小鼠膀胱上皮的中间层细胞中存在Uroplakins

本文运用超薄切片、冰冻蚀刻及免疫胶体金标记等多种电镜技术并结合免疫组化,免疫荧光染色技术,直观地显示出小鼠膀胱上皮的中间层细胞存在Ｕｒｏｐｌａｋｉｎｓ,并在梭形泡膜上形成了与表细胞类似的ＡＵＭ结构,而且梭形泡的ＡＵＭ结构也结合在中间纤维上,白质免疫印迹反应进一步证实中间层细胞含有与表层细胞相同的ＵｒｏｐｌａｋｉｎＩ和ＵｒｏｐｌａｋｉｎⅢ等ＡＵＭ蛋白的主要成分,从而为ＡＵＭ的发生及其与细胞分化关系的     

无斑肥螈消化道五羟色胺免疫活性细胞的分布与形态学观察

用S－P免疫组织化学的方法对无斑肥塬消化道五－羟色胺（5－HTIR）免疫活性细胞的分布及形态进行了观察。结果表明,5－HTIR细胞从食道到直肠的消化道各段均有分布。其中,幽门部密度最高,十二指肠次之,直肠最低。5－HTIR细胞形态多样,有圆形、梭形、纺锤形或烧瓶形,梭形、纺锤形或烧瓶细胞有较长的胞突,有时胞突可见明显分叉。同时讨论了5－HRIR细胞的分点特点、形态与功能的关系。     

异位错构瘤性胸腺瘤2例报道及文献复习

目的探讨异位错构瘤性胸腺瘤(ectopic hamartomatous thymoma,EHT)的临床病理学特征。方法收集2例EHT,结合临床病史及相关文献复习,对其进行分析。结果两例分别发生于30岁和49岁的男性,各因发现左颈部肿块1年和5年余就诊。组织学上,肿瘤由梭形细胞、上皮细胞和脂肪细胞3种成分组成,可见多量薄壁中、小血管,梭形细胞在两例中所占比例最大(50%),成熟的脂肪细胞多呈灶状分布。免疫表型上皮细胞与梭形细胞均弥漫表达CK、CK5/6、p63,此外梭形细胞还可部分表达CD10、CD34、calponin、vimentin、SMA,上皮细胞部分表达EMA,TTF-1、S-100两者均未见表达,Ki-67增殖指数均3%。结论 EHT为一种罕见的良性肿瘤,好发于成年男性,认识其独特的发病部位及临床病理学特征为其诊断与鉴别诊断的关键。     

蝘蜓消化道5-羟色胺细胞的免疫组织化学定位

应用5-羟色胺(5-Hydroxytryptamine 5-HT)特异性抗血清对蝘蜓(Sphenomorphus iudieus)消化道5-HT细胞的分布及形态进行了免疫组织化学研究。结果表明,5-HT阳性细胞在堰蜓消化道各段均有分布,其中以胃幽门部位分布密度最高,食道与直肠部位其次,空肠部位分布密度最低。消化道各段5-HT细胞形态多样,有圆形、椭圆形、梭形、楔形、不规则形,其中梭形细胞多具有胞突。文中对堰蜓消化道5-HT细胞的分布、形态与功能相适应的特点进行了讨论。     

无蹼壁虎消化道5-羟色胺细胞的免疫组织化学定位

目的研究无蹼壁虎(Gekkoswinhonis)消化道内5-HT免疫反应阳性内分泌细胞的分布以及形态。方法应用显示5-HT的免疫组织化学方法。结果5-HT阳性细胞在无蹼壁虎消化道各段均有分布,其中以十二指肠部位分布密度最多,食道和直肠其次,回肠部位分布密度最低。消化道各段5-HT细胞形态多样,有圆形、椭圆形、梭形、不规则形,其中梭形细胞多具有胞突。结论无蹼壁虎的消化道5-HT细胞的分布、形态与其功能相适应。     

中华花龟消化道5-羟色胺免疫活性内分泌细胞的分布与形态学观察

应用5-HT抗血清,以ABC（avidin-biotin-peroxidase complex）免疫组织化学方法,探究中华花龟消化道内5-HT免疫反应阳性内分泌细胞的分布及形态。结果表明,5-HT阳性细胞从食管到直肠的消化道各段均有分布,以十二指肠部位分布密度为最高,胃幽门部次之,食管部最低;5-HT阳性细胞广泛分布于上皮细胞之间、上皮基部、腺泡上皮细胞之间、腺泡之间以及固有膜内,形态多样呈圆形、椭圆形、锥体形、梭形等,以锥体形为主。认为中华花龟消化道5-HT阳性细胞具有内、外分泌两种作用途径,并且5-HT阳性细胞的密度分布可能与其食性、生活环境等有关。     

