本文選題：胎兒胸腺 + CD80�。� 參考：《鄭州大學(xué)》2007年碩士論文

【摘要】： 背景 胸腺是T細(xì)胞發(fā)育最適宜的微環(huán)境。T細(xì)胞在胸腺內(nèi)發(fā)育成熟需要經(jīng)歷嚴(yán)格的陽性選擇和陰性選擇。胸腺細(xì)胞的陰性選擇不僅需要胸腺細(xì)胞的TCR信號，還需要胸腺細(xì)胞與抗原遞呈細(xì)胞(antigen presenting cell，APC)表面結(jié)合提供第二信號(自身肽-MHC分子)的共刺激分子。共刺激信號在陰性選擇中發(fā)揮必要的作用。共刺激信號可以增強(qiáng)也可以終止免疫應(yīng)答，即也具有免疫調(diào)整作用。已證實(shí)B7-1(CD80)與CD28是最基本的共刺激分子。胸腺內(nèi)B7-1主要表達(dá)于樹突狀細(xì)胞(dendritic cell，DC)、巨噬細(xì)胞和某些網(wǎng)狀上皮細(xì)胞(epithelioreticlar cells，ERC)上。 依據(jù)分布和功能廣義的胸腺上皮細(xì)胞可分為6型，其中包含有狹義的網(wǎng)狀上皮細(xì)胞和樹突狀細(xì)胞(又名為交錯突細(xì)胞)，皆可表達(dá)CD80。現(xiàn)今認(rèn)為DC是功能最強(qiáng)大的抗原遞呈細(xì)胞(antigen presenting cell，APC)，它呈遞抗原的能力是巨噬細(xì)胞的100-1000倍。 癌胚抗原(carinoembryonic antigen，CEA)是一種分子量約為180 kDa的糖蛋白分子，胚胎時期的正常表達(dá)分泌，對胚胎的發(fā)育、組織形成具有重要的作用。臨床上，CEA已用于相關(guān)腫瘤的檢測和治療。關(guān)于胸腺細(xì)胞發(fā)育成熟的規(guī)律和胸腺基質(zhì)細(xì)胞對其調(diào)節(jié)作用及分子機(jī)制研究等，多是以動物胸腺為研究對象。目前關(guān)于CDSO及CEA分子在胎兒胸腺的表達(dá)定位的研究國內(nèi)外尚未見報道。本研究應(yīng)用胎兒胸腺組織冰凍切片的HE染色、ANAE(α-naphthyl acidic esterase，α-萘酚酸性酯酶)組化染色、CD80和CEA免疫組化染色，并用Trizol法提取胎兒胸腺蛋白質(zhì)，以Western blotting和ELISA技術(shù)分別對早期、晚期胎兒胸腺觀察了早期、晚期胎兒的CD80和CEA免疫反應(yīng)性(immunoreactivity，IR)的表達(dá)和分布情況。 材料與方法 1．取孕婦自愿提供的意外懷孕、水囊引產(chǎn)的胎兒12例，包含早期胎兒胸腺3-4個月的4例(Ⅰ組)和晚期胎兒胸腺的5-8個月的8例(Ⅱ組)。 2．將標(biāo)本等分為兩份，一份制備冰凍組織切片，進(jìn)行常規(guī)HE染色、ANAE組織化學(xué)染色、CDSO和CEA免疫組織化學(xué)染色，油鏡下各觀察其信號定位。 3．另一份標(biāo)本以Trizol提取蛋白質(zhì)進(jìn)行Western blotting和CEA-ELISA檢測，用Shimadu薄層層析掃描儀掃描CD80及β-actin條帶的OD值，計算早期胎兒胸腺和晚期胎兒胸腺的CD80／β-actin的比值；用酶標(biāo)儀檢測兩組胎兒胸腺CEA含量的OD值。 3．應(yīng)用SPSS 10.0軟件，進(jìn)行ANOVA統(tǒng)計學(xué)分析，以α=0.05檢測兩組間差異顯著性。 結(jié)果 1 HE染色早期胎兒胸腺體積較小，皮質(zhì)和髓質(zhì)發(fā)育差，髓質(zhì)網(wǎng)孔空隙較小。晚期胎兒胸腺體積較大，皮質(zhì)和髓質(zhì)逐漸發(fā)育，髓質(zhì)的網(wǎng)孔空隙較大。晚期標(biāo)本胸腺髓質(zhì)內(nèi)可見典型的哈氏小體(Hassall’s corpuscle)。 2 CD80免疫組化染色CDSO免疫反應(yīng)性(immunoreactivity，IR)CD80-IR呈棕色細(xì)顆粒，廣義的網(wǎng)狀上皮細(xì)胞(epithelioreticfar cells，ERC)均呈CD80陽性反應(yīng)。早期胎兒胸腺CD80-IR細(xì)胞不顯著；晚期胎兒胸腺CD80-IR細(xì)胞明顯增多，且髓質(zhì)內(nèi)顯著多于皮質(zhì)。ERC可分為6型：①Ⅰ型ERC：定位于皮質(zhì)，網(wǎng)狀上皮細(xì)胞呈扁平狀，位于被膜和小梁的內(nèi)面；②Ⅱ型ERC：分布于皮質(zhì)內(nèi)，細(xì)胞呈小星形，突起也很少；③Ⅲ型及Ⅳ型ERC：呈扁平狀，定位于皮質(zhì)髓質(zhì)交界處；④Ⅴ型ERC：分布于髓質(zhì)內(nèi)，細(xì)胞呈大星形狀，突起多且細(xì)長；⑤Ⅵ型ERC：位于髓質(zhì)內(nèi)哈氏小體。 