RESUMO
Caspases (cysteine-containing aspartate-specific proteases) are at the core of the cell's suicide machinery. These enzymes, once activated, dismantle the cell by selectively cleaving key proteins after aspartate residues. The events culminating in caspase activation are the subject of intense study because of their role in cancer, and neurodegenerative and autoimmune disorders. Here we present a mechanistic mathematical model, formulated on the basis of newly emerging information, describing key elements of receptor-mediated and stress-induced caspase activation. We have used mass-conservation principles in conjunction with kinetic rate laws to formulate ordinary differential equations that describe the temporal evolution of caspase activation. Qualitative strategies for the prevention of caspase activation are simulated and compared with experimental data. We show that model predictions are consistent with available information. Thus, the model could aid in better understanding caspase activation and identifying therapeutic approaches promoting or retarding apoptotic cell death.
Assuntos
Apoptose , Proteínas de Arabidopsis , Caspases/fisiologia , Modelos Teóricos , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Simulação por Computador , Ativação Enzimática , Ácidos Graxos Dessaturases/metabolismo , Genes p53/genética , Humanos , Cinética , Modelos Biológicos , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X , Receptor fas/metabolismoRESUMO
The eukaryotic cell cycle is regulated by a complex network of many proteins. Effective reprogramming of this complex regulatory apparatus to achieve bioprocess goals, such as cessation of proliferation at high cell density to allow an extended period of high production, can require coordinated manipulation of multiple genes. Previous efforts to establish inducible cell-cycle arrest of Chinese hamster ovary (CHO) cells by regulated expression of the cyclin-dependent kinase inhibitor (CDI) p21 failed. By tetracycline-regulated coexpression of p21 and the differentiation factor CCAAT/enhancer-binding protein alpha (which both stabilizes and induces p21), we have achieved effective cell-cycle arrest. Production of a model heterologous protein (secreted alkaline phosphatase; SEAP) has been increased 10-15 times, on a per cell basis, relative to an isogenic control cell line. Because activation of apoptosis response is a possible complication in a proliferation-arrested culture, the survival gene bcl-xL was coexpressed with another CDI, p27, found to enable CHO cell-cycle arrest predominantly in G1 phase. CHO cells stably transfected with a tricistronic construct containing the genes for these proteins and for SEAP showed 30-fold higher SEAP expression than controls.
Assuntos
Fosfatase Alcalina/biossíntese , Células CHO/citologia , Ciclo Celular/genética , Proteína Oncogênica p21(ras)/biossíntese , Inibidores da Síntese de Proteínas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Células CHO/metabolismo , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Cricetinae , Genes bcl-2/efeitos dos fármacos , Genes bcl-2/genética , Engenharia Genética , Proteína Oncogênica p21(ras)/genética , Tetraciclina/farmacologiaRESUMO
Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.
Assuntos
Antibacterianos/metabolismo , Regulação da Expressão Gênica , Técnicas Genéticas , Virginiamicina/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Eritropoetina/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Músculos/citologia , Músculos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Streptomyces/genética , Fatores de Tempo , Transfecção , Virginiamicina/farmacologiaRESUMO
Controlling gene activity in space and time represents a cornerstone technology in gene and cell therapeutic applications, bioengineering, drug discovery as well as fundamental and applied research. This chapter provides a comprehensive overview of the different approaches for regulating gene activity and product protein formation at different biosynthetic levels, from genomic rearrangements over transcription and translation control to strategies for engineering inducible secretion and protein activity with a focus on the development during the past 2 years. Recent advances in designing second-generation gene switches, based on novel inducer administration routes (gas phase) as well as on the combination of heterologous switches with endogenous signals, will be complemented by an overview of the emerging field of mammalian synthetic biology, which enables the design of complex synthetic and semisynthetic gene networks. This article will conclude with an overview of how the different gene switches have been applied in gene therapy studies, bioengineering and drug discovery.
Assuntos
Regulação da Expressão Gênica/genética , Genes de Troca/genética , Animais , Redes Reguladoras de Genes/genética , Genoma/genética , Transcrição Gênica/genéticaRESUMO
We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27(Kip1) in sense (p27(Kip1)S) and antisense (p27(Kip1)AS) orientation. Exclusive expression of p27(Kip1)S resulted in complete G(1)-phase-specific growth arrest, while expression of only p27(Kip1)AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27(Kip1) expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27(Kip1) was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27(Kip1) failed to provide the expected G(1)-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.
