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1.
Gen Comp Endocrinol ; 284: 113243, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408625

RESUMO

The suppressor of cytokine signaling 1 (SOCS1) is an essential feedback regulator extensively involved in many different cytokine signaling pathways, such as regulation of the immune system and growth of organism. However, the molecular and functional information on socs1 genes in freshwater fish is unclear. In the present paper, we identified and characterized the full-length closely related but distinct socs1 genes (socs 1a and -1b) in blunt snout bream (Megalobrama amblycephala). The bioinformatic analysis results showed that duplicated socs1s shared majority conserved motifs with other vertebrates. Both socs1a and -1b mRNAs were detected throughout embryogenesis, and gradually increase and then constantly expressed after 16 hpf. Whole-mount in situ hybridization demonstrated that socs1a and socs1b mRNAs were detected in the brain at 12hpf and 24hpf, and in the notochord and brain at 36hpf. In adult fish, the socs1a mRNA were strongly expressed in the heart, eye, kidney, spleen and gonad, but were found to be relatively low in the intestine and liver. On the other hand, the expression of socs1b mRNA was significantly high in the muscle, eye and spleen, and relatively low in the intestine, liver, skin and heart. The results of hGH treatment experiment showed that socs1a and 1b mRNAs were upregulated markedly in the kidney, muscle and liver. Overexpression of socs1s significantly inhibit the GH and JAK/STAT factor stat3 and the inhibitory effect of SOCS1s on GH may be involved in JAK-STAT signaling pathway. These results indicate that SOCS1 plays an important role in regulating growth and development.


Assuntos
Cyprinidae/genética , Duplicação Gênica , Proteína 1 Supressora da Sinalização de Citocina/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Cyprinidae/embriologia , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/metabolismo , Janus Quinases/metabolismo , Modelos Moleculares , Filogenia , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/química , Transcrição Gênica
2.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(2): 200-6, 2011 03.
Artigo em Chinês | MEDLINE | ID: mdl-21488218

RESUMO

OBJECTIVE: To determine the role of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 (HMGB-1) in the pathogenesis of lupus nephritis. METHODS: Serum levels of anti-dsDNA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Renal morphologic features were examined by light microscopy, electron microscopy, and immunohistologic analyses. The mRNA expression of HMGB-1 and monocyte chemoattractant protein-1 (MCP-1) was detected by RT-PCR. RESULT: MRL/lpr mice demonstrated characteristic alterations of serum immune parameters, with progressively increased anti-dsDNA antibodies with age, compared with age-matched C57BL/6J mice. MRL/lpr mice showed progressive development of renal damage, starting at 12 weeks of age and reached the peak at 20 weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. Higher expression of HMGB-1 mRNA was found in MRL/lpr mice than what in C57BL/6J mice. Expression of HMGB-1 was positively correlated with that of MCP-1 mRNA. CONCLUSION: The results demonstrate that the higher expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis.


Assuntos
Proteína HMGB1/metabolismo , Nefrite Lúpica/metabolismo , Animais , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Proteína HMGB1/genética , Rim/metabolismo , Rim/patologia , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , RNA Mensageiro/genética
3.
Yi Chuan ; 32(4): 348-52, 2010 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-20423888

RESUMO

PC-1(Prostate and colon gene 1) gene belongs to TPD52 (Tumor Protein D52) gene family. The expression of PC-1 is found to promote androgen-independent progression. This study was conducted to assess the mechnism of promotion of androgen-independent progression in PC-1 gene. The c-myc gene expression was tested by RT-PCR and Western blotting analyses in the LNCaP-pc-1 and LNCaP-zero cell line. After separation of cytoplasm and nulear proteins of the LNCaP-pc-1 and LNCaP-zero cell line, the beta-catenin protein was detected by Western blotting. C4-2 cell line was used to examine the effects of 10058-F4 on the PC-1 gene expression. The results of RT-PCR and Western blotting indicated that PC-1 enhanced c-myc gene expression in prostate cancer cells, PC-1 was also found to enhance beta-catenin expression in nuclear. Furthermore, a small-molecule c-Myc inhibitor, 10058-F4 represses PC-1 gene expression in C4-2 cell line. Our findings suggest that PC-1 enhances c-myc gene expression in prostate cancer cells through the Wnt/beta-catenin pathway. Meanwhile, c-myc plays a feed-forward role in enhancing PC-1 driven c-myc gene expression, and promotes prostate an-drogen-independent progression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Masculino , Neoplasias da Próstata/genética , beta Catenina/metabolismo
4.
Yi Chuan ; 32(3): 235-41, 2010 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-20233700

