共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
In this study, we evaluated the feasibility of converting cardiac fibroblasts into skeletal muscle cells by forced expression of the MyoD gene, one of the basic helix-loop-helix myogenic factors. Primary cardiac fibroblasts, isolated from newborn rats, were infected with retrovirus-carrying sense or antisense MyoD gene. Ten days after infection, expression of MyoD protein was demonstrated in 95% of cells infected with sense MyoD virus by intense nuclear immunostaining with a MyoD polyclonal antibody. In contrast, none of the cells infected with antisense MyoD virus showed staining. On withdrawal of serum, 95% of MyoD positive cells became elongated and, in the presence of appropriate cell density, fused to form multinucleated myotubes, morphologically similar to striated muscle cell. Expression of downstream myogenic differentiation markers, myosin heavy chain and myocyte-specific enhancer factor 2, in 95% of these myotubes were detected by intense cytoplasmic and nuclear immunostaining, respectively, with specific antibodies. In contrast, no detectable staining was noted in MyoD negative cells. Spontaneous contractile movements were noted in a few clusters of myotubes. In summary, cardiac fibroblasts were able to be converted into bonafide potentially functional skeletal myocytes as shown by definitive morphologic and biochemical changes. Further studies with in vivo models are needed to explore this unique molecular strategy to treat patients with chronic heart failure. 相似文献
3.
4.
A Fassati A Bardoni M Sironi DJ Wells N Bresolin G Scarlato M Hatanaka S Yamaoka G Dickson 《Canadian Metallurgical Quarterly》1998,9(17):2459-2468
5.
6.
Studies geared towards understanding the interaction between skeletal muscle and biomaterials may provide useful information for the development of various emerging technologies, ranging from novel delivery vehicles for genetically modified cells to fully functional skeletal muscle tissue. To determine the utility of elastomeric materials as substrates for such applications, we asked whether skeletal myogenesis would be supported on a commercially available polyurethane, Tecoflex SG-80A. G8 skeletal myoblasts were cultured on Tecoflex two-dimensional solid thin films fabricated by a spin-casting method. Myoblasts attached, proliferated, displayed migratory activity and differentiated into multinucleated myotubes which expressed myosin heavy chain on solid thin films indicating that Tecoflex SG-80A was permissive for skeletal myogenesis. Porous three-dimensional (3-D) cell scaffolds were fabricated in a variety of shapes, thicknesses, and porosities by an immersion precipitation method, and where subsequently characterized with microscopic and mechanical methods. Mechanical analysis revealed that the constructs were elastomeric, recovering their original length following 100% elongation. The 3-D substrates were seeded with muscle precursors to determine if muscle differentiation could be obtained within the porous network of the fabricated constructs. Following several weeks in culture, histological studies revealed the presence of multinucleated myotubes within the elastomeric material. In addition, immunohistochemical analysis indicated that the myotubes expressed the myosin heavy chain protein suggesting that the myotubes had reached a state of terminal differentiation. Together the results of the study suggest that it is indeed feasible to engineer bioartificial systems consisting of skeletal muscle cultivated on a 3-D elastomeric substrate. 相似文献
7.
8.
9.
10.
11.
12.
D Larocca A Witte W Johnson GF Pierce A Baird 《Canadian Metallurgical Quarterly》1998,9(16):2393-2399
Filamentous bacteriophages represent one of nature's most elegant ways of packaging and delivering DNA. In an effort to develop novel methods for ligand discovery via phage gene delivery, we conferred mammalian cell tropism to filamentous bacteriophages by attaching basic fibroblast growth factor (FGF2), transferrin, or epidermal growth factor (EGF) to their coat proteins and measuring CMV promoter-driven reporter gene expression in target cells. In this system, FGF2 was a more effective targeting agent than transferrin or EGF. The detection of green fluorescent protein (GFP) or beta-galactosidase (beta-Gal) activity in cells required FGF2 targeting and was phage concentration dependent. Specificity of the targeting for high-affinity FGF receptors was demonstrated by competing the targeted phage with FGF2, by the failure of FGF2-targeted bacteriophage to transduce high-affinity FGF receptor-negative cells, and by their ability to transduce these same cells when stably transfected with FGFR1, a high-affinity FGF receptor. Long-term transgene expression was established by selecting colonies for G418 resistance, suggesting that with the appropriate targeted tropism, filamentous bacteriophage can serve as a vehicle for targeted gene delivery to mammalian cells. 相似文献
13.