Assuntos
Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , RNA Antissenso/genética , Tetraciclina/farmacologia , Proteínas Supressoras de Tumor , Virginiamicina/farmacologia , Animais , Western Blotting , Células CHO , Cricetinae , Inibidor de Quinase Dependente de Ciclina p27 , Citometria de Fluxo , Imunofluorescência , Vetores Genéticos/genética , Humanos , Cinética , Proteínas Associadas aos Microtúbulos/biossíntese , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Genetically modified mammalian cells are the preferred system for the production of recombinant therapeutic glycoproteins. Other applications include engineering of cell lines for drug screening and cell-based therapies, and the construction of recombinant viruses for gene therapy. This article highlights contemporary core genetic technologies and emerging strategies for genetically engineering mammalian cells for optimal recombinant-protein expression.
Assuntos
Glicoproteínas/biossíntese , Biologia Molecular/métodos , Engenharia de Proteínas/métodos , Apoptose/genética , Regulação da Expressão Gênica , Genes/genética , Glicosilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Recombinação Genética , Transcrição GênicaRESUMO
The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.
Assuntos
Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos , Piruvato Quinase/genética , Zymomonas/genética , Regiões 5' não Traduzidas/genética , Regulação Alostérica , Sequência de Bases , Clonagem Molecular , Escherichia coli/enzimologia , Dados de Sequência Molecular , Piruvato Quinase/biossíntese , Piruvato Quinase/metabolismo , Análise de Sequência de DNA , Zymomonas/enzimologiaRESUMO
In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.
Assuntos
Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Transformação Bacteriana , Variação Antigênica , Proteínas da Membrana Bacteriana Externa/metabolismo , Evolução Biológica , DNA Bacteriano/metabolismo , Proteínas de Fímbrias , Fímbrias Bacterianas/imunologia , Humanos , Modelos Genéticos , Neisseria gonorrhoeae/imunologiaRESUMO
We previously identified and genetically characterized several factors essential for the natural competence of transformation in Neisseria gonorrhoeae. Here we analyse the sequential action of these factors and dissect the overall transformation process into three distinct steps, (i) the sequence-specific uptake of transforming DNA into a DNase-resistant state, (ii) the transfer of DNA to the cytosol and (iii) the processing and recombination of the incoming with the resident DNA. While two pilus-associated factors, PilE and PilC, were previously implicated in the early DNA uptake event, we show here that three competence factors unrelated to pilus biogenesis, ComA, ComL and Tpc, are not essential for DNA uptake and rather act in a subsequent step. The respective mutants, however, lack the characteristic nucleolytic processing observed with the incoming DNA in both wild-type and non-transformable RecA-deficient N. gonorrhoeae, indicating that they are blocked in the processing and/or the delivery of DNA to the cytoplasm. A hypothetical model proposing a sequential action of the known gonococcal competence factors is presented.
Assuntos
Neisseria gonorrhoeae/genética , Transformação Genética , Sequência de Bases , Transporte Biológico Ativo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Genes Bacterianos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Neisseria gonorrhoeae/metabolismoRESUMO
Regulation of heterologous gene expression is of prime importance for a wide variety of basic and applied biological research areas including functional genomics, tissue engineering, gene therapy, and biopharmaceutical manufacturing. Initial gene regulation strategies employed endogenous responsive elements, which resulted in pleiotropic interference of transgene expression with host regulatory networks. Current regulation systems are binary and consist of chimeric transactivators and responsive target promoters of heterologous bacterial or insect origin, or they contain artificially designed components. Regulation of generic systems is based on binding of a transactivator to its cognate promoter, which is modulated by specific molecules such as antibiotics or hormones and brings the transactivation domain into contact with a minimal promoter, thereby inducing target gene expression. Binary gene regulation concepts have been significantly refined in recent years with a focus to improve their regulation performance and their compatibility with human-therapeutic use. In this review we present a detailed analysis of currently available mammalian gene regulation systems and document progress that has pioneered the use of such systems in various aspects of human therapy.
Assuntos
Regulação da Expressão Gênica , Terapia Genética , Genoma , Metabolismo , Animais , Humanos , MamíferosRESUMO
Regulation of the cell cycle and of programmed cell death (apoptosis) is essential for mammalian development and homeostasis. Furthermore, this regulation is fundamental to successful cell culture technology and tissue engineering. Therefore the molecular networks which regulate these processes are critical targets for drug development, gene therapy, and metabolic engineering. This review summarizes the genes, proteins, and interactions presently known to control apoptosis and cell-cycle progression. Knowledge of the networks summarized here and access to the component genes and proteins have already been applied successfully to guide research and development in bioprocess technology and medical treatment.
