胎儿胸腺内CD80和CEA的表达及定位

发布时间：2018-05-31 13:26

本文选题：胎儿胸腺 + CD80　；参考：《郑州大学》2007年硕士论文

【摘要】： 背景胸腺是T细胞发育最适宜的微环境。T细胞在胸腺内发育成熟需要经历严格的阳性选择和阴性选择。胸腺细胞的阴性选择不仅需要胸腺细胞的TCR信号，还需要胸腺细胞与抗原递呈细胞(antigen presenting cell，APC)表面结合提供第二信号(自身肽-MHC分子)的共刺激分子。共刺激信号在阴性选择中发挥必要的作用。共刺激信号可以增强也可以终止免疫应答，即也具有免疫调整作用。已证实B7-1(CD80)与CD28是最基本的共刺激分子。胸腺内B7-1主要表达于树突状细胞(dendritic cell，DC)、巨噬细胞和某些网状上皮细胞(epithelioreticlar cells，ERC)上。依据分布和功能广义的胸腺上皮细胞可分为6型，其中包含有狭义的网状上皮细胞和树突状细胞(又名为交错突细胞)，皆可表达CD80。现今认为DC是功能最强大的抗原递呈细胞(antigen presenting cell，APC)，它呈递抗原的能力是巨噬细胞的100-1000倍。癌胚抗原(carinoembryonic antigen，CEA)是一种分子量约为180 kDa的糖蛋白分子，胚胎时期的正常表达分泌，对胚胎的发育、组织形成具有重要的作用。临床上，CEA已用于相关肿瘤的检测和治疗。关于胸腺细胞发育成熟的规律和胸腺基质细胞对其调节作用及分子机制研究等，多是以动物胸腺为研究对象。目前关于CDSO及CEA分子在胎儿胸腺的表达定位的研究国内外尚未见报道。本研究应用胎儿胸腺组织冰冻切片的HE染色、ANAE(α-naphthyl acidic esterase，α-萘酚酸性酯酶)组化染色、CD80和CEA免疫组化染色，并用Trizol法提取胎儿胸腺蛋白质，以Western blotting和ELISA技术分别对早期、晚期胎儿胸腺观察了早期、晚期胎儿的CD80和CEA免疫反应性(immunoreactivity，IR)的表达和分布情况。材料与方法 1．取孕妇自愿提供的意外怀孕、水囊引产的胎儿12例，包含早期胎儿胸腺3-4个月的4例(Ⅰ组)和晚期胎儿胸腺的5-8个月的8例(Ⅱ组)。 2．将标本等分为两份，一份制备冰冻组织切片，进行常规HE染色、ANAE组织化学染色、CDSO和CEA免疫组织化学染色，油镜下各观察其信号定位。 3．另一份标本以Trizol提取蛋白质进行Western blotting和CEA-ELISA检测，用Shimadu薄层层析扫描仪扫描CD80及β-actin条带的OD值，计算早期胎儿胸腺和晚期胎儿胸腺的CD80／β-actin的比值；用酶标仪检测两组胎儿胸腺CEA含量的OD值。 3．应用SPSS 10.0软件，进行ANOVA统计学分析，以α=0.05检测两组间差异显著性。结果 1 HE染色早期胎儿胸腺体积较小，皮质和髓质发育差，髓质网孔空隙较小。晚期胎儿胸腺体积较大，皮质和髓质逐渐发育，髓质的网孔空隙较大。晚期标本胸腺髓质内可见典型的哈氏小体(Hassall’s corpuscle)。 2 CD80免疫组化染色CDSO免疫反应性(immunoreactivity，IR)CD80-IR呈棕色细颗粒，广义的网状上皮细胞(epithelioreticfar cells，ERC)均呈CD80阳性反应。早期胎儿胸腺CD80-IR细胞不显著；晚期胎儿胸腺CD80-IR细胞明显增多，且髓质内显著多于皮质。ERC可分为6型：①Ⅰ型ERC：定位于皮质，网状上皮细胞呈扁平状，位于被膜和小梁的内面；②Ⅱ型ERC：分布于皮质内，细胞呈小星形，突起也很少；③Ⅲ型及Ⅳ型ERC：呈扁平状，定位于皮质髓质交界处；④Ⅴ型ERC：分布于髓质内，细胞呈大星形状，突起多且细长；⑤Ⅵ型ERC：位于髓质内哈氏小体。 3 ANAE染色已知ANAE组化染色可标记T淋巴细胞及其亚型。胸腺成熟T细胞可分为点样型(代表CD4~+)和散粒型(代表CD8~+)。点样型细胞内可见1-3个边界清楚的深红色小点。散粒型细胞内呈多数弥散小颗粒。早期胎儿胸腺标本ANAE标记细胞不明显；晚期胎儿胸腺髓质内ANAE染色的CD4~+或CD8~+细胞各呈丛状分布。胎儿胸腺髓质内哈氏小体及巨噬细胞ANAE染色呈强阳性的火焰型。 4．CEA免疫组化染色早期胎儿胸腺内CEA免疫反应阳性定位于髓质网状上皮细胞和哈氏小体。晚期胎儿胸腺髓质内CEA免疫反应阳性更强，多集中于哈氏小体。 5 Western Blotting早期胎儿胸腺CD80与β-actin的OD比值为0.66±0.09。晚期胎儿胸腺CD80与β-actin的OD比值为0.89±0.11。两组相比较，差异具有显者性，p＜0.05。 6．ELISA法检测CEA早期胎儿胸腺蛋白提取液中CEA含量的OD值为0.181±0.03，晚期胎儿胸腺蛋白提取液中CEA含量的OD值为0.302±0.04，两组相比较，差异具有显著性，p＜0.05。结论 1．晚期胎儿胸腺标本的CD80免疫印迹和CEA-ELISA的检测值均显著高于早期胎儿。这表明晚期胎儿胸腺的CD80及CEA表达水平显著高于早期胎儿的胸腺。 2．早期胎儿胸腺发育差。晚期胎儿胸腺内CD80免疫反应性细胞依据分布可分为6型，其中Ⅰ型ERC，，Ⅲ型及Ⅳ型ERC均具有隔离屏障作用。皮质内Ⅱ型ERC和位于髓质内Ⅴ型ERC皆为狭义的DC。Ⅵ型ERC定位于哈氏小体。这提示胸腺微环境又可分隔为若干亚微环境。 3．早期组CEA阳性分布于髓质上皮细胞和哈氏小体，晚期组CEA阳性更集中于哈氏小体。哈氏小体的CEA-IR结合CD80-IR以及ANAE强阳性染色，这提示哈氏小体可具有一定的生物学功能。 4．晚期胎儿胸腺的髓质内ANAE的点样型(代表CD4~+)或散粒型(代表CD8~+)各呈丛状分布，提示其各位于不同网孔的亚微环境内。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】