DQ明胶原位酶谱分析方法在检测乳腺癌组织中明胶酶活性的应用

基质金属蛋白酶家族（MMPs）在肿瘤的侵袭和转移中具有重要的作用．其中最重要的是明胶酶类,包括MMP-2和MMP-9。由于能特异性的降解基底膜的主要成分Ⅳ型胶原而发挥作用,因此又称为Ⅳ型胶原酶。明胶酶以酶原形式分泌,在细胞外被活化后才能发挥作用,促进肿瘤细胞的侵袭和转移。但是以往在明胶酶的检测方法中,未能既检测其活性又能对其进行定位．本实验中所采用的DQ明胶原位酶谱分析的方法可以兼顾这两个方面。     

Clarification of the active gelatinolytic sites in human ovarian neoplasms using in situ zymography

Tissues from 26 human ovarian common epithelial tumors were examined to determine where and how gelatinolytic activity was present, in relation to tumor-stromal interaction and histologic types. For this purpose, we used in situ zymography, a newly developed technique using gelatin-coated film. Gelatinolytic activity was evident in ovarian carcinomas and in borderline tumors. Benign tumors had no or only weak activity. Four tissue localization patterns of gelatinolysis were identified: pattern A, tumor cytoplasm; pattern B, tumor-stromal junction; pattern C, stroma; and pattern D, cystic fluid. Mucinous cystadenocarcinomas showed A and/or D patterns. One mucinous and one serous adenocarcinoma and one mucinous borderline tumor had a B pattern. Most serous and all clear cell adenocarcinomas showed strong gelatinolysis of C pattern, especially in the desmoplastic stroma, an area where the tumor cells were dispersed. Immunohistochemically in 12 adenocarcinomas and 3 borderline tumors, the tumor cytoplasm was positive for matrix metalloproteinases (MMP-2) (5 cases), MMP-7 (9 cases), and MMP-9 (6 cases). Stromal components were positive for MMP-2 in 5 cases and for MMP-9 in 3 cases, but they were not positive for MMP-7. MMP antigens were mostly distributed in an almost identical pattern consistent with that seen with in situ zymography. In situ zymography clarified the cellular localization of active gelatinolysis in human ovarian neoplasms, a finding which supports the view that interaction between tumor and stroma is critical for tumor growth. This newly developed method contributes to a better understanding of biologic features of ovarian malignancies.     

In situ analysis of gelatinolytic activity in human dentin

Matrix metalloproteinases (MMPs) such as gelatinases are differentially expressed in human tissues. These enzymes cleave specific substrates involved in cell signaling, tissue development and remodeling and tissue breakdown. Recent evidences show that gelatinases are crucial for normal dentin development and their activity is maintained throughout the entire tooth function in the oral cavity. Due to the lack of information about the exact location and activity of gelatinases in mature human dentin, the present study was designed to examine gelatinolytic levels in sound dentin. In situ zymography using confocal microscopy was performed on both mineralized and demineralized dentin samples. Sites presenting gelatinase activity were identified throughout the entire biological tissue pursuing different gelatinolytic levels for distinct areas: predentin and dentinal tubule regions presented higher gelatinolytic activity compared to intertubular dentin. Dentin regions with higher gelatinolytic activity immunohistochemically were partially correlated with MMP-2 expression. The maintenance of gelatinolytic activity in mature dentin may have biological implications related to biomineralization of predentin and tubular/peritubular dentinal regions, as well as regulation of defensive mechanisms of the dentin-pulp complex.     

Simultaneous dehydrochlorination and film formation of a vinylidene chloride copolymer by aqueous-organic interface reaction

The interface reaction of a THF solution of a vinylidene chloride copolymer with an aqueous alkaline solution in the presence of a quaternary ammonium halide as a phase transfer catalyst affords a dehydrochlorinated polymer film with conjugated polyene structure. The degree of the reaction is dependent on the catalyst structure, and tetrabutylammonium bromide is the best among those used. KOH was found to be more effective than NaOH. The effects of the base and polymer concentration and of the nature of the solvent were also studied. The results are discussed in terms of the change of the rate of formation of ammonium hydroxide at the interface and of its diffusion in the organic phase. The doping of the obtained films with iodine vapor increases the electric conductivity up to 7 · 10^?4 S · cm^?1.     