Assuntos
Apoptose , Biotecnologia , Ciclo Celular , Animais , Linhagem Celular , Engenharia Genética/métodos , Humanos , MamíferosRESUMO
We constructed stable Chinese hamster ovary (CHO) cell lines which conditionally and coordinately express the model product gene secreted alkaline phosphatase (SEAP) and one of the cytostatic genes p21, p27, and p53175P, a p53 mutant deficient in apoptotic but not cell-cycle arrest function. The use of dicistronic expression technology allowed the conditional expression of the model product gene and the cytostatic gene in a coordinated fashion from a single expression unit under the control of the tetracycline-responsive promoter PhCMV-1. Due to the presence of a cytostatic gene in the multicistronic expression unit, the growth behavior of the engineered CHO cell lines could be controlled by the addition or withdrawal of the exogenous agent tetracycline to or from the cell culture medium. Withdrawal of tetracycline resulted in sustained growth arrest of the stable cell lines for a prolonged period. The growth arrest of such cell lines was found to be accompanied by a 10-15-fold increase in their production of SEAP per cell. This controlled proliferation technology allows the design of a novel two-stage production process which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain growth-arrested and increase cell-specific production of a heterologous protein.
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Supressoras de Tumor , Fosfatase Alcalina/genética , Animais , Biotecnologia , Células CHO , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Cricetinae , Inibidor de Quinase Dependente de Ciclina p27 , Inibidores Enzimáticos/metabolismo , Expressão Gênica , Genes , Engenharia Genética , Vetores Genéticos , Proteínas Associadas aos Microtúbulos/biossíntese , Regiões Promotoras Genéticas , Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Tetraciclina/farmacologia , TransfecçãoRESUMO
Regulated expression of a cloned gene often provides much higher final expression of the gene product. Also, regulated expression of an activity can enable optional metabolic engineering and simplify functional genomic research. We constructed di-, tri-, and quattrocistronic mammalian expression vectors which allow the simultaneous, coordinated, and adjustable expression of up to two product genes. A single, tetracycline-regulatable promoter, PhCMV*-1, drives high-level expression of a multicistronic expression unit, containing the product gene(s), the gene for tetracycline-responsive transactivator (tTA), and, in the case of pQuattro-tTA, also the neomycin resistance gene. This autoregulatory genetic configuration retains a very low basal transcription activity in the presence of tetracycline, thereby reducing or eliminating possible toxic effects of tTA expression. However, upon withdrawal of tetracycline, a positive feedback regulation loop is activated which leads to higher levels of tTA expression and consequently also to higher expression levels of all other cistrons encoded on the multicistronic expression unit. Since such multicistronic expression vectors combine all genetic elements necessary for high-level expression as well as regulation in a single multicistronic expression unit, they alleviate limitations of previously reported tetracycline-regulatable vector systems and allow straightforward, one-step genetic engineering of eucaryotic cells to give an adjustable phenotype under strict control of the external stimulus, here tetracycline. Because the expression vectors described here were used for the expression for several heterologous product genes such as the green fluorescent protein and the tumor suppressor gene p21 in several cell lines (CHO-K1, BHK-21, and HeLa), we expect these multicistronic, positive feedback regulation vectors to function in a wide variety of eucaryotic cells and to be useful for basic as well as for applied research applications. Other vectors based upon the same autoregulation and multicistronic expression concepts can be constructed using other regulator gene-regulated promoter elements.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica , Genes , Vetores Genéticos , Animais , Células CHO , Linhagem Celular , Cricetinae , Retroalimentação , Citometria de Fluxo , Imunofluorescência , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Rim , Proteínas Luminescentes/genética , Mapeamento por Restrição , Tetraciclina/farmacologia , Transativadores/genética , TransfecçãoRESUMO
We have previously shown that Chinese hamster ovary (CHO) cells capable of growing in medium free of exogenous proteins die by apoptosis during all stages of a batch culture (Zanghi et al., 1999). On the basis of the hypothesis that extracellular death factors might be important in apoptosis under these conditions, we examined the effect of the growth factor inhibitor and antitumor agent suramin on CHO cell growth and apoptosis in serum-free culture. Suramin protected against apoptosis during exponential growth, as indicated by the absence of DNA laddering and an increase in cell viability from roughly 70% to above 95%. Suramin also effectively dispersed cell aggregates so that single-cell suspension culture was possible. However, suramin did not protect against apoptosis during the death phase, in contrast to serum, suggesting that antiapoptotic factors in the serum remain to be discovered. The increased viable cell yield following suramin supplementation resulted in a 40% increase in product yield, based on results with cells expressing recombinant secreted alkaline phosphatase. Polysulfated compounds dextran sulfate and polyvinyl sulfate worked nearly as well as suramin in dispersing cell clumps and increasing viable cell yield, which implies that suramin's high sulfate group density may be responsible for its effects in cell culture. In addition, suramin was beneficial for long-term adaptation of CHO cells to protein-free media suspension culture, and the compound was synergistic with insulin in accelerating this adaptation time.