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1.
Nanoscale ; 15(28): 11777-11800, 2023 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-37404024

RESUMO

Transition metal nitrides (TMNs) have become excellent substitutes for precious metals such as Pt and Ir in the field of electrocatalysis because of their excellent electrocatalytic performance, high conductivity, good corrosion resistance and stability. As we all know, the commonly utilized carbon-based materials corrode easily during electrocatalysis, which will lead to catalyst falling off and agglomeration. Compared with carbon-based materials, TMNs have stronger corrosion resistance and higher stability. In the metal nitrides, a variety of chemical bonds (metal bond, ionic bond and covalent bond) coexist, among which the ionic bond between metal atoms and nitrogen atoms can make the d-band shrink and narrow, which leads to TMNs having characteristics similar to precious metals in the electrocatalytic process; thus, they can be used as a substitute for precious metal catalysts. In this paper, the synthesis method and catalytic principle of transition metal nitrides and their applications in the fields of hydrogen evolution reaction (HER), oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) are discussed, and the shortcomings of TMNs as a catalyst, the challenges faced in catalyst research and the developments and prospects for the future are pointed out.

2.
J Zhejiang Univ Sci B ; 21(10): 767-778, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33043643

RESUMO

RNA helicases, the largest family of proteins that participate in RNA metabolism, stabilize the intracellular environment through various processes, such as translation and pre-RNA splicing. These proteins are also involved in some diseases, such as cancers and viral diseases. Autophagy, a self-digestive and cytoprotective trafficking process in which superfluous organelles and cellular garbage are degraded to stabilize the internal environment or maintain basic cellular survival, is associated with human diseases. Interestingly, similar to autophagy, RNA helicases play important roles in maintaining cellular homeostasis and are related to many types of diseases. According to recent studies, RNA helicases are closely related to autophagy, participate in regulating autophagy, or serve as a bridge between autophagy and other cellular activities that widely regulate some pathophysiological processes or the development and progression of diseases. Here, we summarize the most recent studies to understand how RNA helicases function as regulatory proteins and determine their association with autophagy in various diseases.


Assuntos
Autofagia , Carcinogênese , Sistema Imunitário/fisiologia , Neoplasias/metabolismo , RNA Helicases/metabolismo , Animais , Antivirais/farmacologia , Proteína Beclina-1/metabolismo , Sobrevivência Celular , Proteína DEAD-box 58/metabolismo , Progressão da Doença , Regulação da Expressão Gênica , Homeostase , Humanos , Splicing de RNA , Receptores Imunológicos/metabolismo
3.
World J Gastroenterol ; 22(36): 8161-7, 2016 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-27688657

RESUMO

A growing body of epidemiologic research has demonstrated that metabolic derangement exists in patients with hepatitis B virus (HBV) infection, indicating that there are clinical associations between HBV infection and host metabolism. In order to understand the complex interplay between HBV and hepatic metabolism in greater depth, we systematically reviewed these alterations in different metabolic signaling pathways due to HBV infection. HBV infection interfered with most aspects of hepatic metabolic responses, including glucose, lipid, nucleic acid, bile acid and vitamin metabolism. Glucose and lipid metabolism is a particular focus due to the significant promotion of gluconeogenesis, glucose aerobic oxidation, the pentose phosphate pathway, fatty acid synthesis or oxidation, phospholipid and cholesterol biosynthesis affected by HBV. These altered metabolic pathways are involved in the pathological process of not only hepatitis B, but also metabolic disorders, increasing the occurrence of complications, such as hepatocellular carcinoma and liver steatosis. Thus, a clearer understanding of the hepatic metabolic pathways affected by HBV and its pathogenesis is necessary to develop more novel therapeutic strategies targeting viral eradication.


Assuntos
Hepatite B Crônica/metabolismo , Fígado/metabolismo , Transdução de Sinais , Ácidos e Sais Biliares/química , Glicemia/análise , Fígado Gorduroso/metabolismo , Glucose/metabolismo , Vírus da Hepatite B , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos , Fígado/patologia , Redes e Vias Metabólicas , Ácidos Nucleicos/análise , Ácidos Nucleicos/química , Vitaminas/química , Vitaminas/metabolismo
4.
Drug Des Devel Ther ; 10: 1243-55, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27069355