3 ANAE染色已知ANAE組化染色可標(biāo)記T淋巴細(xì)胞及其亞型。胸腺成熟T細(xì)胞可分為點(diǎn)樣型(代表CD4~+)和散粒型(代表CD8~+)。點(diǎn)樣型細(xì)胞內(nèi)可見1-3個邊界清楚的深紅色小點(diǎn)。散粒型細(xì)胞內(nèi)呈多數(shù)彌散小顆粒。早期胎兒胸腺標(biāo)本ANAE標(biāo)記細(xì)胞不明顯；晚期胎兒胸腺髓質(zhì)內(nèi)ANAE染色的CD4~+或CD8~+細(xì)胞各呈叢狀分布。胎兒胸腺髓質(zhì)內(nèi)哈氏小體及巨噬細(xì)胞ANAE染色呈強(qiáng)陽性的火焰型。 4．CEA免疫組化染色早期胎兒胸腺內(nèi)CEA免疫反應(yīng)陽性定位于髓質(zhì)網(wǎng)狀上皮細(xì)胞和哈氏小體。晚期胎兒胸腺髓質(zhì)內(nèi)CEA免疫反應(yīng)陽性更強(qiáng)，多集中于哈氏小體。 5 Western Blotting早期胎兒胸腺CD80與β-actin的OD比值為0.66±0.09。晚期胎兒胸腺CD80與β-actin的OD比值為0.89±0.11。兩組相比較，差異具有顯者性，p＜0.05。 6．ELISA法檢測CEA早期胎兒胸腺蛋白提取液中CEA含量的OD值為0.181±0.03，晚期胎兒胸腺蛋白提取液中CEA含量的OD值為0.302±0.04，兩組相比較，差異具有顯著性，p＜0.05。 結(jié)論 1．晚期胎兒胸腺標(biāo)本的CD80免疫印跡和CEA-ELISA的檢測值均顯著高于早期胎兒。這表明晚期胎兒胸腺的CD80及CEA表達(dá)水平顯著高于早期胎兒的胸腺。 2．早期胎兒胸腺發(fā)育差。晚期胎兒胸腺內(nèi)CD80免疫反應(yīng)性細(xì)胞依據(jù)分布可分為6型，其中Ⅰ型ERC，，Ⅲ型及Ⅳ型ERC均具有隔離屏障作用。皮質(zhì)內(nèi)Ⅱ型ERC和位于髓質(zhì)內(nèi)Ⅴ型ERC皆為狹義的DC。Ⅵ型ERC定位于哈氏小體。這提示胸腺微環(huán)境又可分隔為若干亞微環(huán)境。 3．早期組CEA陽性分布于髓質(zhì)上皮細(xì)胞和哈氏小體，晚期組CEA陽性更集中于哈氏小體。哈氏小體的CEA-IR結(jié)合CD80-IR以及ANAE強(qiáng)陽性染色，這提示哈氏小體可具有一定的生物學(xué)功能。 4．晚期胎兒胸腺的髓質(zhì)內(nèi)ANAE的點(diǎn)樣型(代表CD4~+)或散粒型(代表CD8~+)各呈叢狀分布，提示其各位于不同網(wǎng)孔的亞微環(huán)境內(nèi)。
[Abstract]:background
The thymus gland is the most suitable microenvironment for the development of T cells, the development of.T cells in the thymus needs strict positive selection and negative selection. The negative selection of thymus cells requires not only the TCR signal of thymic cells, but also the combination of thymus cells and antigen presenting cell (APC) cells (antigen presenting cell, APC) to provide a signal (self peptide). Co stimulators of -MHC molecules. Co stimulation signals play a necessary role in negative selection. Co stimulatory signals can be enhanced and immune responses can be terminated, that is, immune adjustment. B7-1 (CD80) and CD28 are the most basic co stimulators. B7-1 is mainly expressed in dendritic cells (dendritic cell, DC), and giant macrophages in the thymus. Cells and some epithelioreticlar cells (ERC).