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Suramina/farmacologia , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Meios de Cultura Livres de Soro , Ciclina E/metabolismo , Sulfato de Dextrana/farmacologia , Polivinil/farmacologiaRESUMO
We present an update on the pTRIDENT multicistronic mammalian expression vectors and their implications in various metabolic engineering and therapeutic applications. The pTRIDENT vector family has been expanded by construction of a new set of pTRIDENT-based vectors containing constitutive promoters of human origin (ubiquitin C and EF-1alpha promoters) and selectable markers (zeocin resistance) and expressing different reporter genes (secreted alkaline phosphatase (SEAP) and the secreted single-chain urokinase-type plasminogen activator (low-M(r) u-PA)). In addition, we have constructed pTRIDENT derivatives with novel streptogramin-repressible and streptogramin-inducible promoters for simultaneous and adjustable expression of three different transgenes. Streptogramin-inducible and tetracycline-repressible pTRIDENT derivatives were used to simultaneously control expression of three fluorescent proteins in mammalian cells: the enhanced cyan fluorescent protein (CFP), the recently isolated red fluorescent protein (RFP, also designated dsRed), and the enhanced yellow fluorescent protein (YFP). Owing to their modular structure, the pTRIDENT vector family represents a construction kit for the design of novel multicistronic expression constructs.
Assuntos
Vetores Genéticos , Regiões Promotoras Genéticas , Virginiamicina/farmacologia , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genéticaRESUMO
Screening and development of new antibiotic activities to counteract the increasing prevalence of multidrug-resistant (MDR) human pathogenic bacteria has once again become a priority in human chemotherapy. Here we describe a novel mammalian cell culture-based screening platform for the detection of streptogramin antibiotics. Quinupristin-dalfopristin (Synercid), a synthetically modified streptogramin, is presently the sole effective agent in the treatment of some MDR nosocomial infections. A Streptomyces coelicolor transcriptional regulator (Pip) has been adapted to modulate reporter gene expression (SEAP, secreted alkaline phosphatase) in Chinese hamster ovary cells (CHO) in response to streptogramin antibiotics. This CHO cell-based technology was more sensitive in detecting the production of the model streptogramin pristinamycin, from Streptomyces pristinaespiralis, than antibiogram tests using a variety of human pathogenic bacteria as indicator strains. The reporter system was able to detect pristinamycin compound produced by a single S. pristinaespiralis colony. The assay was rapid (17 hours) and could be carried out in a high-throughput 96-well plate assay format or a 24-well transwell set-up. This novel mammalian cell-based antibiotic screening concept enables detection of bioavailable and non-cytotoxic representatives of a particular class of antibiotics in a single assay and represents a promising alternative to traditional antibiogram-based screening programs.
Assuntos
Antibacterianos/análise , Proteínas de Bactérias , Avaliação Pré-Clínica de Medicamentos/métodos , Virginiamicina/análise , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/patogenicidade , Disponibilidade Biológica , Células CHO , Cricetinae , Cricetulus , Resistência Microbiana a Medicamentos , Quimioterapia Combinada , Humanos , Peptídeo Sintases/efeitos dos fármacos , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/metabolismo , Virginiamicina/farmacologiaRESUMO
Experimental and clinical studies investigated whether urinary incontinence can be effectively treated with transurethral ultrasound-guided injections of autologous myoblasts and fibroblasts.This new therapy was performed in eight female pigs. It could be shown that the injected cells survived well and that new muscle tissue was formed. Next, 42 patients (29 women, 13 men) suffering from urinary stress incontinence were treated. The fibroblasts were mixed with a small amount of collagen as carrier material and injected into the urethral submucosa to treat atrophies of the mucosa. The myoblasts were directly injected into the rhabdosphincter to reconstruct the muscle and to heal morphological and functional defects. In 35 patients urinary incontinence could be completely cured. In seven patients who had undergone multiple surgical procedures and radiotherapy urinary incontinence improved. No side effects or complications were encountered postoperatively. The experimental as well as the clinical data clearly demonstrate that urinary incontinence can be treated effectively with autologous stem cells. The present data support the conclusion that this new therapeutic concept may represent a very promising treatment modality in the future.