RESUMO

INTRODUCTION: Hepatocellular carcinoma is currently the second leading cause of cancer-related deaths worldwide with an increasing incidence. OBJECTIVE: The objective of this study is to investigate the effect of vascular endothelial growth factor small interfering RNA (VEGF-siRNA) on rabbit VX2 carcinoma cell viability in vitro and the effect of transarterial embolization (TAE)-mediated VEGF-siRNA delivery on the growth of rabbit VX2 liver-transplanted model in vivo. METHODS: Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot technologies were used to detect the expression level of VEGF. TAE and computed tomography scan were used to deliver the VEGF-siRNA and detect the tumor volume in vivo, respectively. Microvessel density was detected by immunohistochemistry with CD34 antibody. A biochemical autoanalyzer was used to evaluate the hepatic and renal toxicity. RESULTS: The designed VEGF-siRNAs could effectively decrease the expression levels of VEGF mRNA and protein in vitro and in vivo. In vitro, the viability of rabbit VX2 carcinoma cells was reduced by 38.5%±7.3% (VEGF-siRNA no 1) and 30.0%±5.8% (VEGF-siRNA no 3) at 48 hours after transfection. Moreover, in rabbit VX2 liver-transplanted model, the growth ratios of tumors at 28 days after TAE-mediated siRNA delivery were 155.18%±19.42% in the control group, 79.67%±19.63% in the low-dose group, and 36.09%±15.73% in the high-dose group, with significant differences among these three groups. Microvessel density dropped to 34.22±4.01 and 22.63±4.07 in the low-dose group and high-dose group, respectively, compared with the control group (57.88±5.67), with significant differences among these three groups. Furthermore, inoculation of VX2 tumor into the liver itself at later stage induced significant increase in alanine aminotransferase and aspartate aminotransferase, indicating an obvious damage of liver functions, while treatment of VX2 tumor via TAE-mediated VEGF-siRNA had no toxicity to the livers and kidneys of rabbits, and VEGF-siRNA had the ability to protect liver damage induced by tumor growth. CONCLUSION: This is the first study to demonstrate that targeting VEGF via TAE-mediated siRNA delivery may become a powerful new option for effective treatment of hepatocellular carcinoma in the clinic.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Sistemas de Liberação de Medicamentos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Coelhos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
5.
Int J Dermatol ; 47(5): 448-56, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18412860

RESUMO

BACKGROUND: Matrine is a traditional Chinese medicine with significant inhibitory activity against malignant tumors. Its effects on the invasiveness and metastasis of malignant tumors have rarely been reported. AIM: To investigate whether matrine can inhibit the metastasis-related activities of the human malignant melanoma cell line A375 in vitro. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and Annexin-V-fluorescein isothiocyanate/propidium iodide (Annexin-V-FITC/PI) affinity assay were used to examine the effects of matrine on the proliferation and apoptosis induction of A375 cells. The morphologic changes of A375 cells were observed by light and electron microscopy. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to evaluate the expression of heparanase mRNA and protein. The effect of matrine on the adhesion ability and invasiveness of treated A375 cells was tested by cell-Matrigel adhesion assay and Matrigel invasion assay, respectively. RESULTS: Matrine showed significant inhibition of the proliferation of A375 cells in a dose- and time-dependent manner. It also induced apoptosis in a dose-dependent manner. Compared with the control group, the levels of heparanase mRNA and protein expression of A375 cells treated with different concentrations of matrine were decreased significantly, as were their adhesion ability and invasiveness. CONCLUSIONS: These findings indicate that matrine inhibits the invasiveness and metastasis of A375 cells in vitro. The mechanisms may be linked to the inhibition of cellular proliferation, induction of apoptosis, and downregulation of heparanase mRNA and protein expression.


Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Melanoma/patologia , Quinolizinas/farmacologia , Análise de Variância , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Glucuronidase/genética , Glucuronidase/metabolismo , Humanos , Medicina Tradicional Chinesa , Melanoma/enzimologia , Melanoma/fisiopatologia , Melanoma/ultraestrutura , Microscopia Eletrônica de Transmissão , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/prevenção & controle , Fitoterapia , Plantas Medicinais/química , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sophora/química , Matrinas
6.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 36(6): 581-7, 2007 11.
Artigo em Chinês | MEDLINE | ID: mdl-18067232

RESUMO

OBJECTIVE: To construct heparanase gene-targeted small interfering RNA(siRNA) and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant melanoma A375 cell. METHODS: Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strand was synthesized and inserted into pRNATU6.1 vector, which was then identified by PCR and sequencing. Human malignant melanoma A375 cells were transfected with the constructed vector using lipofectamine method. Semi-quantitative PCR was performed to evaluate the heparanase-mRNA expression levels, and Western blot was performed to evaluate the expression of heparanase protein. RESULTS: The vector containing siRNA was identified by PCR and sequencing; the results of semi-quantitative PCR and Western blot showed that the expression levels of both heparanase RNA and protein in transfected A375 cells were decreased significantly(P<0.05). CONCLUSION: The heparanase gene-targeted siRNA and its vector were successfully constructed, which can reduce the heparanase gene and protein expression in transfected cells.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Glucuronidase/genética , Melanoma/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Sequência de Bases , Linhagem Celular Tumoral , Glucuronidase/metabolismo , Humanos , Melanoma/patologia , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção
7.
Zhong Yao Cai ; 29(3): 253-6, 2006 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-16850724

