斜带石斑鱼胸腺的显微和超微结构

本文通过解剖及组织切片技术、光学显微镜、透射和扫描电子显微镜技术,对斜带石斑鱼(Epinephelus coioides)胸腺器官组织进行了观察研究。结果表明:斜带石斑鱼胸腺实质主要由胸腺细胞(淋巴细胞)和网状上皮细胞构成。鱼体从Ⅰ龄之后,其胸腺发生明显的变化,与幼鱼有所不同,主要是胸腺可明显区分为三个区域:胸腺外皮质区、内皮质区和髓质区。外皮质区主要由网状上皮细胞、黏液细胞、成纤维细胞和少量淋巴细胞构成,细胞排列疏松;内皮质区主要由密集的淋巴细胞和网状上皮细胞组成,以含有大量的淋巴细胞为特征;髓质区主要由淋巴细胞和较多的网状上皮细胞构成,总体特征是淋巴细胞数量比内皮质区的少,且细胞排列较疏松。外皮质区、内皮质区相当于高等脊椎动物的皮质;髓质区相当于高等脊椎动物的髓质。髓质区之下有结缔组织,在Ⅱ龄以上的成体出现胸腺小体(Hassall's corpuscles)或类似胸腺小体的结构,而且随着年龄的增加,胸腺外皮质区增厚,结缔组织增加,还表现在内皮质区和髓质区组织逐渐萎缩变薄,胸腺的细胞组成类型和淋巴细胞数量上有所变化等等。这些现象在Ⅱ龄鱼开始出现,即胸腺呈现退化迹象,在Ⅲ龄以上鱼体呈现明显的退化和萎缩。胸腺表面扫描电镜结果表明:其上皮细胞表面具有微嵴以及由微嵴组成的指纹状结构,有一些微孔分布。透射和断面扫描电镜的结果进一步表明:胸腺组织内的细胞成分复杂,除了淋巴细胞和网状上皮细胞外,还具有巨噬细胞、肥大细胞、肌样细胞、浆细胞、指状镶嵌细胞和纤维细胞等。     

小鼠胸腺皮质外位腺苷三磷酸酶的细胞化学定位

对探索细胞之间相互作用的机制,对ＢＡＬＢ／ｃ小鼠胸腺皮质内腺苷三磷酸酶进行了细胞化学定位。结果表明该酶活性主要位于毛细血管基膜,内此细胞皮膜和吞饮小泡;上皮性网状细胞和胸腺细胞的质膜外层,特别是二者的相邻面。巨噬细胞及上皮性网状细胞的溶酶和囊泡也具有此酶活性。本文对外位腺苷三磷酸酶有抑制腺苷三磷酸诱发胸腺细胞凋落死亡的可能性进行了讨论。     

人胎胸腺上皮细胞的培养及其上清液中胸腺激素的测定

人胎儿胸腺组织在体外培养,30天内基本为上皮细胞生长。其主要依据是电镜下可见细胞浆内普遍存在张力纤维,细胞之间有桥粒连接。用脐血花环法测定胸腺激素活性,结果表明,在所收集的胸腺细胞培养上清液中确有胸腺激素活性存在。     

外泌体——癌症的“双刃剑”

正外泌体定义外泌体(exosome)是细胞对外分泌的小囊泡,常为30~120纳米大小的膜包裹结构,可特异性地包裹一些蛋白质、脂质或核酸等物质,具有生物活性,能够被受体细胞吸收,实现细胞间的物质运输和信息传递。20世纪80年代初研究人员在体外培养的正常细胞或者肿瘤细胞内发现细胞外泌现象,细胞向其培养基中分泌带有细胞膜特征的囊泡结构。1983年Rose M.Johnstone等发现体外培养的绵羊网格红细胞在成熟过程中会向外分泌     

中华鳖胸腺显微和亚显微结构及其在进化上的意义

应用透射电子显微镜观察了中华鳖胸腺的显微和亚显微结构。发现中华鳖胸腺从结构上可分为皮质和髓质,皮质富含淋巴细胞,髓质富含上皮性网状细胞。在各发育期中华鳖胸腺皮质、髓质交界处和髓质区有明显的囊状胸腺小体和交错突细胞。在２００ｇ以上的成年鳖胸腺内发现有同心圆状胸腺小体。电镜下,胸腺内淋巴细胞分为大、中、小３型。上皮性网状细胞从亚显微结构上分为支持型和分泌型。胸腺囊包括细胞内囊和细胞间囊,以细胞内囊居多     

