Bone morphogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice

Secondary cartilages including mandibular condylar cartilage have unique characteristics. They originate from alkaline phosphatase (ALP)-positive progenitor cells of the periosteum, and exhibit characteristic modes of differentiation. They also have a unique extracellular matrix, and coexpress type I, II and X collagens. We have previously shown that there is a total absence of secondary cartilages in Runx2-deficient (Runx2-/-) mice. To clarify whether Runx2 is essential for chondrocytic differentiation of secondary cartilages, we performed an organ culture system using mandibular explants derived from Runx2-/- mice at embryonic day 18.0. Since mRNA for bone morphogenetic protein 2 (BMP2) was strongly expressed in osteoblasts of condylar anlagen in wild-type mice, and was down-regulated in those of Runx2-/- mice, we chose to investigate BMP2 effects on secondary cartilage formation. Condensed mesenchymal cells of mandibular condylar anlagen in precultured explants were ALP-positive and expressed type I collagen and Sox9. After culture with recombinant human (rh) BMP2, chondrocytic cells showing ALP activity and expressing Sox5, Sox9, and type I and II collagens, appeared from mesenchymal condensation. This expression profile was comparable with the reported pattern of chondrocytes in mouse secondary cartilages. However, chondrocyte hypertrophy was not observed in the explants. These findings indicate that BMP2 partially rescued chondrocyte differentiation but not chondrocyte hypertrophy in secondary cartilage formation in Runx2-/- mice. Runx2 is required for chondrocyte hypertrophy in secondary cartilage formation, and it is likely that BMP2, which is abundantly secreted by osteoblasts in condylar anlagen, contributes to the early process of secondary cartilage formation.     

Expression of alkaline phosphatase in the mature mouse placenta visualized by in situ hybridization and enzyme histochemistry

Alkaline phosphatases (APs) are a family of cell surface glycoproteins that are expressed in a variety of tissues. Their physiologcial functions are still unclear. Three different AP genes have been found to be expressed in mice, and AP cloned from the placenta is of the tissue non-specific (TNAP) type. We have in investigated the location of TNAP mRNA and active AP in mature mouse placenta, using in situ hybridization and enzyme histochemistry on serial sections. Digital image analysis was used to estimate relative amounts of TNAP mRNA. Tissue non-specific alkaline phosphatase messenger was detected only in the placental labyrinth, whereas active AP was present both in the labyrinth and in a zone of cells at the margin of the decidua basalis, bordering the myometrium and the metrial gland. This latter location of AP activity has not been described previously. The AP-positive zone of the decidua had a condensed appearance and a central defect in the zone was visible on sections taken from the middle of the placenta. No TNAP messenger was found in the zone of AP-positive decidual cells.     

Changes in the pattern of expression of alkaline phosphatase in the mouse uterus and placenta during gestation

The development of the rodent chorio-allantoic placenta is a complicated process that results in the formation of a transport system capable of sustaining embryonic and fetal growth and development. Intimately linked to this process is alkaline phosphatase (AP), a cell-surface glycoprotein that possibly functions as a transport protein. In the present study, we have mapped the location of AP-expressing cells in the mouse utero-placental unit during the development of the chorio-allantoic placenta by use of enzyme histochemistry and in situ hybridization histochemistry. We found that at implantation the expression of the tissue non-specific AP (TNAP) gene is located exclusively in the decidua and that most of this decidual expression ceases as the placenta starts to form. One exception is a mesometrially located marginal zone of the decidua, which continues to express the TNAP gene until day 12 and the active protein until at least day 16. Trophoblasts of the chorion already express AP before the time of fusion with the ectoplacental cone, after which AP is expressed by trophoblasts of the resulting ectoplacental plate. AP expression in the mature chorio-allantoic placenta is localized in the placental labyrinth and spongy zones. In the latter zone, expression ceases on about day 14. Giant trophoblasts start to express AP on about day 10, with some cells still positive for AP at day 16. The yolk sac does not express AP at any developmental stage. The results show that AP expression during placental development is neither restricted to cells known to be involved in transport, nor expressed in all cells thought to be involved in this transport. This may indicate that AP is not merely a transport protein but has additional functions.     

