RESUMEN
To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Asunto(s)
Infecciones por Coxsackievirus/virología , Enterovirus Humano B/genética , Miocarditis/virología , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Infecciones por Coxsackievirus/tratamiento farmacológico , Regulación hacia Abajo , Enterovirus Humano B/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Miocarditis/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , ARN Viral/genética , Replicación ViralRESUMEN
OBJECTIVE: To study the effect of Itk down regulation on Jurkat cell proliferation and inflammatory cytokines production, and provide useful data for Itk as an attractive target for potential drugs. METHODS: Three shRNAs against different region of Itk were constructed and cotransfected with pEGFP-C1-hItk. The shRNA, which can knock down Itk, was selected and packed into lentivirus. After Jurkat cells were transfected with shRNA lentivirus, the change of Itk protein expression, cell proliferation and cytokines production was observed. RESULTS: Itk mRNA was reduced about 55% in Jurkat cells transfected with Itk-shRNA1, compared with that in control cells shRNAnon (P < 0.05). Knocking down Itk expression had a profound inhibitory effect on Jurkat cell proliferation. In addition, there was a substantial decrease in level of cytokines, such as IL-2, IL-5, IL-10 and IFN-gamma, produced by cell transfected with Itk-shRNA1. CONCLUSION: Knocking down Itk expression can inhibit Jurkat cell proliferation and inflammatory cytokines production.
Asunto(s)
Citocinas/genética , Regulación hacia Abajo , Células Jurkat/enzimología , Proteínas Tirosina Quinasas/inmunología , Animales , Proliferación Celular , Citocinas/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-5/genética , Interleucina-5/inmunología , Células Jurkat/citología , Células Jurkat/inmunología , Ratones , Proteínas Tirosina Quinasas/genéticaRESUMEN
OBJECTIVE: By using the RNAi method to inhibit Itk protein expression specificity, to observe lymphocytes proliferation and cytokines production, verify its function as a drug target. METHODS: Designed siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA. RESULTS: Itk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-gamma. They all have statistical difference (P < 0.05). CONCLUSION: Itk has an important immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines.This can supply an experimental basis to regard Itk as drug target for inflammation therapy.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Linfocitos/citología , Proteínas Tirosina Quinasas/inmunología , Bazo/citología , Animales , Citocinas/genética , Citocinas/inmunología , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Tirosina Quinasas/genética , Bazo/inmunologíaRESUMEN
OBJECTIVE: To evaluate the possibility of short interfering RNA (siRNA) inhibiting Coxsackievirus B3 (CVB3) infection in vitro, and discover the mechanism initially. METHODS: We obtained proper effective dosage of siRNA by observing cytopathic effect (CPE). Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR. RESULTS: Results showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77% and its effect can last for 48 h stably in cells. 0.6 micromol/L siRNA-3753 got a high inhibiting effect of virus and didn't show any toxicity to cells. So we consider this concentration as the experimental concentration. siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R (PKR). CONCLUSION: The data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.
Asunto(s)
Infecciones por Coxsackievirus/terapia , Enterovirus Humano B/genética , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/metabolismo , Células HeLa , Humanos , ARN Interferente Pequeño/genética , ARN Viral/genéticaRESUMEN
To study the inhibitory effect and function characteristics of small interfering RNA (siRNA) on cosxackievirus B3(CVB3) infection by RNA interference technique, siRNA-2B against the viral 2B region was synthesized and transfected into HeLa cell, which was then infected with CVB3. The efficiency of siRNA transfection was examined by FCM, the cell toxicity of siRNA-2B by MTT, and the antiviral ability of siRNA-2B by cytopathic effect (CPE), plaque reduction assay and RT-PCR. The results showed that siRNA-2B could be transfected efficiently into HeLa cell and lasted at least 48h. High concentration of siRNA-2B didn't show any sign of toxicity to cells. siRNA-2B exhibited a significant protective effect on cell viability by specific inhibition of viral replication. It showed a close relationship between the concentrations of siRNA-2B and the antiviral effects. siRNA-2B led to dramatical reduction of viral titers in supernatant of cell culture and weakened the reinfection ability of the virus. These data proposed that siRNA-2B, targeting 2B protein, can effectively inhibit CVB3 infection in HeLa cell and exhibits its transfection efficiency, viral inhibition specificity and adose-dependant manner, suggesting its potential role in prevention and treatement of CVB3 infection.