HB Burch F Shakir TR Fitzsimmons DP Jaques CD Shriver 《Canadian Metallurgical Quarterly》1998,8(10):871-880
Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species. 相似文献
14.
15.
Myotome and sclerotome precursor cells are derived, respectively, from cells in the dorsomedial and ventromedial regions of the somite. To assay changes in the specification of myotomal precursor cells during somite maturation, we implanted dorsomedial quadrant fragments, from staged quail somites, next to the notochords of host chick embryos, and superimposed two additional notochords on these implants. In this notochord signalling environment, dorsomedial quadrant cells that are developmentally plastic are expected to differentiate as cartilage, while cells determined to a myogenic fate are expected to differentiate as skeletal muscle. Large numbers of differentiated chondrocytes developed from dorsomedial quadrant grafts of all stages of paraxial mesoderm development tested, indicating that persistent chondrogenic potential in cells fated to form muscle and dermis can be elicited by notochord signals. Differentiated myocytes, however, appeared in two somite-stage-dependent phases. In the first phase, dorsomedial quadrants from segmental plate and early stage somites (II and IV) form small, disorganized clusters of individual myocytes. The frequency of first-phase myocluster formation increases as myogenic factor expression begins in the dorsomedial quadrant, indicating that myogenic determination assayed by this method is closely linked to the expression of myogenic factors in the dorsomedial quadrant. In the second phase, dorsomedial quadrants from somite stages XI-XIII consistently form morphologically organized muscle tissue containing large numbers of parallel-oriented, multinucleated myotubes. Mitotic labelling demonstrated that muscle precursors were determined to the muscle phenotype prior to withdrawal from the cell cycle. Thus, myogenic determination in cells of the dorsomedial quadrant is acquired at earlier stages of somite maturation than the ability to proliferate and form muscle tissue. These results are consistent with the hypothesis that successive lineages of myotome precursor cells with different mitotic and morphogenetic properties arise in the dorsomedial quadrant during somite maturation. 相似文献
16.
17.
UM Wewer K Iba ME Durkin FC Nielsen F Loechel BJ Gilpin W Kuang E Engvall R Albrechtsen 《Canadian Metallurgical Quarterly》1998,200(2):247-259
Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis. 相似文献
18.
The chloride channel CIC-1 is required to maintain a normal excitability of mature muscle fibers; its blockade leads to hyperexcitability, the hallmark of the disease myotonia. In mouse and rat myotubes, representing the embryonic stage of muscle, CIC-1 mRNA is not detectable by Northern blotting. From neonatal to adult, CIC-1 expression increases at least fourfold. Using RT-PCR and hybridization on cultured myotubes we found CIC-1 mRNA at a level of 0.4-1.1% of that in mature mouse muscle, and < or = 0.01% in myoblasts, at stages when desmin mRNA levels are already high. The level of CIC-1 mRNA is thus a sensitive and specific indicator of the maturation of skeletal muscle cells. 相似文献
19.
20.
MJ Cooper 《Canadian Metallurgical Quarterly》1996,23(1):172-187
Gene therapy provides a significant opportunity to devise novel strategies for the control or cure of cancer. Success of this modality will ultimately depend on the ability to express a therapeutic gene of interest at high levels, and specific gene delivery to targeted tumor cells will minimize toxicities. Although current gene therapy trials typically use viral-based, infectious vectors to express suitable target genes in human cancer cells, these vectors have significant limitations in their expression characteristics, lack of specificity in targeting tumor cells for gene transfer, and safety concerns regarding induction of secondary malignancies and recombination to form replication-competent virus. These limitations have refocused efforts to develop noninfectious gene transfer technologies for in vivo gene delivery of plasmid-based expression vectors. This article reviews recent developments in non-infectious gene transfer techniques, including liposome and receptor-mediated methods, which can efficiently deliver plasmid vectors into tumor cells in vivo. Additionally, strategies are reviewed for efficiently expressing target genes in tumor cells, including use of tissue-specific promoters, inducible promoters, and replication-control sequences to regulate extrachromosomal amplification of vector DNA in human tumor cells. Optimal coupling of these noninfectious gene transfer and expression technologies have the potential to yield safe and effective gene therapies for patients with cancer. 相似文献