Age-related changes in gene expression patterns of matrix metalloproteinases and their collagenous substrates in mandibular condylar cartilage in rats

Matrix metalloproteinases (MMPs) have been implicated in physiological cartilage matrix remodelling as well as in pathological and invasive extracellular matrix remodelling of tissue. Age-related changes in the gene expression patterns of MMPs in mandibular condylar cartilages (MCCs) were analysed. We examined the gene expression patterns of Mmp-8 and -13 and their substrates, Col1a1, Col2a1 and Col10a1, in MCC of growing and ageing rats. Temporomandibular joints of male Wistar rats aged 4, 8, 16 and 32 weeks were subjected to in situ hybridization analysis. Histologically, MMCs showed characteristics of growth plate cartilage at ages 4 and 8 weeks, and more closely resembled articular cartilage thereafter. Mmp-8 was expressed in the cells in all cartilaginous cell layers at ages 4 and 8 weeks, and then was localized only in the mature cells at ages 16 and 32 weeks. Whereas Mmp-13 expression was limited to the lowermost hypertrophic chondrocytes in the growth stage, mature chondrocytes instead of hypertrophic chondrocytes expressed Mmp-13 in adult non-hypertrophic MCC. Because Mmp-8 and -13 expression overlapped with Col2a1 and Col10a1, chondrocytes could play a pivotal role in degradation as well as production of the cartilaginous matrix in MCC.     

Development and ageing of the articular cartilage of the rabbit knee joint: distribution of the fibrillar collagens

The development of the articular cartilage of the rabbit knee joint from the 17-day fetus to the 2-year adult rabbit has been examined. At 17 days, the developing femur and tibia are separated by the interzone. Cavitation occurs around 25 days; the cells of the intermediate layer flatten and move onto those of the chondogenous layers to create the articular surfaces. After birth, growth of the cartilage is mainly the result of matrix production. Ossification of the epiphyses is complete by 6 weeks postpartum. Horizontal zones can be distinguished in the articular cartilage; the superficial cells are aligned parallel to the surface, but in the deep layers the cells are in columns. The tidemark is first seen at 12–14 weeks. The matrix of the interzone in the 17-day fetus contains types I, III and V collagens, but no type II. After cavitation at 25 days, the surface layer of the articular cartilage still contains type I, but no type II collagen. From 6 weeks postnatal onwards, type II collagen is present throughout the cartilage and type I disappears. Type III collagen is initially in the interterritorial matrix, but later it is mainly pericellular. Type V collagen is pericellular both in the chondrogenous layers and later in the articular cartilage, but is not present in the epiphyseal cartilage below. From 6 weeks onwards, types III and V collagens create a capsule around all the chondrocytes above the tidemark. The relationship of types V and XI collagens is discussed. It is concluded that the articular chondrocytes form a unique subset of cells from the earliest stages of joint formation in the fetal rabbit.     

CoREST基因在骨关节炎软骨组织中的表达、分布和意义

目的: 观察RE-1元件辅助沉默因子(CoREST)基因在正常和骨关节炎(OA)膝关节软骨组织中的表达和分布,分析CoREST基因表达下调对OA的病理意义。方法: 取材膝关节正常(n=10)和OA(n=12)软骨组织,提取软骨细胞并包埋制备切片,应用real-time PCR技术对比CoREST基因在正常和OA软骨细胞中表达水平的总体差异,应用原位杂交技术观察CoREST基因在正常/OA软骨表、中、深各层组织中的表达和分布的特点。结果: CoREST基因在OA软骨细胞中的表达水平显著降低64.6%(P<0.01)。CoREST基因广泛表达于正常软骨组织的表层、中层和深层,以中层表达最为显著,其次为深层和表层。在OA软骨组织中,CoREST基因在浅层细胞簇部分细胞中表达水平升高,在深层潮线状细胞柱中表达水平普遍降低。结论: CoREST基因在正常和OA软骨组织细胞中的差异表达和分布与文献报道的X型胶原基因的组织学表达一致。结合前期研究成果,我们认为CoREST的生物功能可能与OA软骨细胞的终末分化相关。     

横纹肌肉瘤中MDM2及p53基因表达的原位杂交检测

目的观察ＭＤＭ２、ｐ５３癌基因在横纹肌肉瘤（ＲＭＳ）发病中的作用及其与临床病理、预后间的关系。方法对确诊并有随访的３１例ＲＭＳ标本,用原位杂交技术进行ＭＤＭ２、ｐ５３定位观察。结果发现ＭＤＭ２及ｐ５３癌基因表达阳性率分别为７７４％（２４／３１例）和６６７％（２１／３１例）,其阳性率与年龄、性别和ＲＭＳ组织类型无明显关联（Ｐ＞０．０５）,但阳性率及其强度与ＲＭＳ分化程度（Ⅰ级与Ⅲ级）,转移与否及存活率差异有显著性（Ｐ＜０．０５）。结论ＭＤＭ２、ｐ５３阳性检出率有助于判断ＲＭＳ恶性程度及预测肿瘤的转移和预后。     