Asunto(s)
Enterovirus/genética , ARN Interferente Pequeño/genética , Proteínas no Estructurales Virales/genética , Enterovirus/crecimiento & desarrollo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
OBJECTIVE: To evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA). METHODS: Antiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR. RESULTS: Eight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection. CONCLUSION: SiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.
Asunto(s)
Infecciones por Coxsackievirus/terapia , Enterovirus/genética , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Infecciones por Coxsackievirus/virología , Efecto Citopatogénico Viral/efectos de los fármacos , Enterovirus/fisiología , Células HeLa , Humanos , ARN Interferente Pequeño/genética , Replicación Viral/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the feasibility and safety of autologous peripheral blood hematopoietic stem cell transplantation (auto-PBHSCT) and its therapeutic effect on refractory rheumatism among preschool children. METHODS: Three boys with juvenile rheumatoid arthritis (JRA), juvenile systemic lupus erythematosus (JSLE) and juvenile dermatomyositis (JDM) respectively, 3 to 6 years old with the mean age of 5 years with 3.5 to 22 months course of disease with 14 months on average, received auto-PBHSCT. Their conditions were so severe that conventional therapy failed to control the diseases. The changes of both clinical manifestations and immunologic indexes were observed before and after transplantation with long term following up at specialty clinic of rheumatism. RESULT: The time when neutrophil count >or= 0.5 x 10(9)/L in the 3 children was days +9, +13 and +11 respectively, that of platelet count >or= 20 x 10(9)/L was days +14, +18 and +13 respectively. The cellular immune function remained abnormal with CD4 cells at a low level and CD4/CD8 being inverted. As to the JDM child, the skin rash had disappeared and his muscle tone was improved to grade 5 within one month after the transplantation. The EMG and serum creatase level returned to normal and muscle MRI findings were improved greatly within 2 months after the transplantation. As to the JSLE child, skin rash and proteinuria had disappeared, MRI of brain showed that the pathological changes had been absorbed and EEG returned to normal 3 months after the transplantation, all the autoantibodies turned to negative within 8 months after transplantation. As to the JRA child, the arthritis had been improved remarkably within 3 weeks after auto-PBHSCT. There was no swelling of joints nor movement limitation 3 months post transplantation. The steroids and immunosuppressive drugs were discontinued post transplantation. Cushing syndrome disappeared. Their body heights increased by 10 to 15 cm in the past 18 months, and they all returned to school. There was no relapse during follow-up periods of 25 - 27 months. CONCLUSION: The therapy with auto-PBHSCT for refractory rheumatism among preschool children was remarkably effective in a short-term, yet the safety and long-term effect still need to be further studied.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Trasplante de Células Madre de Sangre Periférica , Enfermedades Reumáticas/terapia , Niño , Humanos , Masculino , Trasplante Autólogo , Resultado del TratamientoRESUMEN
OBJECTIVE: In this study, the authors investigated inhibition of coxsackievirus B (CVB) gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon and structural protein coding sequences and also observed the dose-response of the sequence specific inhibition of CVB plaque formation by antisense oligonucleotides. METHODS: Antiviral activities of these oligonucleotides were evaluated by using plaque reduction assay, yield reduction assay, cytopathic effect (CPE) and Western blot analysis. The cells were treated with random oligonucleotides as a specificity control. RESULTS: At a screening concentration of 5 micromole, 6 of the phosphorothioate oligonucleotide demonstrated some reduction of virus replication relative to untreated cells. 70%-90% inhibition of virus at 0.1 MOI (multiplicity of infection), 50% inhibition of virus infection at 10 MOI. The levels of the VP1 were reduced in CVB-infected cells treated with Scb561 and Scb733. VP1 was significantly reduced after treatment with 0.625 micromole Scb561 and almost undetectable in cells treated with 2.5 micromole Scb561. Dose response experiments implied that sequence specific oligonucleotide doses were related to effect on inhibition of CVB3 infection. When oligonucleotide doses were increased from 1.25 to 5 micromole, 75% to 90% inhibition were observed with Scb561 and 65% to 80% inhibition with Scb733, whereas random control failed to inhibit CVB replication (8% inhibition for each). CONCLUSION The present studies showed that antisense oligonucleotides against internal ribosome entry site (IRES) and translation initiation codon were capable of specifically inhibiting the synthesis of viral protein and subsequent productive CVB replication.The selective inhibition using antisense oligonucleotide might lead to development of an effective antiviral agent for future clinical evaluation.