Assuntos
Técnicas de Cultura de Células/métodos , Fibroblastos/transplante , Mioblastos/transplante , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Incontinência Urinária/diagnóstico , Incontinência Urinária/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Fibroblastos/patologia , Rejeição de Enxerto/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mioblastos/patologia , Transplante de Células-Tronco/efeitos adversos , Engenharia Tecidual/efeitos adversos , Resultado do TratamentoRESUMO
In the last years preclinical studies have paved the way for the use of adult muscle derived stem cells for reconstruction of the lower urinary tract. Between September 2002 and October 2004, 42 women and 21 men suffering from urinary stress incontinence (age 36-84 years) were recruited and subsequently treated with transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. In parallel, 7 men and 21 women (age 39-83 years) also diagnosed with urinary stress incontinence were treated with standard transurethral endoscopic injections of collagen. Patients were randomly assigned to both groups. After a follow-up of 12 months incontinence was cured in 39 women and 11 men after injection of autologous myoblasts and fibroblasts. Mean quality of life score (51.38 preoperatively, 104.06 postoperatively), thickness of urethra and rhabdosphincter (2.103 mm preoperatively, 3.303 mm postoperatively) as well as contractility of the rhabdosphincter (0.56 mm preoperatively, 1.462 mm postoperatively) were improved postoperatively. Only in two patients treated with injections of collagen incontinence was cured. The present clinical results demonstrate that, in contrast to injections of collagen, urinary incontinence can be treated effectively with ultrasonography-guided injections of autologous myo- and fibroblasts.
Assuntos
Materiais Biocompatíveis/administração & dosagem , Colágeno/administração & dosagem , Endossonografia/métodos , Implantação de Prótese/métodos , Transplante de Células-Tronco/métodos , Incontinência Urinária/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas/transplante , Cistoscopia , Feminino , Fibroblastos/citologia , Fibroblastos/transplante , Seguimentos , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Mioblastos/citologia , Mioblastos/transplante , Próteses e Implantes , Estudos Retrospectivos , Transplante Autólogo , Resultado do Tratamento , Uretra , Bexiga Urinária , Incontinência Urinária/diagnóstico por imagemRESUMO
Recordings have been performed with a CMOS-based microelectrode array (MEA) featuring 11'016 metal electrodes and 126 channels, each of which comprises recording and stimulation electronics for extracellular, bidirectional communication with electrogenic cells. The important features of the device include (i) high spatial resolution at (sub) cellular level with 3'200 electrodes per mm(2) (diameter 7 microm, pitch 18 microm), (ii) a reconfigurable routing of the electrodes to the 126 channels, and (iii) low noise levels. Recordings from neonatal rat cardiomyocytes forming confluent layers and microtissues are shown. Moreover, signals from dissociated rat hippocampal neurons and from neurons in an acute cerebellar slice preparation are presented.
Assuntos
Dispositivos Lab-On-A-Chip , Miócitos Cardíacos/fisiologia , Neurônios/fisiologia , Animais , Galinhas , Eletrodos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Procedimentos Analíticos em Microchip/métodos , Microeletrodos , Ratos , Ratos Long-EvansRESUMO
Cartilage replacement is a challenging issue in reconstructive surgery. In the past few years, tissue engineering has been tested as a means of cartilage reconstruction. Tissue engineering of cartilage depends on the use of adequate polymers. In addition to several natural and synthetic polymers, fibrin gel has been tested for cartilage reconstruction. However, fibrin is intrinsically unstable. The purpose of this study was to stabilize fibrin by increased fibrinolytic inhibition and to test these preparations for cartilage reconstruction with human nasal septum chondrocytes. Increased fibrinolytic inhibition was achieved with aprotinin and tranexamic acid. Stabilized fibrin-chondrocyte constructs were cultivated for 4 weeks in vitro and compared with constructs made of standard, commercially available fibrin gel. The effect of several cell densities on stability, and the production of extracellular matrix components, were assessed on the basis of histology and immunohistochemistry. In contrast to constructs made of standard fibrin gel, stabilized constructs were stable for the entire observation period and demonstrated no or only minor shrinkage. Cells in these constructs appeared to be viable, and an extracellular matrix could be demonstrated in all constructs. The authors conclude that fibrin-chondrocyte constructs stabilized by increased fibrinolytic inhibition could be an adequate tool for cartilage reconstruction.