东方蝾螈消化道5-羟色胺免疫活性细胞的分布与形态学观察,前插2

采用ABC免疫组织化学方法对东方蝾螈Cynops orientalis消化道内5-羟色胺免疫活性内分泌细胞的分布及形态进行观察.结果显示,5-羟色胺(5-HT)细胞从食管到直肠各段均有分布,其中十二指肠的分布密度最高,空肠次之,幽门和直肠部最低.5-HT细胞形态多样呈圆形、锥体形和梭形等,根据其形态认为东方蝾螈消化道5-HT免疫活性细胞具有内分泌、外分泌两种功能.     

肾脏黏液性小管状和梭形细胞癌三例报道并文献复习

目的探讨肾脏黏液性小管状和梭形细胞癌(mucinous tubular and spindle cell carcinoma,MTSCC)的临床病理学特点。方法对3例MTSCC进行光镜、特殊染色、免疫组织化学染色及荧光原位杂交(FISH)检测,并复习临床资料及相关文献。结果 3例MTSCC中,2例为男性,1例女性,年龄分别为50、71、75岁(中位71岁),例2临床表现为腰痛,3例均无肉眼血尿。肿瘤长径分别为2.0cm、3.5cm、6.0cm(中位3.5cm),肿瘤切面灰白色,与周围肾实质界限清晰,例2局部伴出血、坏死。组织学上肿瘤由温和一致的立方细胞紧密排列成狭长的小管结构及梭形细胞两种成分构成,例1、例3部分肿瘤细胞胞质透明,例1黏液性间质稀少;例2、例3间质内见泡沫细胞聚集。免疫表型:3例均表达AMCAR、CK7、CK19、EMA、NSE等,Ki-67低于5%。FISH结果:3例均无乳头状肾细胞癌的染色体异常:3、7、17染色体扩增及Y染色体丢失。结论MTSCC为低级别肾细胞癌,形态学谱系广泛,免疫表型表达远曲小管上皮标记(AMCAR、CK7、CK19、EMA、NSE等),主要需与乳头状肾细胞癌相鉴别,一般预后较好,术后仍须密切随访。     

枕纹锦蛇消化道5-羟色胺免疫活性内分泌细胞的分布与形态学观察

采用ABC免疫组织化学法,应用5—羟色胺(5-HT)特异性抗血清,对枕纹锦蛇(Elaphe dione)消化道内含有的5—HT内分泌细胞进行了免疫组织化学的定位研究和形态学观察。结果显示,5-HT细胞在消化道各部位的分布密度呈倒“V”形,以十二指肠最高,胃贲门部最低。其形态多样,上段(食管、胃)多为圆形和椭圆形,主要分布于上皮基部和腺泡上皮之间;中段(十二指肠、空肠、回肠)以细长锥体形、梭形、圆形为主,主要分布于上皮基部和上皮细胞之间;下段(直肠)为圆形,分布于上皮基部。锥体形细胞常有一个长突起伸入到固有膜或肠腔,行使内或外分泌功能;梭形细胞有两个细长突起,一个指向固有膜,另一个指向肠腔,表明这种细胞可能具有内、外分泌的双重功能。     

植物细胞外囊泡及其分析技术的进展

细胞外囊泡是细胞在生理和病理条件下通过胞吐作用释放的具有磷脂双分子层结构的纳米级囊泡。细胞外囊泡作为蛋白质、核酸、脂质和代谢物等物质的载体,能够在细胞与细胞之间穿梭,行使物质传递、信息交流的功能,是细胞间通讯的重要媒介。近年来,植物中细胞外囊泡的研究也不断深入,其研究和分析技术也取得了很大进展。本文介绍了细胞外囊泡的组成,综述了植物中细胞外囊泡的生物学功能,分析了细胞外囊泡分离与富集方法的优缺点,以及原位成像技术的应用,最后对植物细胞外囊泡研究技术发展的重点进行了展望。     

Assembly of urothelial plaques: tetraspanin function in membrane protein trafficking