人正常食管鳞状上皮不同原代培养方法的比较

该研究通过比较人正常食管鳞状上皮不同的原代培养方法,以期为不同的实验目的提供不同的培养方法。实验用到的正常食管粘膜上皮来源于食管癌患者手术切除的标本,采用组织块法和酶消化法,分别用DMEM/F12混合培养基和K-SFM无血清培养基进行培养。通过直接观察、细胞形态学观察和免疫细胞化学方法观察细胞的生长情况、细胞形态学特征及鉴定所得到的细胞,比较不同方法与不同培养基组合中原代培养细胞的生长状况。用组织块法,在DMEM/F12混合培养基中人正常食管上皮细胞生长较好,细胞融合较快,成纤维细胞污染较少,15～17天上皮细胞铺满瓶底的70%～80%,获得的细胞数量大,但细胞传代后成纤维细胞污染严重。用酶消化法,在K-SFM无血清培养基中人正常食管上皮细胞生长好,细胞融合快,成纤维细胞污染基本消除,细胞纯度高,10～12天细胞便可以铺满瓶底的70%～80%,这种方法培养的细胞可以冻存、复苏和传代。其余各种培养方法所得细胞无论在生长状态、培养周期、成纤维细胞污染和传代方面均较前两种方法差。以上各种方法培养的细胞经免疫细胞化学染色鉴定证实细胞呈广谱细胞角蛋白阳性,确定是食管上皮来源的细胞。酶消化法加K-SFM无血清培养基是本实...     

重组人kallistatin蛋白在毕赤酵母中的高效表达及生物活性分析

为了研究kallistatin(Kal)的生物活性,本实验构建了可分泌表达Kal的毕赤酵母菌株。首先通过PCR方法从pAAV-Kal中扩增出KalcDNA,并克隆至酵母表达载体pPIC9,得到甲醇酵母分泌型表达载体pPIC9-Kal,然后将载体线性化并电击转化毕赤酵母GS115(his4),通过MD平板筛选出阳性表达菌株。阳性表达菌株在BMMY培养基(pH7.0)中29℃培养,经2%甲醇诱导表达96h,摇瓶表达量可达14mg/L。表达上清经PhenylSuperose、Heparin SepharoseFF分离纯化,目的蛋白纯度达到98%,分子量为58kDa。生物活性实验显示,所得到的Kal蛋白具有较好的抗氧化活性,过氧化物酶活性达到(163±4)U/(mg·min),可有效降低H2O2对LX-2细胞的氧化损伤。另外,重组产生的Kal还能抑制HUVEC细胞的增殖。本研究首次成功地利用毕赤酵母表达系统分泌表达了有生物活性的Kal,为继续开展其抗肿瘤活性奠定了基础。     

酰胺化酶基因导入非内分泌细胞并表达

群马大学内分泌研究所教授竹内利行等使用非内分泌细胞合成了肽激素活性分子,这一结果在4月京都日本医学会总会上发表.在载体上搭载降钙素(CT)基因和酰胺化酶基因,导入中国仓鼠卵巢细胞(CHO),产生酰胺化的活性型CT. 内分泌细胞首先作为生物合成前体肽激素,使经处理和酰胺化形成的活性型积集在分泌颗粒上,经外界刺激被释放.在淋巴系细胞和上皮细胞、成纤维细胞、血管内皮系细胞等易培养的非分泌系细胞直接表达活性肽,并大量生产有很多优点.竹内等打算开发用皮肤细胞代替人工胰脏等.     

分泌表达bFGF的中国仓鼠卵巢(CHO)细胞的构建及其特性分析

CHO细胞在无血清或无蛋白培养条件下培养通常会遇到贴壁能力差,细胞活力差等问题。通过构建分泌型bFGF基因,克隆到pIRESneo3表达载体上,转染CHO细胞,通过MTT法间接检测细胞培养上清中bFGF表达,并在无蛋白培养基中观察细胞的生长。结果显示转染的CHO细胞表达bFGF,且分泌的bFGF有生物活性;转染的CHO细胞在无蛋白培养基中较未转染的CHO细胞的贴壁能力和活力强。成功改造了CHO细胞,为CHO细胞在无血清或无蛋白条件下大规模培养提供了基础。     