The thymic epithelial cells in the broad sense of distribution and function can be divided into 6 types, including narrow sense reticular epithelial cells and dendritic cells (also known as interstaggered cells), which can express CD80. today that DC is the most powerful antigen presenting cell (antigen presenting cell, APC), and its ability to deliver antigen is 100-1000 of macrophages. Double.
Carinoembryonic antigen (CEA) is a glycoprotein molecule with a molecular weight of about 180 kDa, the normal expression and secretion of embryo, which plays an important role in the development of embryo and tissue formation. In clinical, CEA has been used for the detection and treatment of related tumors. The research on the regulatory and molecular mechanisms is mostly based on the animal thymus. The research on the expression of CDSO and CEA molecules in the fetal thymus has not been reported at home and abroad. This study uses HE staining in frozen section of fetal thymus, ANAE (alpha -naphthyl acidic esterase, alpha naphthol acid esterase), CD80 and C EA immunohistochemical staining was used and Trizol method was used to extract the fetal thymus protein. The expression and distribution of CD80 and CEA immunoreactivity (immunoreactivity, IR) in the early and late fetuses were observed by Western blotting and ELISA technique respectively.
Materials and methods
1. unwanted pregnancy voluntarily provided by pregnant women, 12 cases of fetus induced by water sac, 4 cases of early fetal thymus (group I) and 8 cases of late fetal thymus in 5-8 months (Group II).
2. the specimens were divided into two parts and one portion was prepared for frozen tissue section. Routine HE staining, ANAE histochemical staining, CDSO and CEA immunohistochemical staining were performed, and the signal location was observed under the oil microscope.
3. the other specimens were detected by Trizol extraction of protein for Western blotting and CEA-ELISA, and the ratio of CD80 / beta -actin in the early fetal thymus and late fetal thymus was calculated by Shimadu TLC scanner. The ratio of CD80 / beta -actin in the early fetal thymus and the late fetal thymus was calculated, and the OD value of the thymus CEA content of the two groups was detected by the enzyme labeling instrument.