RESUMO

Our research intends to obtain extra-cellular proteinogram of cell lines representing different advancement stages of prostate cancer and to test whether screened differential expression proteins can be secreted and used as serum biomarkers for prostate cancer. By examining differential expression spots in two extra-cellular protein 2D-PAGE gels and mass spectrum, candidate molecules were obtained. The expressions of these candidate molecules in eight cell lines and response to androgen stimulus in LNCaP were analyzed by RT-PCR. By constructing eukaryotic expression vectors and western-blotting with anti tags antibodies, the candidate molecules were tested to understand whether they can be expressed in transfected 293T cell culture fluid. Two overexpressed molecules-triosephosphate isomerase 1 (TPI1) and syndecan bind-ing protein, syntenin (ST1)-in extra-cellular proteinogram of C4-2 were screened out; both of them are secretary proteins. On transcriptional level, both proteins were up-regulated with the malignancy of prostate cancer cell lines and ST1 was dose-dependently inhibited by androgen. Considering cellular level results, both TPI1 and ST1 have their potential as serum biomarkers for indicating the developmental stage of prostate cancer.


Assuntos
Neoplasias da Próstata/metabolismo , Sinteninas/metabolismo , Triose-Fosfato Isomerase/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Masculino , Metribolona/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinteninas/genética , Triose-Fosfato Isomerase/genética
5.
Prostate ; 69(11): 1176-87, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19415690

RESUMO

BACKGROUND: Androgen independent prostate cancer (AIPC) is not responsive to androgen ablation therapy. The biomarkers of AIPC are lack. Numerous proteomics studies have focused on finding new markers of AIPC and exploring their possible functions, but little is known about the difference between conditioned medium (CM) from AIPC and androgen dependent prostate cancer (ADPC) cells. METHODS: We performed a proteome analysis of CM from LNCaP, C4-2, and C4-2B cells by a two dimensional electrophoresis based technology. Western blots and immunohistochemical studies were employed to explore the expression pattern of the identified protein in prostate cancer cell lines and clinical specimens, respectively. Then we examined the possible roles and mechanisms of the ubiquitous mitochondrial creatine kinase (uMtCK) in vitro. RESULTS: Besides prostate specific antigen (PSA) and insulin-like growth factor binding protein-2 (IGFBP2), uMtCK was identified in the CM of AIPC cells. uMtCK was up-regulated in AIPC cells and in human prostate cancer tissues at WHO grade III. Stably transfected exogenous uMtCK showed a growth promoting effect rather than mock vector in LNCaP cells, with or without bicalutamide in culture medium. Further assays showed that higher degrees of ROS generation and Akt signaling pathway activation in LNCaP-uMtCK than in LNCaP-neo cells. CONCLUSIONS: We showed that uMtCK could be easily detected in CM of LNCaP lineaged AIPC cells. Exogenous uMtCK in LNCaP cells surprisingly contributed to overproduction of ROS, activation of Akt signaling pathway and more aggressive phenotypes including androgen independence development.


Assuntos
Adenocarcinoma/metabolismo , Androgênios/metabolismo , Creatina Quinase Mitocondrial/metabolismo , Meios de Cultivo Condicionados/metabolismo , Progressão da Doença , Neoplasias da Próstata/metabolismo , Adenocarcinoma/patologia , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Creatina Quinase Mitocondrial/análise , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Dados de Sequência Molecular , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo
6.
Cancer Res ; 67(18): 8906-13, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17875733

RESUMO

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.