骨肿瘤MDM2和p53基因的改变

目的从基因水平研究ＭＤＭ２和ｐ５３基因在骨肿瘤中的表达，探讨其在骨肿瘤发生发展中的作用。方法用地高辛标记原位杂交技术研究了３８例骨肿瘤（骨肉瘤１２例，软骨肉瘤１０例，骨巨细胞瘤１４例，软骨母细胞瘤２例）ＭＤＭ２和ｐ５３的表达情况，并分析两种基因表达之间的相互关系。结果ＭＤＭ２在骨肉瘤、软骨肉瘤和骨巨细胞瘤中的阳性率分别为４１．７％、５０．０％和３５．７％。ｐ５３的阳性率分别为５８．３％、４０．０％和２１．４％。２例软骨母细胞瘤ＭＤＭ２和ｐ５３均为阳性。ＭＤＭ２与ｐ５３基因表达呈显著正相关（Ｐ＜０．００５）。结论ＭＤＭ２和ｐ５３基因改变是骨肿瘤的一种常见现象，可能参与骨肿瘤的发生与发展。     

Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage

The present study was designed to investigate whether or not chondrocytes in articular cartilage express type I collagen in vivo under physiological conditions. Expressions of the gene and the phenotype of type I collagen were examined in rat tibial articular cartilage in the knee joint during development. Knee joints of Wistar rats at 1, 5, and 11 weeks postnatal were fixed in 4% paraformaldehyde with or without 0.5% glutaraldehyde and decalcified in 10% EDTA. After the specimens were embedded in paraffin and serial sections made, adjacent sections were processed for immunohistochemistry and in situ hybridization for type I collagen. The epiphysis of the tibia was composed of cartilage in week-1 rats. Formation of articular cartilage was in progress in week 5 as endochondral ossification proceeded and was completed in week 11. Anti-type I collagen antibody stained only the superficial area of the epiphysis in week 1, but the immunoreactivity was expanded into the deeper region of the articular cartilage with development in weeks 5 and 11. Hybridization signals for pro-alpha 1 (I) collagen were seen in some of chondrocytes in the epiphysis of the week-1 tibia. The most intense signals were identified in chondrocytes in week 5 and the signals appeared weaker in week 11. The present study demonstrated that chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of the articular cartilage.     

层黏连蛋白受体在小鼠睾丸和附睾中的表达

目的 探讨层黏连蛋白受体(LAMR1)在小鼠睾丸和附睾中的表达.方法收集3只正常成年昆明小鼠睾丸和附睾.采用原位杂交和免疫组织化学方法,检测LAMR1 mRNA及蛋白在成年小鼠睾丸和附睾中的分布.结果 LAMR1 mRNA在附睾头和附睾尾表达最强;在睾丸生精细胞中也有表达.免疫组织化学结果显示,LAMR1蛋白从附睾头到...     

Cloning and localization of immediate early response 2 (ier2) gene in the brain of medaka

Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3′-, 5′- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.     

A comparative study of congenital and postnatally acquired human cytomegalovirus infection in infants: lack of expression of viral immediate early protein in congenital cases

Postmortem tissues from infants with congenital and postnatally acquired human cytomegalovirus (HCMV) infection were examined by routine histology, immunohistochemistry (IHC) and in situ hybridization (ISH) to determine the dynamics of viral replication in vivo. Histologically, infants in both groups showed characteristic inclusion-bearing cells most commonly in lung, kidney, liver and pancreas. IHC for late proteins using a rabbit polyclonal antibody and ISH for viral genomes detected most of the infected cells as nuclear and/or cytoplasmic signals. However, immunostaining with a monoclonal antibody against viral immediate early (IE) proteins was variable depending on the stage of viral replication within an individual infected cell. In tissues of infants with postnatal HCMV infection, many cells harboured IE antigens, while in tissues from congenital cases most of the affected cells lacked IE antigens and only a few showed cytoplasmic staining. The difference was not caused by the antigenic diversity among viral strains as confirmed by in vitro study. Our findings suggested that congenital infections exhibited uniformly late stage proteins with inactive viral replication at death, while acquired ones remained active. The different viral activity may reflect the immune status of congenital and acquired HCMV infections.     