Identification of activated matrix metalloproteinase-2 (MMP-2) as the main gelatinolytic enzyme in malignant melanoma by in situ zymography

The involvement of extracellular matrix-degrading enzymes, such as matrix metalloproteinases and serine proteases, during tumour progression and metastasis is well established. In particular, the activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP has been shown to contribute to the invasive abilities of various tumours. This study presents evidence that in tissue of malignant melanomas, increased effective gelatinolytic activity is mainly located at sites where melanoma cells interact with the surrounding extracellular matrix. Forty-one primary melanomas (30 superficial spreading and 11 nodular type) and six lymph node metastases were investigated by a modified technique of gelatin in situ zymography. This technique localizes areas of effective proteolytic activity within tissue sections. In 28/41 (68%) primary melanomas and in 6/6 (100%) metastases, considerable proteolysis was detected at the invading part of the tumour and especially at sites of tumour-stroma interactions, whereas no or only weak proteolytic activity was localized within the centres of solid nests of tumour cells. Zymographic analysis of extracts obtained from different areas of microdissected melanoma specimens identified activated MMP-2 as the enzyme responsible for this activity. Immunohistochemical analysis detected strong staining for MMP-2 and MT1-MMP, even in areas in which no proteolytic activity was found by in situ zymography, emphasizing the importance of more functional techniques for the investigation of balanced proteolytic systems. This technology makes it possible to draw conclusions regarding the balance between activated proteases and inhibitors, which are frequently found to be present together in close proximity in vivo.     

In situ detection of gelatinolytic activity in developing craniofacial tissues

Frozen tissue sections of developing and adult rat heads were incubated on a film coated with a gelatin-containing colloidal silver emulsion in order to detect gelatinolytic activity present in the different tissues. The method, termed film in situ zymography, is based on the ability of the thiol group of the propeptide released from the degraded gelatin to induce a structural change in the colloidal silver and thereby a visible change in color. The frozen tissue sections mounted on the coated film were incubated at 37°C overnight. Gelatinolytic activity was detected as a color change from yellow to red. The activity of gelatinase was completely blocked by phenanthroline, which inhibits matrix metalloproteinases. Gelatinolytic activity was widely present in the oral epithelium, tooth buds, tongue, Meckel’s cartilage, salivary glands, and other tissues. The intensity of the gelatinolytic activity varied among the different tissue types. The present study demonstrated gelatinolytic activity in both developing and adult craniofacial tissues. These results suggest that gelatinolytic activity plays an important role in normal turn-over in several tissues. Whereas some of the activity also in the developing rats may be related to this turn-over process, some of it is probably directly associated with developmental changes. Accepted: 3 March 1999     

The immunohistochemical demonstration of major histocompatibility antigens in the human kidney using monoclonal antibodies

The distribution of HLA-ABC and DR antigens in human kidneys was studied using monoclonal antibodies to monomorphic determinants and a 4-layer immunoperoxidase technique applied to frozen and paraffin sections. Enzyme digested paraffin sections provided the best localization. HLA-ABC antigens were located on all renal endothelium, Bowman's capsule and in proximal tubules. HLA-DR antigen was restricted to capillary and venous endothelium plus, at low density, in proximal tubules. Minor differences in distribution were found with different monoclonal antibodies, but dendritic cells were not detected using monoclonal antibodies to HLA-ABC, HLA-DR or leukocyte-common antigens.     

Duration of gelatinolytic activity of acrosomal proteinases of rabbit spermatozoa

The gelatinolytic activity of acrosomal proteinases of rabbit spermatozoa can be observed maximally up to about 100 h after washing and incubation of the spermatozoa in Brakett's defined medium at 37 degrees C. It is independent of sperm motility and seems to be provable as long as the acrosomal membranes remain intact. In a defined in vitro system this phenomenon appears to be a function of time and temperature.     