Assuntos
Diester Fosfórico Hidrolases/metabolismo , Neoplasias da Próstata/metabolismo , Pirofosfatases/metabolismo , Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Animais , Linhagem Celular Tumoral , DNA Antissenso/genética , Progressão da Doença , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Nitrilas/farmacologia , Proteína Oncogênica v-akt/metabolismo , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Pirofosfatases/biossíntese , Pirofosfatases/genética , Transdução de Sinais , Compostos de Tosil/farmacologia , Transfecção
7.
Oncol Rep ; 40(4): 2408-2416, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066881

RESUMO

Eph receptor tyrosine kinases and their ephrin ligands, mediate an important cell communication system both in normal and oncogenic development, and play central roles in a series of processes including angiogenesis, stem cell maintenance and cancer metastasis. Eph receptor A3 (EphA3), commonly overexpressed in a broad range of cancers, including gastric cancer (GC), is related to tumor progression. Our previous study revealed that EphA3 may play important roles in tumorigenesis and angiogenesis in GC. However, its exact role and the mechanisms underlying its function in GC remain unclear. In the present study, lentivirus­mediated RNA interference was employed to knock down the expression of EphA3 in GC HGC­27 cells. Functional analyses indicated that depletion of EphA3 expression inhibited the cell growth and tumorigenicity of HGC­27 cells in vitro and in vivo. Furthermore, knockdown of the expression of EphA3 in HGC­27 cells inhibited tube formation and migration of HUVEC endothelial cells. Tumor angiogenesis in vivo was also inhibited upon EphA3 knockdown in HGC­27 cells, with reduced microvessel density (MVD) in xenograft models. We further revealed that EphA3 depletion inhibited tumor angiogenesis and migration through the signal transducer and activator of transcription 3/vascular endothelial growth factor (STAT3/VEGF) signaling pathway. These results indicated that EphA3 may be an effective prognostic indicator and a potential target for GC therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neovascularização Patológica/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias Gástricas/irrigação sanguínea , Neoplasias Gástricas/patologia , Animais , Apoptose , Ciclo Celular , Feminino , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/metabolismo , Receptor EphA3 , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 36(4): 412-6, 2007 07.
Artigo em Chinês | MEDLINE | ID: mdl-17717837

RESUMO

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB1) is originally identified as a DNA-binding protein that functions as a structural co-factor. HMGB1 is actively secreted by macrophage/monocytes via inflammatory stimuli. The extracellular HMGB-1 acts as a mediator of acute and chronic systematic inflammation. In this article we briefly review its role in rheumatic diseases: arthritis, polymyositis and dermatomyositis, lupus erythematosus and Sjogren's syndrome. Increased cytoplasmic expression and extracellular deposition of HMGB1 are found in the affected tissues of those diseases, especially stronger in/around focal infiltrates of mononuclear cells. TNFalpha and IL-1beta are co-expressed in areas of extracellular HMGB1. HMGB1 together with TNF alpha and IL-1beta may form a proinflammatory loop promoting the chronic inflammations. The new findings of HMGB1 as a cytokine provide a better understanding of rheumatiod diseases, and could become a clinically relevant therapeutic target that might be more efficient than other known cytokines.


Assuntos
Proteína HMGB1/metabolismo , Polimiosite/metabolismo , Doenças Reumáticas/metabolismo , Humanos , Interleucina-1beta/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
J Zhejiang Univ Sci B ; 7(12): 1006-14, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17111471