RESUMO

OBJECTIVE: To investigate the effect of matrine on the invasiveness and expression of heparanase-mRNA in human malignant melanoma cell line A375. METHODS: The A375 cells were treated by matrine in different concentration. The total RNAs were extracted from the cells 48 hours after treatment and then semi-quantitative RT-PCR were performed to evaluate the heparanase-mRNA expression levels. Effect of matrine on adhesion of treated A375 cells was tested by cell-Matrigel adhesion assay. The invasiveness of treated A375 cells was measured by Matrigel invasion assay. RESULTS: The hepanase-mRNA expression, adhesion and invasiveness of A375 cells treated with matrine of different final concentrations significantly decreased compared with that of the controls (p < 0.01). Besides, the inhibitory effects were signifcantly different when the cells treated with matrine of different concentrations (P < 0.01). CONCLUSION: By down-regulating the expression of heparanase-mRNA, matrine has a significant inhibitory effect on the adhesion and invasiveness of human malignant melanoma cell line in a dose-dependent manner.


Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Glucuronidase/biossíntese , Melanoma/patologia , Quinolizinas/farmacologia , Sophora/química , Alcaloides/administração & dosagem , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , Glucuronidase/genética , Humanos , Melanoma/enzimologia , Invasividade Neoplásica , Plantas Medicinais/química , Quinolizinas/administração & dosagem , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Matrinas
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 35(2): 154-60, 2006 03.
Artigo em Chinês | MEDLINE | ID: mdl-16610081

RESUMO

OBJECTIVE: To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs). METHODS: Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR. RESULT: Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration. CONCLUSION: The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , RNA Interferente Pequeno , RNA de Transferência de Valina/genética , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos E da Hepatite B/biossíntese , Antígenos E da Hepatite B/genética , Humanos , Rim/citologia , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Viral/genética , Transfecção
10.
Zhonghua Gan Zang Bing Za Zhi ; 14(2): 109-13, 2006 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-16494780

RESUMO

OBJECTIVE: To explore the mutated KLF6 gene in hepatocellular carcinoma (HCC) and to characterize its behavior in human hepatocellular carcinoma cell line HepG2. METHODS: We analyzed the DNA isolated from 23 hepatocellular carcinoma tissues and their adjacent nontumor tissues by polymerase chain reaction (PCR). Direct sequencing was used to establish the incidence of mutation in exon2 of the KLF6 gene. Loss of growth suppressive function of the HCC-derived KLF6 mutants was characterized by in vitro analyzing alteration of cell cycle and MTT assay. Expression of p21WAF1, a possible downstream gene of KLF6, was detected in human hepatocellular carcinoma cell line HepG2 transiently transfected with KLF6 genes. RESULTS: Mutations of KLF6 were found in 2 of the 23 (8.7%) hepatocellular carcinomas. The two mutations were located in the transactivation domain and one of them resulted in single amino acid substitution of TGG (W) by GGG (G) at codon 162. Unlike the wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21WAF1 following transfection into culture cells. CONCLUSIONS: Mutations of the KLF6 gene may play a role in the pathogenesis of HCC, but are not the dominating mechanism resulting in inactivation of KLF6 functions. KLF6 suppresses hepatocellular carcinoma cell proliferation partly through upregulating expression of the p21WAF1 gene.


Assuntos
Carcinoma Hepatocelular/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Hepáticas/genética , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/fisiologia , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/fisiologia , Análise de Sequência de DNA
11.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 34(2): 104-9, 2005 03.
Artigo em Chinês | MEDLINE | ID: mdl-15812881

RESUMO

OBJECTIVE: To inhibit HBV core antigen gene expression with plasmid-based RNAi. METHODS: The shRNA expression vector targeting HBV core antigen gene was designed and constructed. Human embryonic kidney cell line AD293 was co-transfected with HBcAg-EGFP fusion protein expression vector and shRNA expression vector transiently, and the cells without shRNA-transfection and with non-specific shRNA transfection were used as controls. Inhibitory effect of RNAi was detected by fluorescence-activated cell sorting (FACS) and real-time fluorescence quantificational RT-PCR. RESULTS: HBV core antigen gene expression in AD293 was inhibited by shRNA, with the maximal inhibition rate of 76 % measured by FACS and of 63.1 % by real-time PCR. CONCLUSION: Effective inhibition of HBV core antigen gene expression by plasmid-based RNAi provides an alternative for anti-HBV study in vitro, which has potential clinical application.