Histochemical and immunohistochemical morphology of carcinoid heart disease

Histochemical and immunohistochemical investigations were performed on tissue obtained from the right heart side in three patients subjected to valve, replacement operations because of severe carcinoid heart disease. Extensive fibrotic changes were present on the endocardium of the right atrium, the papillary muscles of the tricuspid valve and the leaflets of the tricuspid and pulmonic valves of all patients. The main constituent of the lesions was a stroma with abundant acid mucopolysaccharides and collagen but devoid of stainable elastic components. The lesions were in some areas sharply delineated from the normal endocardium, but often also extended into the endocardium and myocardium. Small to medium sized vessels were demonstrated histochemically in the lesions and confirmed by positive immunoreaction against endothelial and smooth muscle cells. The moderate number of mesenchymal cells within the lesions had immunoreactivity consistent with muscle cells which seemed to have a very low proliferating activity. The histochemical and immunohistochemical techniques used confirmed some earlier observations in carcinoid heart disease but also rendered new information contradicting previous findings. The infiltrative nature of the carcinoid plaque gives a new dimension to the carcinoid heart disease. The etiology still remains obscure and well known growth factors for connective tissue such as platelet derived growth factor (PDGF) do not seem to be directly involved in the process.     

Simultaneous demonstration of two antigens on single T cells using antibodies with contrasting labels

We describe a double labeling method for the discrimination of 2 antigens on single cells. It consists of a combination of 3H-immunoautoradiography and immunocytochemistry applied to cells previously fixed on poly-L-lysine (PLL)-coated multispot slides. The method has been applied to various mouse cells contemporaneously labeled with 2 different monoclonal antibodies. In order to distinguish the attached antibodies unambiguously, they were labeled with contrasting markers. One of the antibodies was marked with tritium blackening the photographic film that covers the slide. The other was detected with the peroxidase-anti-peroxidase (PAP) method forming a reddish precipitate. The contrast between the reddish reaction product of the PAP-labeled antibody and the black silver grains allows cells, specifically labeled with both antibodies, to be distinguished from cells labeled with only one or neither of the antibodies. Tritium-labeled antibodies were introduced because of their advantage over antibodies labeled with iodine in the closer localization of the silver grains to the bound antibody and their much longer halflife (60 days versus 12 years). In this study we applied a tritium-labeled anti-Thy-1.1 together with anti-Lyt-1 monoclonal antibody for studying the distribution of the corresponding antigens on lymphocytes in the mouse thymus and lymph node cells.     

Early acquisition of gelatinolytic activity in carcinogenesis of the uterine cervix.

Film in situ zymography is a newly developed technique for detecting in situ gelatinolytic activity. Using the film in situ zymography method to stamp preparations, we evaluated the gelatinolytic activity in early stage cervical neoplasia and invasive squamous cell carcinoma. To determine the sensitivity of film in situ zymography for detecting gelatinase expression, slides made from stamps of 50 specimens resected from the uterine cervix, including early invasive carcinoma (FIGO Ia1 and Ib1) and cervical intraepithelial neoplasia (CIN) were examined by film in situ zymography. The specimens were also examined immunohistochemically with regard to mitotic activity and gelatinase expression. Gelatinolytic activity was subdivided into two patterns, the homogeneous pattern and the heterogeneous pattern. The homogeneous pattern consisted of circumscribed areas around atypical cell clusters; these areas were composed of homogenously digested full-thickness collagen, whereas the heterogeneous pattern consisted of spottily digested areas of superficial collagen around atypical cell clusters. All invasive carcinomas (8/8 cases) and carcinoma in situ (14/14 cases) were positive for gelatinolytic activity, and 33.3%(5/15 cases) of the specimens of CIN-1, CIN-2, and severe dysplasia were also positive. All invasive carcinomas and 6 of 14 carcinoma in situ (43%) showed homogenous pattern, and the other positive specimens showed heterogenous pattern. The MIB-1 index was 33.8% in invasive carcinoma, increasing stepwise from dysplasia to carcinoma. Matrix metalloproteinase-2 immunostaining was positive in 4 of 8 cases of invasive carcinoma and generally stained the stromal area around the tumor nest. These results indicated that matrix metalloproteinases are functionally activated even in carcinoma in situ and in cervical intraepithelial neoplasia of the uterine cervix not showing invasive growth histologically. Film in situ zymography can detect with sensitivity the invasive potential of carcinoma in situ and cervical intraepithelial neoplasia. An analysis combining cytological examination and film in situ zymography is a potentially useful tool for estimating invasive activity.     