Differential expression of interleukin-5 mRNA+ cells and eosinophils in Nippostrongylus brasiliensis infection in resistant and susceptible strains of mice

Interleukin (IL)-5 is produced by both T cells and eosinophils and has been implicated in lymphocyte and eosinophil differentiation and maturation. The extent to which differences in IL-5 expression contribute to genetic variability in parasite immunity was investigated by comparing eosinophilia, IgE production, mastocytosis and IL-5 mRNA⁺ cells following Nippostrongylus brasiliensis infection of resistant (BALB/c) and susceptible (C57BL/6) mice. In uninfected mice, IL-5 mRNA⁺ cells detected by in situ hybridization were distributed throughout the lamina propria and crypt regions of the small intestine in both strains, but were 1.5-fold higher in BALB/c than in C57BL/6 mice. Following N. brasiliensis infection, the numbers of IL-5 mRNA⁺ cells in BALB/c mice continued to increase until day 11 post-infection at which time they were more than 4-fold more numerous than in uninfected control mice of the same strain. In C57BL/6 mice, IL-5 mRNA⁺ cells reached peak numbers on day 7 post-infection, only 1.5-fold higher than uninfected controls, but the numbers began to decline thereafter. At all time points after day 5, the numbers of IL-5 mRNA⁺ cells in the gut of C57BL/6 mice were significantly lower than BALB/c mice. The differences in numbers of IL-5 mRNA⁺ cells induced by infection in each strain of mice correlated with changes in blood and intestinal eosinophilia, mastocytosis and IgE production and was reflected in differences in worm expulsion and egg counts. Although numbers of intestinal IgA-containing cells increased in both strains after infection, there was no difference between strains except at day 11 when there were significantly higher numbers in BALB/c mice than in C57BL/6 mice. These results suggest that IL-5 is an important regulatory factor determining host immunity to parasite infection and that differential regulation of IL-5 expression explains in part the observed strain differences with respect to parasite resistance.     

MMP-2、MT-MMP-2在实验性肝纤维化形成和逆转过程中表达的动态研究

目的 探讨基质金属蛋白酶-2(MMP-2)、膜型基质金属蛋白酶-2(MT-MMP-2)在实验性肝纤维化形成和逆转过程中的动态表达及意义。方法 建立大鼠实验性CCL中毒性肝纤维化模型，以正常大鼠为对照。采用核酸原位杂交和免疫组化法检测了实验性肝纤维化形成和自然逆转过程中MMP-2、MT-MMP-2 mRNA及相关抗原的来源细胞、时序和量的变化。结果 MMP-2、MT-MMP-2 mRNA及相关抗原在间质细胞和部分肝细胞中表达，以纤维间隔及汇管区最为明显。在肝纤维化形成过程中，MMP-2、MT-MMP-2表达逐渐增强；逆转过程中，MMP-2、MT-MMP-2表达先增强后减弱。结论 MT-MMP-2在肝脏中表达，肝脏间质细胞是MMP-2、MT-MMP-2的主要来源细胞，MMP-2、MT-MMP-2基因和蛋白表达水平与肝纤维化程度密切相关，在肝纤维化形成和逆转过程中起重要作用。     

显色原位杂交和免疫组织化学检测乳腺癌HER2/neu基因状况和蛋白表达的对照性研究

目的探讨显色原位杂交（CISH）在检测乳腺癌中HER2／neu基因扩增上的作用。方法挑选乳腺浸润性导管癌患者组织石蜡蜡块（回顾性255例,前瞻性271例）,进行免疫组织化学（IHC）、CISH检测。15例回顾性标本送往德国HERA检测中心进行FISH检测。结果（1）回顾性病例中IHC阳性3＋者CISH基因扩增率为91．6％（120／131）,IHC2＋者CISH基因扩增率为56．5％（39／69）,IHC与CISH检测结果符合率为81．2％（207／255）,两者明显相关（P〈0．01）。（2）前瞻性病例中IHC蛋白过表达率31．7％．CISH基因扩增率27．3％。IHC3＋者CISH基因扩增率为91．4％（53／58）,IHC2＋者CISH基因扩增率为46．4％（13／28）,IHC与CISH检测结果符合率为89．7％（243／271）,两者明显相关（P〈0．01）。（3）经德国检测中心荧光原位杂交（FISH）检测的15例中14例和CISH结果完全一致,1例检测失败,而CISH为无扩增。（4）CISH检测基因扩增与雌激素受体（ER）、孕激素受体（PR）表达明显负相关（P值均〈0．01）。结论CISH检测HER2基因扩增结果与IHC检测蛋白过表达及FISH结果高度一致,CISH是检测HER2基因扩增的一项新技术。