人食管癌上皮细胞系(EC-56)及其类上皮与梭状细胞克隆系的建立与鉴定

从一名35岁男性食管鳞癌病人的手术切除的癌组织标本,经体外培养建立了一个食管癌上皮细胞系(EC-56),并由此分离出两个细胞克隆系,分别命名为EC-56 C-2及C-5,进行了一系列鉴定。C-2系由梭状细胞组成,而C-5系则由类上皮细胞组成。这两个细胞克隆系都具有恶性细胞系的一般特性:例如能在体外长期连续传代;具有非整倍体核型;有丰富的微绒毛以及能被刀豆球蛋白A凝集等。C-2和C-5系除形态不同外,在生物学特性上也有差别。C-5系能在半固体琼脂培养基中形成集落,并能在以抗胸腺免疫血清处理的乳小鼠体内形成肿瘤,而C-2系列缺乏上述两种能力。 在电镜下,这两个细胞克隆系的细胞膜上均可发现桥粒,进一步表明它们具有上皮细胞的特征。本实验结果表明,从人食管癌细胞系中可分离到恶性程度不同的克隆系,而且有可能把它们用于肿瘤细胞的痛变和去恶化研究。     

Establishment of a mouse thymic epithelial cell line, IT-76MHC and a brief review on cultured thymic epithelial cells.

Interactions between thymocytes and thymic stromal cells are essential for thymocyte differentiation, but little evidence has been presented to directly show in vivo functions or interactions of the stromal cells. Among the stromal cells, the thymic epithelial cell has been considered to have profound effect on thymocyte differentiation and maturation. The calcium-depleted medium, originally developed for the culture of mouse epidermal cells, was applied for the culture of the mouse thymic epithelial cells, and successfully, an epithelial cell line, IT-76MHC was obtained from the mouse thymus. IT-76MHC cells were identified as distinct mouse thymic epithelial cells by 1/ mosaic-like arrangement, 2/ presence of well-developed desmosome and 3/ tonofilaments, 4/ positivity for cytokeratin, and 5/ induced expression of MHC class I and II by IFN-gamma treatment. IGF-1, IGF-2, oxytocin and vasopressin were also detected immunohistochemically in IT-76MHC cells. Furthermore, the IT-76MHC thymic epithelial cells, when injected intrathymically in the allogeneic mouse, prolonged the survival of skin graft from the same donor strain that IT-76MHC cells were derived. These results demonstrate that the thymic epithelial cell line IT-76MHC produces modest thymocyte survival factors as well as a growth suppressor, and that IT-76MHC cells have the ability to induce transplantation tolerance probably through their expression of MHC class I and II molecules. Taken altogether, the IT-76MHC thymic epithelial cells have been proved to be useful tools to better understand the in vivo functions of thymic epithelial cells, and to gain a deep insight into their involvement in the critical selection process of thymocytes which still remains obscure. Finally and additionally, literatures so far reported on thymic epithelial cells in culture, especially lines and clones, are reviewed and their identity as well as their functions are discussed.     

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line. TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.     

Effects of cholinergic agonists on the proliferation and protein synthesis in a cultured thymic epithelial cell line

Thymic epithelial cells appear to release the humoral factors endowing precursors of T cells (thymus-dependent lymphocytes) with the capacity to differentiate and maturate into relatively mature T cells. We have separated the polypeptide fractions containing these factors from the culture supernatant of thymic epithelial cell line. Thymus is reported to be innervated by autonomic nervous system from the prenatal to the pubertal period. But the physiological significance of the nervous system in this lymphoid organ remains obscure. And the modulator of the epithelial cell functions, namely, production and release of the bioactive polypeptides have never been clarified. We show here that acetylcholine (Ach) or carbamylcholine (Cch) enhanced the proliferation of thymic epithelial cells from the TAD3 cell line at preconfluent state, and the protein synthetic activity at confluent state. This phenomenon was completely suppressed by the pretreatment of alpha-bungarotoxin (alpha-BTx). These results suggest that nicotinic Ach-receptors exist on the epithelial cell surface membrane and that the differentiation and maturation of thymic lymphocytes are indirectly regulated by the activated functions of thymic epithelial cells stimulated with cholinergic agonists.     

The effects of thymic epithelial monolayer-conditioned medium on suppressor cell function following chemotherapy in pediatric patients

Summary Human thymic epithelial monolayer-conditioned medium (TEM-CM) enhanced concanavalin A (ConA)-induced suppressor T-lymphocyte activity in 15 of 17 studies of fractionated light-density bone marrow mononuclear cells (LD-BMMC) obtained from pediatric cancer patients within 7 days of chemotherapy (P<0.001). However, TEM-CM depressed ConA-induced suppressor T-lymphocyte activity in 14 of 18 studies of LD-BMMC obtained from patients who had received their chemotherapy 14–21 days previously (P<0.05). In studies of LD-BMMC from normal subjects, TEM-CM did not show any significant effect on suppressor cell activity, nor did TEM-CM significantly affect spontaneous suppressor cell activity in patients or normals. The effect of direct culture on thymic epithelial monolayers was equivalent to the effect of TEM-CM in both ConA-induced and spontaneous suppressor cell assays. These data demonstrate thymic factor-mediated changes in suppressor T-cell activity of pediatric cancer patients and suggest a postchemotherapy alteration in the bone marrow population of inducible prethymic T cells.     