RESUMO

Currently there is considerable interest among oncologists to find anticancer drugs in Chinese herbal medicine (CHM). In the past, clinical data showed that some herbs possessed anticancer properties, but western scientists have doubted the scientific validity of CHM due to the lack of scientific evidence from their perspective. Recently there have been encouraging results, from a western perspective, in the cancer research field regarding the anticancer effects of CHM. Experiments showed that CHM played its anticancer role by inducing apoptosis and differentiation, enhancing the immune system, inhibiting angiogenesis, reversing multidrug resistance (MDR), etc. Clinical trials demonstrated that CHM could improve survival, increase tumor response, improve quality of life, or reduce chemotherapy toxicity, although much remained to be determined regarding the objective effects of CHM in human in the context of clinical trials. Interestingly, both laboratory experiments and clinical trials have demonstrated that when combined with chemotherapy, CHM could raise the efficacy level and lower toxic reactions. These facts raised the feasibility of the combination of herbal medicines and chemotherapy, although much remained to be investigated in this area.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos como Assunto , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos
10.
Yi Chuan ; 28(1): 71-7, 2006 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-16469720

RESUMO

Genes lacZ, lacY and lacA in the lac opron of E. coli chromosome were respectively substituted with gene luc by using plasmid pBR322-Red, selection-counterselection system kan/sacB and various strategies of Red homologous recombination including Red mediated linearized double-stranded DNA homologous recombination and Red mediated recombineering with overlapping single stranded DNA oligonucleotides. Then, a series of new strains, CWL2, CWL4 and CWL6, were constructed and we found that they can express protein Luc efficiently. To further study the expression of exogenous genes at the site of lacZ, we have constructed a strain named CWD1 by knockin the cholera toxin B subunit(ctxb) gene at the lacZ site, then we found that CWD1 can express protein CTB efficiently and CTB was secreted out of the cell. So we assured that the sites of structure genes in the lac operon of Escherichia coli chromosome were suitable for expressing foreign genes.


Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Técnicas de Introdução de Genes/métodos , Plasmídeos/genética , Toxina da Cólera/genética , Óperon Lac/genética
11.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 28(2): 230-3, 2006 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-16733910

RESUMO

OBJECTIVE: To explore the effect of polymorphism in codon Ala54Thr of human intestinal fatty acid-binding protein gene (IFABP) on the therapeutic efficacy of fenofibrate. METHODS: Totally 147 patients with hyperlipidemia [72 men mean age: (56.2 +/- 8.63) years; 75 women mean age: (58.4 +/- 9.12) years] were enrolled. IFABP genotypes were detected by polymerase chain reaction, Hha I digestion, and sequencing. Four weeks before and after treatment, the levels of fasting serum total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A I (apoA I) and apolipoprotein B (apoB) were detected with biochemical techniques. RESULTS: The frequency of IFABP genotype was 0.47 for A/A, 0.37 for A/T, and 0.16 for T/T, and the allelic frequency was 0.65 for A and 0.35 for T. No significant different was found in lipid levels in every genotype before treatment (P > 0.05). After 4 weeks of treatment, the levels of TC, TG, LDL-C, and apoB significantly decreased (P < 00.01), and the levels of HDH-C and apoA I significantly increased (P < 0.01). The total therapeutic efficacy on A54A and A54T were 97% and 95%, respectively. In the patients with T54T genotype after treatment, no significant difference in lipids levels was found except TG (P < 0.05), and the total efficacy was only 38%. The total therapeutic efficacies of fenofibrate on A54A and A54T were higher than those of T54T, and there was significant different between A54A and T54T (P < 0.01). CONCLUSION: The polymorphism of human IFABP gene in hyperlipidemia is related with the therapeutic efficacy of fenofibrate, and the T54T IFABP genotype may have strong negative effect on such efficacy.


Assuntos
Proteínas de Ligação a Ácido Graxo/genética , Fenofibrato/uso terapêutico , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/genética , Hipolipemiantes/uso terapêutico , Polimorfismo Genético , Idoso , Apolipoproteínas/sangue , Feminino , Frequência do Gene , Genótipo , Humanos , Hiperlipidemias/sangue , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
12.
Yi Chuan Xue Bao ; 32(5): 533-7, 2005 May.
Artigo em Chinês | MEDLINE | ID: mdl-16018266

RESUMO

Using lambda phage Red recombinase mediated in vivo homologous recombination system, a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique, and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria. To confirm the recombination functions of pBR322-Red, a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T-->G mutation in galK gene on the bacterial chromosome. The result demonstrated that a new lambda Red-mediated recombineering system based on pBR322-Red was successfully established.


