大鼠脉络丛上皮细胞四种酶的超微定位研究

发布时间：2018-06-26 10:47

本文选题：碱性磷酸酶 + 酸性磷酸酶　；参考：《河北医科大学》2007年硕士论文

【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。
[Abstract]:Objective: the choroid plexus in the ventricle can be seen as a vascular plexus that is tightly wrapped around the skin, and then forms the interface between the blood and the cerebrospinal fluid. The secretion of the cerebrospinal fluid is the main characteristic of the choroid plexus epithelium, which is the result of the exchange of many inorganic ions across the choroid plexus epithelium, and its main driving force comes from the sodium and potassium of the epithelial cell surface of the brain cells. Pump and carbonic anhydrase. This study will apply 4 kinds of enzymes closely related to cell function (adenosine ATPase - energy supply, acid phosphatase - lysosome marker enzyme;) electron microscopic cytochemical ultramicro localization technique to study choroid plexus epithelial cells in order to further understand the localization of enzyme in choroid plexus epithelial cells, and discuss Its significance in the formation, absorption and circulation of cerebrospinal fluid provides a morphological basis for further understanding of the anatomy and physiology of the brain. At the same time, it has a broad application prospect for the in-depth study of the pathogenesis of hydrocephalus, meningitis, neurodegenerative diseases and other diseases.
Methods: 80 normal adult male SD rats, weight 200-350g, normal feeding and natural light, were randomly divided into 4 groups of 20 groups, used for the electron microscopic enzyme histochemical observation of four enzymes. After chloral hydrate intraperitoneal anesthesia, the lateral ventricle, the three ventricle, and the four ventricle vein plexus were quickly cut. The lead citrate method was used as the AKPase enzyme, the lead nitrate method ACPase, U The usitalo-Karnovsky method was done with 5 '-NTase and Wachstein-Meisel method for histochemical staining of Mg2+-ATPase. After taking the material in the corresponding fixed solution and fixed in short time, it was put in the same buffer solution at 4 C for the night, incubated in the temperature box at 37 C, and after the cold buffer solution was fully washed, then the cold 1% four oxidizing starved for 1 hours, the gradient acetone dehydrated, and the bag was dehydrated. The embedding agent was soaked, embedded and polymerized, ultrathin sections were stained with uranyl acetate, and JEM-1230 was observed by transmission electron microscope.
Enzyme cytochemistry control experiment: (1) no specific substance was added to the action liquid; (2) 30 min was used before treatment for 60 degrees; (3) the specific inhibitor (AKPase was inhibited by levamisole).
Results: (1) morphological observation: the structure of the choroid plexus under transmission electron microscope has three components, with the capillary network as the center, the surrounding connective tissue and the choroid plexus epithelial cells. The epithelial cells are single layer cubic, the nucleus is located in the basal part of the cell and is round or oval, and the mitochondria in the cytoplasm and the small vacuolar endoplasmic reticulum are more in the cytoplasm, Golgi S. Rich in body, they are distributed at the top of the cell, rich in free ribosome and scattered in the cytoplasm. The free surface of the cells is densely microvilli. The plasma membrane of the basal part of the cell is folded to form a number of plasma membrane folds, and the outer cell has a basement membrane. The cells have a special junction complex, the capillary is a pore, the matrix connective tissue is elongated. These cells are closely related to the epithelium of the choroid plexus. They are in the microvilli at the top of the epithelial cells. They show the ultrastructural characteristics of the typical macrophages, such as vesicles and lysosomes. (2) the ultrastructure of the acid phosphatase (ACPase) of the choroid plexus epithelium: when used in 2% When mixed with paraformaldehyde and 2% glutaraldehyde, the ACPase activity appeared in the epithelial cell lysosomes under transmission electron microscopy. When mixed with 2% polyformaldehyde and 0.5% glutaraldehyde, the activity of ACPase appeared not only in the lysase of the epithelial cells, but also in the smooth surface endoplasmic reticulum near the Golgi mature surface. There was no significant difference in the distribution of acid phosphatase in the choroid plexus epithelium of each ventricle. There was no significant difference in the distribution of acid phosphatase in the choroid plexus epithelium of each ventricle. (3) the ultrastructure of the alkaline phosphatase (AKPase) in the choroid plexus epithelium: the AKPase activity appeared in the choroid plexus epithelium in the ventricles of the brain. On the microvilli and the plasma membrane of basal folds, epithelial junction, capillary endothelial cells, blood, erythrocyte membrane and interstitial fiber cells,.AKPase activity also appeared on cytoplasm, Golgi body, endoplasmic reticulum, mitochondria, lysosome and other organelles. There was no positive reaction between the three kinds of control specimens. (4) there was no significant difference between the ventricles of the brain. ( The ultrastructure of adenosine triphosphatase (Mg2+-ATPase) in the choroid plexus epithelium: under transmission electron microscope, the activity of Mg2+-ATPase appears in the microvilli surface of the choroid plexus epithelial cells, the basement folds and the junction of the cells; the enzyme activity also appears on the stroma of the choroid plexus and the capillary endothelial cells in the capillary blood tube. There was no significant difference between the ventricles. (5) the ultrastructure of the 5 '- nucleotidase (5' -NT ASE) in the choroid plexus epithelium: under transmission electron microscopy, 5 '-NTase activity appeared in the cell connections and basal folds of the epithelial cells, in the microvilli of the epithelial cells and in the interstitium. The plasma membrane of various organelles in the epithelial cells was also found. No reaction product deposition was observed. No significant difference was observed between the ventricles.
Conclusion: ACPase plays an important role in the removal of substances in cerebrospinal fluid and the maintenance of the balance of the brain environment through the uptake of substances in cerebrospinal fluid and the storage of metabolic products in the cerebrospinal fluid. We speculate that the role of AKPase on the choroid plexus epithelium is to prevent phosphate esters from entering the brain and / or participating in the process of phosphoric acid, and then involved in certain substances. Transmembrane transport and may form an enzyme barrier. The ATPase activity of the choroid plexus epithelium may provide the energy required to enter the ventricles of the cerebrospinal fluid formed by the cytoplasm. The 5 '-NTase in the cell junction on the side of the epithelial cells may contribute to the formation of cell connections and form part of the blood brain barrier and may be produced by the production of adenosine. The 5 '-NTase, located in the epithelial basement folds, may be closely related to nucleic acid degradation metabolism and material transport in order to maintain the stability of nucleic acid substances in the brain.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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