Methods in molecular medicine最新文献

In rheumatology and joint research, as in other fields, a purely descriptional appqoach to morphology cannot satisfy the exactions of modern clinical medicine. Investigators now appreciate the need to gauge pathological changes and their response to treatment by quantifying susceptible structural parameters. But the desired information respecting three-dimensional structures must be gleaned from either actual or virtual two-dimensional sections through the tissue. This information can be obtained only if the laws governing stereology are respected. In this chapter, the stereological principles that must be applied, and the practical methods that have been devised, to yield unbiased estimates of the most commonly determined structural parameters, namely, volume, surface area and number, are summarized.

The development in the techniques for obtaining synovial tissue biopsy, especially through arthroscopy, have resulted in greater access to high-quality synovial tissue. The use of immunohistochemistry in arthritis research has greatly furthered our understanding of the varied immunological and biochemical pathways involved in inflammatory arthropathopies such as rheumatoid and psoriatic arthritis. Immunohistochemistry provides a strikingly visual narrative of the essential elements involved in inflammatory arthritis, from the infiltrating inflammatory cells (e.g., T-cells, macrophages, B-cells, and neutrophils), their products (e.g., cytokines, metalloproteinases) and their varied receptor molecules. This chapter describes the standard three-stage immunoperoxidase technique used in our laboratory and widely in the literature. Some problems that may be encountered and how they may be overcome are commented on. Also described is a method for dual-labeled immunofluoresence staining.

Most current approaches used to analyze gene expression in tissue samples are based on RNA isolated either from cultured synovial cells or from synovial biopsies. However, this strategy does not distinguish between specific gene expression profiles of cells originating from discrete tissue areas. Therefore, we established the combination of laser-mediated microdissection and RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display to analyze profiles of gene expression in histologically defined areas of arthritic tissue. Cryosections derived from synovial tissue were used to obtain cell samples from different tissue areas of both rheumatoid arthritis (RA) and osteoarthritis (OA) patients using a microbeam laser microscope. RNA was isolated and analyzed using nested RNA arbitrarily primed PCR to generate a fingerprint of the expressed gene sequences. Differentially expressed bands were isolated, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry.

Microtubule-binding proteins are conveniently divided into two large groups: MAPs (microtubule-associated proteins), which can stabilize, anchor, and/or nucleate microtubules, and motors, which use the energy of ATP hydrolysis for a variety of functions, including microtubule network organization and cargo transportation along microtubules. Here, we describe the use of Taxol-stabilized microtubules for purification of MAPs, motors, and their complexes from Xenopus egg extracts. Isolated proteins are analysed using sodium dodecyl sulfate gel electrophoresis and identified by various mass spectrometry and database mining technologies. Found proteins can be grouped into three classes: (1) known MAPs and motors; (2) proteins previously reported as associated with the microtubule cytoskeleton, but without a clearly defined cytoskeletal function; (3) proteins not yet described as having microtubule localization. Sequence-similarity methods employed for protein identification allow efficient identification of MAPs and motors from species with yet unsequenced genomes.

Replacement of skin has been one of the most challenging aims for surgeons ever since the introduction of skin grafts in 1871. It took more than one century until the breakthrough of Rheinwald and Green in 1975 that opened new possibilities of skin replacement. The combination of cell culture and polymer chemistry finally led to the field of tissue engineering. Many researchers all over the world have been fascinated by the chance of creating a skin-like substitute ex vivo without any further harm to the patients, especially those with massive burns. Many different approaches to create new substitutes and further improvements in genetical and stem cell research led to today's skin equivalents. But still, the "gold standard" for wound coverage is the autologous split-thickness skin graft. Future research will aim at originating biologically and physiologically equal skin substitutes for the treatment of severe burns and chronic ulcers.

Tissue engineering aims to provide a temporary scaffold for repair at the site of injury or disease that is able to support cell attachment and growth while synthesis of matrix proteins and reorganization take place. Although relatively successful, bladder tissue engineering suffers from the formation of scar tissue at the scaffold implant site partly due to the phenotypic switch of smooth muscle cells (SMCs) from a quiescent contractile phenotype to a synthetic proliferative phenotype, known as myofibroblast. We hypothesize that culturing human SMCs in enzymatically degradable poly(ethylene) glycol (PEG) hydrogels modified with integrin-binding peptides, and in co-culture with human urothelial cells (UCs), will offer some insight as to the required environment for their subsequent differentiation into quiescent SMCs. We have established protocols for isolation, culture, and characterization of human bladder UCs, SMCs, and fibroblasts and investigated co-culture conditions for SMCs and UCs. The optimal PEG hydrogel properties, promoting growth of these cells, have been investigated by varying the amounts of cell adhesion peptide, PEG, and crosslinker and examined using light and fluorescence microscopy. Furthermore, the cell organization within and on top of gels 14 days post seeding has been examined by histology and immunohistochemistry. We have investigated a co-culture model for UCs and SMCs integrated into PEG hydrogels, mimicking a section of the bladder wall for reconstructive purposes that also could contribute to the understanding of the underlying basic mechanisms of SMC differentiation.

Investigating the cell biology of gene expression requires methodologies for localizing RNA relative to proteins involved in RNA transcription, processing, and export. Adenovirus is an important model system for the analysis of eukaryotic gene expression and is also being used to investigate the organization of gene expression within the nucleus. Here are described the combined in situ hybridization and immunofluorescence staining techniques that have been used to study the localization of viral RNA relative to nuclear structures that contain splicing factors.

Owing to their in vivo-like characteristics, three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are gaining increasing popularity as an in vitro model of tumors. A straightforward and simple approach to the cultivation of these MCTS is the hanging-drop method. Cells are suspended in droplets of medium, where they develop into coherent 3D aggregates and are readily accessed for analysis. In addition to being simple, the method eliminates surface interactions with an underlying substratum (e.g., polystyrene plastic or agarose), requires only a low number of starting cells, and is highly reproducible. This method has also been applied to the co-cultivation of mixed cell populations, including the co-cultivation of endothelial cells and tumor cells as a model of early tumor angiogenesis.

Reactive oxygen and nitrogen species are thought to contribute to pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, restenosis, heart failure, and diabetic vascular complications. Some of these reactive oxygen species also play an important role in vascular signaling. In this chapter, we describe various techniques that we have successfully employed to reliably measure superoxide and hydrogen peroxide. Because reactive oxygen species are capable of rapidly inactivating nitric oxide and because endothelial function characterized by nitric oxide bioavailability is an important indicator of vascular health, we have also included novel techniques capable of directly measuring nitric oxide radical from vascular cells and tissues.

Diabetes mellitus is the most common disease in Westernized countries in large part because of the rising prevalence of obesity and physical inactivity. In addition, diabetes mellitus is an important risk factor for both heart failure and ischemic heart disease. As insulin resistance is known as an important pathophysiological feature in the cardiac diseases, understanding the mechanisms responsible for altered metabolism and insulin signaling in the diabetic heart may help identify novel targets in these conditions. Phosphatidylinositol (PI)-3 kinase (PI3K) and Akt are key signaling molecules in insulin and insulin-like growth factor-1 (IGF-1), which induce multiple biological effects in the heart such as cell survival and hypertrophy. Here, we have shown several fundamental techniques to study the role of PI3K and Akt in heart diseases.