capillary wall. We also observed positive reaction for this enzyme in the cytoplasm, the plasma membrane of various cell organelles such as lysosome, Golgi omplexs, mitochondria etc. We see no difference between the three ventricles. (4) Ultracytochemical observation of Mg2+-ATPase on the Rat choroid plexus epithelium: In the transmission electron microscope, we observed that electron dense reaction products are at the sites of the plasma membrane of microvilli and the basal infoldings, at the junctions between epithelial cells. Mg2+-ATPase reaction products are also found at the stroma and the endothelium cells of the capillary vessels. There are no reaction products in the control group, and no differences are seen between the three ventricles. (5) Ultracytochemical observation of 5’-NTase on the Rat choroid plexus epithelium: In the transmission electron microscope, we observed that electron dense reaction products are at the junctions between epithelial cells. There are no reaction products neither at the sites of the plasma membrane of microvilli and the basal infoldings, nor at the stroma and the endothelium cells of the capillary vessels. There are no reaction products in the control group, and no differences are seen between the three ventricles.
Conclusion: We discussed the subcellular localization of ACPase and speculated that the role of acid phosphatase in the choroid plexus is probably in the uptake of materials from the CSF or the storage of the waste products of cell metabolism. ACPase may play a great role in maintain internal environment homeostasis. We speculated that the role of Alkaline phosphatase in the choroid plexus is probably in preventing the entry of phosphate esters into brain tissue and/or participating in transphosphorylation process, and then participating in the active transport of some substances. It can also be considered as part of the enzyme barrier. The magnesium-activated ATPase at the borders of the choroid plexus epithelial cells might be interpreted in terms of its role in providing the energy needed for the passage of CSF formed in the cell cytoplasm to the ventricle of the brain. And the 5’-NTase between the epithelial cells may help to form the cell junction, be considered as part of the blood-cerebrospinal fluid barrier, and is likely to participate in signal transferation by producing adenosine. The 5’-NTase at the basal infoldings may be concerned with the metabolization of nucleic acid and matter transportation, and may play a great role in maintain nucleic acid homeostasis of the brain.
引文
1 Epstein MH, Feldman AM, Brusilow SW, et al. Cerebrospinalfluid production: stimulation by cholera toxin. Science,1977,196:1012-1013
2 Hollway AL, Whitfield M. Cytochemical localization of adenyl cyclase activity in rat islets of Langerhans. Journal of Histochemistry and Cytochemidtry,1972,20:873-879
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