Assuntos
Reparo do DNA/genética , Recombinases/genética , Recombinação Genética , Bacteriófago lambda/enzimologia , Bacteriófago lambda/genética , DNA Recombinante/genética , DNA de Cadeia Simples/genética , Eletroporação , Escherichia coli/genética , Galactoquinase/genética , Galactoquinase/metabolismo , Engenharia Genética/métodos , Óperon , Plasmídeos , Mutação Puntual , Recombinases/metabolismo
13.
Zhonghua Bing Li Xue Za Zhi ; 34(1): 42-6, 2005 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-15796881

RESUMO

OBJECTIVE: To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. METHODS: Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression. RESULTS: NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice. CONCLUSION: Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.


Assuntos
Transformação Celular Neoplásica , Genes Neoplásicos/fisiologia , Proteínas de Neoplasias/biossíntese , Animais , Linhagem Celular Transformada , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Transfecção
14.
Zhonghua Nan Ke Xue ; 11(4): 256-60, 2005 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-15921253

RESUMO

OBJECTIVE: To study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2. METHODS: Recombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells. RESULTS: PC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells. CONCLUSION: RNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.


Assuntos
Diester Fosfórico Hidrolases/biossíntese , Neoplasias da Próstata/genética , Pirofosfatases/biossíntese , Interferência de RNA , Linhagem Celular Tumoral , Regulação para Baixo , Expressão Gênica , Humanos , Masculino , Diester Fosfórico Hidrolases/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Pirofosfatases/genética , RNA Mensageiro/genética , Transfecção
15.
Yi Chuan Xue Bao ; 30(10): 983-8, 2003 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-14669518

RESUMO

Driven by the need of functional genomics, a homologous recombination-based, highly efficient genetic engineering system that termed "recombineering" has recently been developed. Recombineering has been defined as a genetic engineering with phage-encoded recombination function that utilizes short homologies, a convenient term to describe homologous-dependent, recombination-mediated, genetic engineering. The bacteriophage lambda Red recombination system has critical differences from standard E. coli RecA-dependent recombination pathway. The phage systems have unique advantage in that they can catalyze efficient recombination with very short regions of sequence homology (< 50 bp). Recombineering does not require construction of plasmid or phage DNA intermediates containing the appropriately pre-engineered homology segment. All that is required in vitro is the synthesis of standard oligonucleotides or construction of PCR products that provide the homology. Importantly, they function even in the absence of RecA. These approaches do not rely on the presence of suitable restriction site, and can be used to insert, delete, clone or substitute genomic DNA sequences at any desired position on a target molecule in Escherichia Coli. Recombineering also facilitates many kinds of genomic experiments difficult to be carried out. In this article, the bacteriophage lambda Red recombinase system, the progression and applications of this powerful new technique are reviewed according to the data published recently.


Assuntos
Engenharia Genética/métodos , Recombinação Genética , Bacteriófago lambda/genética , DNA Recombinante/genética , Escherichia coli/genética , Modelos Genéticos
16.
Yi Chuan Xue Bao ; 29(11): 983-9, 2002.
Artigo em Chinês | MEDLINE | ID: mdl-12645261

RESUMO

A system used for detecting the transcriptional activating activity of the function-unknown gene products in mammalian cells was developed. Based on the plasmid pTet-Off and the eukaryotic expressing vector pCDNA3.1B(-)/myc-his, firstly, we constructed a set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negative control), pZHO2 (as a positive control). The system also includes the plasmids pTRE-luc (encoding the Firefly luciferase reporter gene) and pRL-TK (encoding Renilla luciferase gene as background control). To confirm the feasibility of the system, the plasmids pZHO1, pZHO2 and pZHO3 (encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was contransfected into such mammalian cells as C4-2, MCF-7, COS7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibility of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.


