本文選題：堿性磷酸酶 + 酸性磷酸酶　； 參考：《河北醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:位于腦室的脈絡(luò)叢可以看作是由緊密連接上皮包繞的血管叢,進(jìn)而形成了血液和腦脊液間的界面。腦脊液分泌是脈絡(luò)叢上皮的主要特點(diǎn),是由許多無(wú)機(jī)離子跨過(guò)脈絡(luò)叢上皮交換的結(jié)果,其主要驅(qū)動(dòng)力來(lái)自上皮細(xì)胞腦脊液面的鈉鉀泵和碳酸酐酶。本研究將應(yīng)用4種與細(xì)胞功能密切相關(guān)的酶(三磷酸腺苷酶—提供能量;酸性磷酸酶—溶酶體的標(biāo)志酶;)的電鏡細(xì)胞化學(xué)超微定位技術(shù)對(duì)脈絡(luò)叢上皮細(xì)胞進(jìn)行研究,以期進(jìn)一步全面了解脈絡(luò)叢上皮細(xì)胞內(nèi)的酶定位,并探討其在腦脊液生成、吸收和循環(huán)中的意義,為人類進(jìn)一步認(rèn)識(shí)腦的解剖和生理提供形態(tài)學(xué)依據(jù)。同時(shí),對(duì)于深入研究腦積水、腦膜炎,神經(jīng)系統(tǒng)變性疾病等疾病的發(fā)病機(jī)理,具有廣闊的應(yīng)用前景。 方法:選取正常成年雄性SD大鼠80只,體重200-350g,常規(guī)飼養(yǎng),自然光照,隨機(jī)分為4個(gè)組每組20只,用于四種酶的電鏡酶組織化學(xué)觀察。水合氯醛腹腔麻醉后,迅速切取側(cè)腦室、三腦室、四腦室脈絡(luò)叢。采用檸檬酸鉛法做AKPase酶,硝酸鉛法做ACPase,Uusitalo-Karnovsky法做5’-NTase,Wachstein-Meisel法做Mg2+-ATPase的組織化學(xué)染色。取材后放入相應(yīng)固定液中短時(shí)固定后,放入同種的緩沖液中4℃下過(guò)夜,在37℃溫箱中孵育,用冷的緩沖液充分洗滌后,再用冷1%四氧化餓后固定1小時(shí),梯度丙酮脫水,包埋劑浸透、包埋和聚合,超薄切片,醋酸雙氧鈾輕染,JEM-1230透射電鏡觀察照相。 酶細(xì)胞化學(xué)對(duì)照實(shí)驗(yàn):(1)作用液不加專一性物;(2)作用前用60℃處理30 min;(3)加特異性抑制劑(AKPase用左旋咪唑抑制)。 結(jié)果:(1)形態(tài)學(xué)觀察:透射電鏡下脈絡(luò)叢的結(jié)構(gòu)有三種成分,以毛細(xì)血管網(wǎng)為中心,周圍為結(jié)締組織,表面為脈絡(luò)叢上皮細(xì)胞.上皮細(xì)胞呈單層立方形,核位于細(xì)胞基底部,呈圓形或橢圓形,胞質(zhì)內(nèi)線粒體和小泡狀滑面內(nèi)質(zhì)網(wǎng)較多,高爾基氏體豐富,它們分布于細(xì)胞頂部,游離核糖體豐富,多分散于胞質(zhì)內(nèi),細(xì)胞的游離面有密集的微絨毛,上皮細(xì)胞基底部質(zhì)膜有折疊形成許多質(zhì)膜內(nèi)褶,胞外有基膜。細(xì)胞間有特殊連接構(gòu)成復(fù)合體,毛細(xì)血管為有孔型,基質(zhì)結(jié)締組織由伸長(zhǎng)的成纖維細(xì)胞分泌的膠原纖維網(wǎng)組成。偶見(jiàn)叢上細(xì)胞,這些細(xì)胞與脈絡(luò)叢上皮緊密相關(guān),形態(tài)多樣。他們位于上皮細(xì)胞頂部微絨毛,顯示出典型激活巨噬細(xì)胞的超微結(jié)構(gòu)特點(diǎn)如小泡和溶酶體。