Familial erythrophagocytic lymphohistiocytosis: immunophenotypic, immunohistochemical, and ultrastructural demonstration of the relation to sinus histiocytes

Familial erythrophagocytic lymphohistiocytosis (FEL), a rare, rapidly fatal childhood disease, is characterized by fever, hepatosplenomegaly, pancytopenia, and widely disseminated lymphohistiocytic infiltrates with prominent erythrophagocytosis. Immunophenotypic, immunohistochemical, and ultrastructural studies of two siblings with FEL were performed in an effort to determine the nature of the proliferating histiocyte of FEL. These studies demonstrated that the FEL histiocytes lack S-100 protein, T6, and Birbeck granules, which are found in Langerhans and interdigitating dendritic cells. The FEL histiocytes express alpha 1-antichymotrypsin, Leu-M3, HLA-DR, and, variably, lysozyme and Leu-M1. Thus, the proliferating histiocyte of FEL is a member of the mononuclear phagocytic system and has a phenotype similar to that of the histiocytes that normally populate the sinuses of benign and reactive lymph nodes. These studies suggest that FEL may represent uncontrolled proliferation of sinusoidal histiocytes.     

颅内原发性生殖细胞瘤的病理和β—HCG的免疫组织化学观察

Clinical pathologic observation and immunohistochemical demonstration on beta--HCG are reported in 13 cases of intracranial primary germinomas, including 7 cases of germinomas in the region of the pineal gland and 6 cases in the hypothalamic area. Among them, there were 11 cases of simple type and 2 cases of beta--HCG--producing tumors with a character of differentiation of trophoblasts in the latter. This paper supports the concept that intracranial primary germinoma is an atypical teratoma arising from the germ cells. The significance of beta--HCG immunohistochemical demonstration in the pathologic diagnosis of germinomas and the mechanism of endocrine disturbance are discussed.     

Localization of plasminogen activator activity within normal and injured lungs by in situ zymography

During inflammatory lung injury, the fibrinolytic activity that is normally present within bronchoalveolar lavage (BAL) fluid (BALF) is often suppressed due to increased levels of inhibitors, including plasminogen activator inhibitor (PAI)-1. Despite this suppression, BALF frequently contains fibrin degradation products, indicating persistence of fibrinolytic activity within the lung. To address this discrepancy and determine the sites where plasminogen activation is occurring, we developed an in situ zymographic technique for frozen sections of lung tissue that localizes plasminogen activator activity at the cellular level. After validating the method using enzyme inhibitors and mice with genetic manipulations of their plasminogen system genes, we applied the technique to lungs of normal and bleomycin-exposed mice. In normal mice, plasminogen activator activity was localized to bronchial epithelial cells, cells of the alveolar walls, and alveolar macrophages. After bleomycin exposure, in situ zymography showed that, despite loss of fibrinolytic activity within BALF, abundant enzymatic activity was associated with aggregates of inflammatory cells. PAI-1-deficient mice that are protected from bleomycin-induced fibrosis had preserved plasminogen activator activity in BALF and increased tissue activity, as determined by in situ zymography. We conclude that analysis of BALF does not adequately reflect the fibrinolytic activity that persists within microenvironments of the lung during inflammation.