Assuntos
Regulação Viral da Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Rim/virologia , Interferência de RNA/fisiologia , Linhagem Celular , Embrião de Mamíferos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Humanos , Rim/citologia , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Transfecção
12.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 34(2): 110-5, 2005 03.
Artigo em Chinês | MEDLINE | ID: mdl-15812882

RESUMO

OBJECTIVE: To develop an effective report gene system to test the effect of small interfering RNA (siRNA). METHODS: HBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR. RESULTS: siRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively. CONCLUSION: This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.


Assuntos
Regulação Viral da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Antígenos de Superfície da Hepatite B/genética , Interferência de RNA , Carcinoma Hepatocelular/patologia , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/patologia , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , Células Tumorais Cultivadas
13.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 34(2): 121-5, 2005 03.
Artigo em Chinês | MEDLINE | ID: mdl-15812884

RESUMO

OBJECTIVE: To explore the inhibitory effect of combination of lamivudine with thymosin alpha1 (Talpha1) on the replication of duck hepatitis B virus (DHBV). METHODS: Peking ducks of 1 d old were challenged with DHBV-positive serum and used as a duck hepatitis B model. After treated with lamivudine for three months, the ducks were randomly grouped and treated with or without Talpha1 for 8 d. Serum DHBV titrate was observed by semi-quantitative PCR, and inflammation and degeneration of hepatocytes were observed by pathology examination. RESULTS: The serum DHBV titrate was significantly reduced (4483.2+/-5193.4 compared with 9351.8+/-5059.6) after lamivudine treatment, and it was reduced more significantly(1692.2+/-589.2) after combination treatment with Talpha1. Lamivudine reduced the degeneration degree of hepatocytes (3.2+/-0.8 compared with 4.6+/-0.5) and the inflammation degree of liver (6.2+/-3.3 compared with 8.6+/-2.8). The combination treatment with Talpha1 increased liver inflammation degree (9.0+/-5.2). CONCLUSION: Both Talpha1 and lamivudine may reduce the replication of DHBV in Peking ducks and combination treatment may have the better anti-virus effect and enhance immune response in liver.


Assuntos
Infecções por Hepadnaviridae/tratamento farmacológico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Lamivudina/uso terapêutico , Timosina/análogos & derivados , Animais , Animais Recém-Nascidos , Antivirais/uso terapêutico , Células Cultivadas , Quimioterapia Combinada , Patos , Vírus da Hepatite B do Pato/genética , Hepatite Viral Animal/tratamento farmacológico , Hepatócitos/virologia , Timalfasina , Timosina/uso terapêutico , Replicação Viral/efeitos dos fármacos
14.
World J Gastroenterol ; 11(4): 498-502, 2005 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-15641133

RESUMO

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method. RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively. CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).


Assuntos
Regulação Viral da Expressão Gênica , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , RNA Interferente Pequeno/genética , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Antígenos E da Hepatite B/genética , Humanos , Técnicas In Vitro , Neoplasias Hepáticas , Plasmídeos , RNA Mensageiro/genética , Transfecção
15.
Zhonghua Gan Zang Bing Za Zhi ; 12(9): 515-8, 2004 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-15387899

RESUMO

OBJECTIVE: To find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors. METHODS: Four shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR. RESULTS: Four shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%. CONCLUSIONS: The results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells


Assuntos
Inativação Gênica , Marcação de Genes , Antígenos de Superfície da Hepatite B/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Regulação Viral da Expressão Gênica , Marcação de Genes/métodos , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Complexo de Inativação Induzido por RNA/genética
16.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 33(4): 300-5, 310, 2004 07.
Artigo em Chinês | MEDLINE | ID: mdl-15269979

RESUMO

OBJECTIVE: To develop a system for quick screening of efficient siRNA targeted HBx mRNA. METHODS: Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis. RESULT: (1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388. CONCLUSION: Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.


Assuntos
RNA Interferente Pequeno/análise , Transativadores/antagonistas & inibidores , Sequência de Bases , Células Cultivadas , Terapia Genética , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Transativadores/genética , Transfecção , Proteínas Virais Reguladoras e Acessórias
17.
World J Gastroenterol ; 10(14): 2050-4, 2004 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15237432

RESUMO

AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.


Assuntos
Regulação para Baixo , Vírus da Hepatite B/genética , Interferência de RNA , RNA Viral/metabolismo , Ribonucleoproteínas/antagonistas & inibidores , Autoantígenos , Linhagem Celular , Humanos , Antígeno SS-B
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