Seeding of the 10-day mouse embryo thymic rudiment by lymphocyte precursors in vitro

The thymic rudiment was removed from the mouse embryo at 10 days of gestation, while it was still included in the 3rd branchial arch. When cultured alone, either in vitro or on the chick chorioallantoic membrane (CAM), it failed to develop as a lymphopoietic organ and remained in an epithelial state. If it was associated in transfilter culture with various types of hemopoietic organs from either embryonic or adult mice (e.g. yolk sac, fetal liver, thymus, bone marrow), it became seeded by lymphoid precursor cells and underwent a normal histogenetic process. If the donor and the receptor explants belonged to different strains of mice, the thymus that developed in culture was chimeric: thymic stroma cells (i.e., epithelial and connective cells) were of the receptor explant type, whereas the lymphoid population was of the donor type. Two genetic markers were used to label the thymic cell types, the Thy-1-1-Thy-1-2 system and the isozymes of the glucose phosphate isomerase.     

Culture of epithelial and stromal cells of guinea-pig endometrium and the effect of oestradiol-17 beta on the epithelial cells

Epithelial and stromal cells of guinea-pig endometrium were separated by enzymic digestion, isolated by successive centrifugation, and maintained in culture as pure cell types for 5 days on growth medium. On Day 5, ultrastructural studies were performed on the two cell types, demonstrating that epithelial cells can grow as a monolayer composed of cohesive groups of polygonal cells (1.3 X 10(5) cells/cm2), while stromal cells were mostly fibroblastic. The effect of hormones was studied on the epithelial cells in culture. The monolayer was cultured into harvest medium for 3 days to ensure the complete removal of endogenous steroids, then these cells were incubated with 2 X 10(-9) M-oestradiol-17 beta for 3 days. There was a rise in the progesterone receptor level, varying from 1.3 to 10.8 times. The three enzymes known to interfere with oestradiol-17 beta metabolism were present in the epithelial cells grown in our culture conditions. By incubation with oestrone sulphate for 3 days it was demonstrated that, in cultured epithelial cells, oestrone sulphate is converted into oestradiol-17 beta sulphate, and oestrogen sulphates are hydrolysed to active oestrogens.     

Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties

The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.     

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.     

小鼠胸腺上皮细胞系MTEC1产生30kD的MCP-1类趋化因子

髓质型小鼠胸腺上皮细胞系MTEC1自发分泌趋化因子(Chemokines),细胞培养上清先经CPG珠(Controlled-pore glass beads)初步浓缩纯化,再依次经肝素-Sepharose柱亲和层析、阳离子交换FPLC及RP-HPLC分离纯化,得到了一个30kD的蛋白质分子,对淋巴细胞和单核巨噬细胞均有趋化活性。此蛋白分子N端封闭,因而先经甲酸水解得到蛋白片段,分析了其中24个氨基酸的序列,发现其与小鼠MCP-1/JE(Monocyte Chemoattractant Pro-tein-1)完全相同。以上结果说明,MTEC1产生的是mMCP-1/JE类趋化因子。     

In vitro growth and maintenance of two morphologically distinct populations of thymic epithelial cells

Cultures of thymic epithelial cells were generated and maintained in valine-free minimum essential medium (MEM) supplemented with 690 mg/liter of D-valine. These cultures have been maintained for 1 year through multiple passages by trypsinization of 60-70% confluent monolayers. Large and small epithelial cells were present in early cultures. They were separated into two stable subpopulations based on (1) their differential growth rates and (2) their differential adherence to the culture substratum. These morphologically distinct cell populations, TECS and TECL, were 100% keratin positive and contained cells with desmosomes and tonofilaments, all characteristics of epithelial cells. Esterase analysis of both cell populations revealed a 1 and 9% esterase-positive cell population in cultures of keratin-positive small (TECS) and large (TECL) cells, respectively. The percentages of esterase-positive cells corresponded to the 2 and 10% populations of TECS and TECL, respectively, that contained both desmosomes and phagolysosomes. These results establish conditions for the long-term propagation of pure thymic epithelial cells. Such cultures can be used to study the functional interactions between epithelial cells and lymphoid cells. Morphologic and histochemical analyses have identified subsets of these cells which may prove to have differential effects on thymocyte proliferative and developmental processes.