(2)脈絡(luò)叢上皮酸性磷酸酶(ACPase)的超微分布:當(dāng)用2%多聚甲醛和2%戊二醛混合固定時(shí),透射電鏡下可見(jiàn)ACPase活性出現(xiàn)在上皮細(xì)胞的溶酶體;當(dāng)用2%多聚甲醛和0.5%戊二醛混合固定時(shí),ACPase活性不僅出現(xiàn)在上皮細(xì)胞的溶酶體內(nèi),還出現(xiàn)在靠近高爾基體成熟面的滑面內(nèi)質(zhì)網(wǎng)上。對(duì)照組的溶酶體、高爾基體及內(nèi)質(zhì)網(wǎng)等細(xì)胞器均未顯示ACPase活性。各腦室脈絡(luò)叢上皮酸性磷酸酶的分布未見(jiàn)明顯差異。各腦室脈絡(luò)叢上皮酸性磷酸酶的分布未見(jiàn)明顯差異。(3)脈絡(luò)叢上皮堿性磷酸酶(AKPase)的超微分布: AKPase活性出現(xiàn)在各腦室脈絡(luò)叢上皮細(xì)胞的微絨毛和基底褶的質(zhì)膜上,上皮連接處、毛細(xì)血管內(nèi)皮細(xì)胞、血液、紅細(xì)胞膜和間質(zhì)纖維細(xì)胞上。AKPase活性還出現(xiàn)在胞質(zhì),高爾基體、內(nèi)質(zhì)網(wǎng)、線粒體、溶酶體等細(xì)胞器的質(zhì)膜上。三種對(duì)照標(biāo)本均未見(jiàn)陽(yáng)性反應(yīng)。各腦室間未見(jiàn)明顯差異。(4)鎂三磷酸腺苷酶(Mg2+-ATPase)在脈絡(luò)叢上皮的超微分布:透射電鏡下可見(jiàn)Mg2+-ATPase活性出現(xiàn)在脈絡(luò)叢上皮細(xì)胞的微絨毛表面、基底皺褶處、細(xì)胞連接處;酶活性還出現(xiàn)在脈絡(luò)叢的間質(zhì)和毛細(xì)血管內(nèi)皮細(xì)胞上。對(duì)照實(shí)驗(yàn)未見(jiàn)酶反應(yīng)產(chǎn)物的沉積。各腦室間未見(jiàn)明顯差異。(5) 5’-核苷酸酶(5’-NT ase)在脈絡(luò)叢上皮的超微分布:透射電鏡下可見(jiàn)5’-NTase活性出現(xiàn)在上皮細(xì)胞的細(xì)胞連接和基底皺褶處,在上皮細(xì)胞微絨毛、間質(zhì)內(nèi)未見(jiàn)反應(yīng)產(chǎn)物的沉積。上皮細(xì)胞內(nèi)各種細(xì)胞器的質(zhì)膜亦未見(jiàn)反應(yīng)產(chǎn)物沉積。各腦室間未見(jiàn)明顯差異。 結(jié)論:ACPase通過(guò)對(duì)腦脊液中物質(zhì)的攝取和細(xì)胞代謝產(chǎn)物的儲(chǔ)存,在參與腦脊液中物質(zhì)的清除和維護(hù)大腦內(nèi)環(huán)境的平衡中起著重要作用。我們推測(cè)脈絡(luò)叢上皮上AKPase的作用是阻止磷酸酯類入腦和/或參與轉(zhuǎn)磷酸過(guò)程,進(jìn)而參與某些物質(zhì)的跨膜轉(zhuǎn)運(yùn),并可能構(gòu)成酶屏障。脈絡(luò)叢上皮緣的ATPase活性可能提供了在細(xì)胞質(zhì)形成的腦脊液進(jìn)入腦室中所需的能量。位于上皮細(xì)胞側(cè)面的細(xì)胞連接處5’-NTase,可能有助于細(xì)胞連接的形成,構(gòu)成血腦液屏障的一部分,并可能通過(guò)產(chǎn)生腺苷參與細(xì)胞間的信息的傳導(dǎo)。而位于上皮基底皺褶處的5’-NTase可能與核酸降解代謝和物質(zhì)運(yùn)輸有密切關(guān)系,以維持腦內(nèi)核酸物質(zhì)的穩(wěn)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R341

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7 朱f趂，

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