Assuntos
Plasmídeos/genética , Proteínas/genética , Ativação Transcricional/genética , Animais , Sítios de Ligação/genética , Células COS , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
17.
Yi Chuan ; 26(5): 739-44, 2004 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-15640095

RESUMO

Prostate-specific antigen (PSA) as a prostate cancer marker is a chymotrypsin-like serine protease which is expressed primarily by both normal prostate epithelium and the vast majority of prostate cancers. PSA expression is tightly regulated by androgen through the activation of androgen receptor. However, in the absence of androgens, PSA gene expression can become elevated. This suggests that either the AR can be activated in the absence of androgen to elevate PSA gene expression or that another transcription factor acting on the PSA promoter is stimulated. This article reviews the research on the structure of the PSA promoter and enhancer and the mechanisms of the PSA expression.


Assuntos
Androgênios/fisiologia , Regiões Promotoras Genéticas/fisiologia , Antígeno Prostático Específico/genética , Neoplasias da Próstata/metabolismo , Animais , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Interleucina-6/farmacologia , Masculino , Metribolona/farmacologia , Antígeno Prostático Específico/biossíntese , RNA Mensageiro/biossíntese , Receptores Androgênicos/fisiologia
18.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 33(4): 281-6, 2004 07.
Artigo em Chinês | MEDLINE | ID: mdl-15269975

RESUMO

OBJECTIVE: To observe the skin regeneration after hair follicle bulb cells were implanted into collagen/chitosan porous scaffolds in vitro. METHODS: The cultured dorsal hair follicle bulb cells of 4d-old C57BL/6J mice were implanted into collagen/chitosan porous scaffolds in vitro. The skin regeneration was observed. RESULT: The skin-like structure was formed on the collagen/chitosan porous scaffolds where were cultured the hair follicle bulb cells before 4th passages. CONCLUSION: The skin-like structure is generated in vitro when early passages of cultured hair bulb cells are implanted into collagen/chitosan porous scaffolds.


Assuntos
Quitina/análogos & derivados , Folículo Piloso/citologia , Pele/citologia , Engenharia Tecidual , Animais , Quitosana , Colágeno , Camundongos , Camundongos Endogâmicos C57BL , Regeneração
19.
Space Med Med Eng (Beijing) ; 16(3): 175-8, 2003 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-12934610

RESUMO

OBJECTIVE: To study the effects of rapid eye movement sleep (REMS) deprivation on the expression of mRNA coding for neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) of hypothalamus in rats. METHOD: Flower pot technique was adopted to deprive the REMS of Sprague-Dawley rats for 24 h, 48 h and 72 h respectively. The expression of mRNA coding for nNOS or iNOS of hypothalamus in rats was assayed by reverse-transcription polymerase chain reaction (RT-PCR). RESULT: The amount of nNOS mRNA was significantly higher in 24 h REMS deprivation group (P<0.01), then the amount was lowered in 48 h deprivation group and became significantly lower in 72 h deprivation group than that in the control group (P<0.05). There was low expression of iNOS mRNA of hypothalamus in rats, and there was no difference in the expression of iNOS mRNA among 24 h, 48 h REMS deprivation and the control groups. But the expression was significantly increased in 72 h REMS deprivation group (P<0.01). CONCLUSION: Deprivation of REMS increased the expression of nNOS and iNOS mRNA of hypothalamus. Excessive nitric oxide (NO) might be a major factor resulting in not only the sleep rebound phenomenon but also the injury of human function caused by sleep loss directly or indirectly by other sleep factors.


Assuntos
Expressão Gênica , Hipotálamo/enzimologia , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/metabolismo , Privação do Sono/enzimologia , Animais , Hipotálamo/fisiologia , Óxido Nítrico Sintase/genética , Ratos , Ratos Sprague-Dawley , Privação do Sono/genética , Privação do Sono/metabolismo , Sono REM/genética , Sono REM/fisiologia , Fatores de Tempo
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