The apical surface of mammalian urothelium is covered by 16-nm protein particles packed hexagonally to form 2D crystals of asymmetric unit membranes (AUM) that contribute to the remarkable permeability barrier function of the urinary bladder. We have shown previously that bovine AUMs contain four major integral membrane proteins, i.e., uroplakins Ia, Ib, II, and IIIa, and that UPIa and Ib (both tetraspanins) form heterodimers with UPII and IIIa, respectively. Using a panel of antibodies recognizing different conformational states of uroplakins, we demonstrate that the UPIa-dependent, furin-mediated cleavage of the prosequence of UPII leads to global conformational changes in mature UPII and that UPIb also induces conformational changes in its partner UPIIIa. We further demonstrate that tetraspanins CD9, CD81, and CD82 can stabilize their partner protein CD4. These results indicate that tetraspanin uroplakins, and some other tetraspanin proteins, can induce conformational changes leading to the ER-exit, stabilization, and cell surface expression of their associated, single-transmembrane-domained partner proteins and thus can function as "maturation-facilitators." We propose a model of AUM assembly in which conformational changes in integral membrane proteins induced by uroplakin interactions, differentiation-dependent glycosylation, and the removal of the prosequence of UPII play roles in regulating the assembly of uroplakins to form AUM.     

What determines differentiation of urothelial umbrella cells?

Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.     

Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder.

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.     

Formation of asymmetric unit membrane during urothelial differentiation

Mammalian urothelium undergoes unique membrane specialization during terminal differentiation making numerous rigid-looking membrane plaques (0.3–0.5 m diameter) that cover the apical cell surface. The outer leaflet of these membrane plaques is almost twice as thick as the inner leaflet hence the name asymmetric unit membrane (AUM). Ultrastructural studies established that the outer leaflet of AUM is composed of 16 nm particles forming two dimensional crystals, and that each particle forms a twisted ribbon structure. We showed recently that highly purified bovine AUMs contain four major integral membrane proteins: uroplakins Ia (27 kD), Ib (28 kD), II (15 kD) and III (47 kD). Studies of the protease sensitivity of the different subdomains of uroplakins and other considerations suggest that UPIa and UPIb have 4 transmembrane domains, while UPII and UPIII have only one transmembrane domain. Chemical Crosslinking studies showed that UPIa and UPIb, which share 39% amino acid sequence, are topologically adjacent to UPII and UPIII, respectively, thus raising the possibility that there exist two biochemically distinct AUM particles, i.e., those containing UPIa/UPII vs. UPIb/UPIII. Bovine urothelial cells grown in the presence of 3T3 feeder cells undergo clonal growth forming stratified colonies capable of synthesizing and processing all known uroplakins. Transgenic mouse studies showed that a 3.6 kb 5-flanking sequence of mouse uroplakin II gene can drive the expression of bacterial LacZ gene to express in the urothelium. Further studies on the biosynthesis, assembly and targeting of uroplakins will offer unique opportunities for better understanding the structure and function of AUM as well as the biology of mammalian urothelium.     

Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum

Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.     

Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, syn-thesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation. Accepted: 19 August 1997     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

The apical surface of mammalian bladder urothelium is covered by large (500-1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin-Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.     

Chromosomal localization of uroplakin genes of cattle and mice

The asymmetric unit membrane (AUM) of the apical surface of mammalian urinary bladder epithelium contains several major integral membrane proteins, including uroplakins IA and IB (both 27 kDa), II (15 kDa), and III (47 kDa). These proteins are synthesized only in terminally differentiated bladder epithelial cells. They are encoded by separate genes and, except for uroplakins IA and IB, appear to be unrelated in their amino acid sequences. The genes encoding these uroplakins were mapped to chromosomes of cattle through their segregation in a panel of bovine x rodent somatic cell hybrids. Genes for uroplakins IA, IB, and II were mapped to bovine (BTA) Chromosomes (Chrs) 18 (UPK1A), 1 (UPK1B), and 15 (UPK2), respectively. Two bovine genomic DNA sequences reactive with a uroplakin III cDNA probe were identified and mapped to BTA 6 (UPK3A) and 5 (UPK3B). We have also mapped genes for uroplakins 1A and II in mice, to the proximal regions of mouse Chr 7 (Upk1a) and 9 (Upk2), respectively, by analyzing the inheritance of restriction fragment length variants in recombinant inbred mouse strains. These assignments are consistent with linkage relationships known to be conserved between cattle and mice. The mouse genes for uroplakins IB and III were not mapped because the mouse genomic DNA fragments reactive with each probe were invariant among the inbred strains tested. Although the stoichiometry of AUM proteins is nearly constant, the fact that the uroplakin genes are unlinked indicates that their expression must be independently regulated. Our results also suggest likely positions for two human uroplakin genes and should facilitate further analysis of their possible involvement in disease.     

Expression and localization of four uroplakins in urothelial preneoplastic lesions

In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.