3. using SPSS 10 software, ANOVA statistical analysis was performed, and alpha =0.05 was used to detect significant difference between the two groups.
Result
1 HE staining early fetal thymus volume is smaller, cortex and medulla development is poor, medullary space gap is smaller. Late fetal thymus volume is larger, the cortex and medulla gradually develop, the medulla space gap is larger. The late specimen of the thymus medulla can be seen in the typical Hassall s corpuscle in the thymus medulla.
2 CD80 immunohistochemical staining CDSO immunoreactivity (immunoreactivity, IR) CD80-IR was brown fine particles, the generalized reticular epithelial cells (epithelioreticfar cells, ERC) showed a positive reaction. The early fetal thymus CD80-IR cells were not significant; the late fetal thymus CD80-IR cell was significantly increased, and the medulla was significantly more than cortical.ERC can be divided into Type 6 type: (1) type I ERC: located in the cortex, the reticular epithelial cells are flat, located in the inner surface of the membrane and trabecula; 2. Type II ERC: in the cortex, the cells are small star shaped, and the protuberances are very few; (3) type III and type IV ERC: flat, located at the junction of cortex medulla; (4) type V ERC: distributed in medulla and large star shape in the medulla Protuberance is many and elongate; type VI ERC: Hamlet bodies located in medulla.
3 ANAE staining known ANAE histochemical staining can mark the T lymphocyte and its subtypes. Thymic mature T cells can be divided into point like type (representing CD4~+) and granular type (representing CD8~+). In the point like cells, there are clear dark red dots with clear boundaries. Most scattered small granules in the granular cells. The early fetal thymus specimens are not marked by ANAE labelled cells. The ANAE staining of CD4~+ or CD8~+ cells in the medulla of the advanced fetal thymus showed a plexiform distribution. The ANAE staining of Hamlet and macrophages in the medulla of the fetal thymus showed a strong positive flame type.
The positive immunoreaction of CEA in the early fetal thymus of 4.CEA immunohistochemical staining was located in the medullary reticular epithelial cells and Harris corpuscle. The CEA immunoreaction in the late thymus medulla was more positive and concentrated in Harris corpuscle.
5 Western Blotting early fetal thymus CD80 and beta -actin OD ratio is 0.66 + 0.09. late fetal thymus CD80 and the OD ratio of beta -actin is 0.89 + 0.11. two groups, the difference is obvious, P < 0.05.
The 6.ELISA method was used to detect the CEA content in the early fetal thymus protein extract of CEA, and the CEA content was 0.181 + 0.03. The CEA content of the advanced fetal thymus protein extract was 0.302 + 0.04. The difference was significant in the two groups, P < 0.05.
conclusion
The detection values of CD80 immunoblotting and CEA-ELISA in the specimens of 1. advanced fetal thymus were significantly higher than those of the early fetus, which showed that the expression level of CD80 and CEA in the advanced fetal thymus was significantly higher than that of the early fetal thymus.
The early fetal thymus development is poor. The CD80 immunoreactive cells in the late fetal thymus can be divided into 6 types according to their distribution, of which type I ERC, type III and type IV ERC have isolation barrier. The type II ERC in the cortex and the type V ERC in the medulla are all DC. VI ERC located in the Hamlet body. This suggests that the thymus microenvironment can be separated to be separated. Several sub microenvironments.
The positive distribution of CEA in the early 3. group was in the medullary epithelial cells and hastelis corpuscle, and the CEA positive in the late group was more concentrated in the hasteli corpuscle. The CEA-IR combined with CD80-IR and the strong positive staining of ANAE, which suggested that Harris corpuscle has some biological function.
4. the point like type (representing CD4~+) or granular type (representing CD8~+) in the medulla of the advanced fetal thymus (representing CD8~+) is in a plexiform distribution, indicating that each of the medulla is located in the submicroenvironment of different meshes.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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