ABSTRACT
Os lentiviros de pequenos ruminantes (LVPR) são responsáveis por enfermidades infecciosas e multissistêmicas causadas pelo Vírus da Artrite Encefalite Caprina (CAEV) e o Vírus da Maedi-Visna (MVV), e se apresentam sob as formas clínicas: articular, mamária, respiratória e nervosa. Desta forma esse trabalho objetivou determinar a ocorrência e avaliar os fatores de risco associados à infecção por LVPR no Estado de Sergipe, Brasil. Foram coletadas amostras sanguíneas de 1200 ovinos e 675 caprinos oriundos respectivamente de 60 e 41 propriedades localizadas em 25 municípios sergipanos no período de 2011 a 2014. Os diagnósticos dos LVPR foram determinados pela técnica sorológica de Imunodifusão em Gel Ágar (IDGA) usando o kit comercial da marca Biovetech®. Os dados das variáveis associadas aos fatores de risco foram obtidos a partir de questionários aplicados aos proprietários dos rebanhos e analisados estatisticamente. As frequências absolutas e relativas foram determinadas por análise estatística descritiva e os fatores de risco por análise univariada das variáveis de interesse pelo Teste de Qui-quadrado de Pearson e Exato de Fisher, quando necessário, e em seguida submetidos à análise de regressão logística. Foi evidenciada uma soropositividade de 5,03% (34/675) em caprinos e 1,50% em ovinos com 26,82% (11/41) e 28,33% (17/60) das propriedades apresentando ao menos um animal positivo respectivamente. Na análise dos fatores de risco, não foram observadas diferenças significantes para os ovinos, enquanto que, para os caprinos, rebanhos acima de 100 animais, que pastejam em áreas comuns com outros rebanhos, em uma distância ≤500 metros entre as propriedades, que adotam medidas biotecnológicas da reprodução e não utilizam agulhas estéreis, são mais susceptíveis à infecção por LVPR. Sendo assim, conclui-se que, há a presença dos LVPR em rebanhos sergipanos, e mesmo que em baixas frequências faz-se necessário a implementação de medidas profiláticas devido a possibilidade de expansão e desenvolvimento da caprinocultura do estado, e o alto padrão genético da raça Santa Inês.(AU)
The lentiviruses of small ruminants are infectious and multisystemic diseases caused by the Caprine Arthritis Encephalitis Virus (CAEV) and the Maedi-Visna Virus (MVV), and present the clinical forms: articular, mammary, respiratory and nervous. This work aimed to determine the occurrence and to evaluate the risk factors associated with lentivirus infection of small ruminants in the State of Sergipe, Brazil. Blood samples were collected from 1200 sheep and 675 goats from 60 and 41 farms respectively, located in 25 Sergipe municipalities from 2011 to 2014. The diagnosis of small ruminant lentiviruses (LVPR) was determined by the serological technique of Immunodiffusion in Gel Agar (IDGA) using the commercial kit of the brand Biovetech®. Data from the variables associated with risk factors were obtained from questionnaires applied to the owners of the herds and analyzed statistically. Absolute and relative frequencies were determined by descriptive statistical analysis and risk factors by univariate analysis of the variables of interest by Pearson's Chi-square test and Fisher's exact test, when necessary. A logistic regression analysis was used, considering as a dependent variable for LVPR infection the reactive or non-reactive result observed in the IDGA. A seropositivity of 5.03% (34/675) was observed in goats and 1.50% in sheep with 26.82% (11/41) and 28.33% (17/60) of the properties had at least one animal positive respectively. The analysis of the risk factors, no significant differences were observed for sheep, while for goats, herds above 100 animals grazing in common areas with other herds, at a distance ≤ 500 meters between the properties, that adopt Biotechnological measures of reproduction and do not use sterile needles, are more susceptible to LVPR infection. Therefore, it´s concluded there is presence of lentiviruses of small ruminants in sergipan herds, and even if at low frequencies it is necessary to implement prophylactic measures due to the possibility of expansion and development of goat breeding of the state and the high genetic standard of the Santa Inês breed.(AU)
Subject(s)
Animals , Ruminants/virology , Lentivirus Infections/diagnosis , Immunodiffusion/veterinaryABSTRACT
Background:Epstein-Barr virus (EBV)is associated with various human lymphoid and epithelial malignancies such as Burkitt's lymphoma,nasopharyngeal carcinoma and gastric carcinoma. LMP2A,a virus-encoded latent membrane protein is expressed in a portion of EBV-associated gastric carcinoma (EBVaGC)and has been shown to be related with the tumorigenesis and progression of EBVaGC. Aims:To explore the effect of LMP2A silencing on growth of EBVaGC cells in vitro by using a lentivirus-mediated RNA interference (RNAi)to inhibit LMP2A gene expression. Methods:A lentivirus vector pGCSIL-LMP2A-shRNA-LV and a negative control vector were constructed and transfected into the EBVaGC cell line GT38. Real-time PCR and Western blotting were used to determine the inhibitory effect of the lentivirus vector;CCK-8 assay,colony formation assay and flow cytometry were employed to assess the cell growth,cell cycle and apoptosis of GT38 cells. Results:In GT38 cells transfected with LMP2A-shRNA-LV,the expression level of LMP2A mRNA was decreased by 65. 4%,and that of LMP2A protein was reduced by 50. 8%;the cell proliferation was inhibited,the colony formation ability was suppressed,the percentage of cells in G0 / G1 phase and apoptotic rate were increased when compared with those transfected with negative control vector or without transfection (P < 0. 05). Conclusions:Lentivirus-mediated RNAi is effective for silencing LMP2A gene expression,subsequently inhibiting the growth of EBVaGC cells,inducing G0 / G1 phase arrest and enhancing cell apoptosis in vitro. LMP2A is supposed to be a potential target for gene therapy of EBVaGC.
ABSTRACT
Objective To study the expression of STIM 1 gene in human hypopharyngeal carcinoma cell line FaDu and its effect on FaDu cell apoptosis .Methods Lentivirus infection was used to knock STIM 1 down in FaDu cells .Group STIM1-siRNA: the expression of STIM1 in FaDu cell was inhibited by STIM 1-siRNA lentivirus .Group control:FaDu cells were infected by negative control siRNA lentivirus . Real-Time PCR was applied to identify the efficacy of lenticirus infection and the expression of STIM 1 in FaDu cells.Western blot was used to identify the expression of STIM 1 protein after lenticirus infection .Flow cytometry assay was performed to detect the apoptosis of FaDu cells in the two groups.The data were statistically analyzed with SPSS 17.0 software.Results Compared with GAPDH (Ct=12.08 ±0.05),the expression of STIM1 in FaDu cells was significant expressed (Ct=22.21 ±0.05,P<0.001).Real-Time PCR analysis the relative mRNA expression of STIM1 in FaDu cells of control group and STIM 1-siRNA group were (1.00 ±0.08) and (0.12 ±0.01) respectively (P<0.001). Western blot showed that the expression of STIM 1 gene and protein in FaDu cells were inhibited significantly after STIM 1-siRNA lentiviral in-fection,which was in accordance with the results of Real-Time PCR analysis.Flow cytometry assay showed that the siRNA-mRNA group had a higher apoptosis percentage (9.81 ±0.56)% compared to the control group (4.36 ±1.32)%,with statistically significant difference (P<0.05).Conclusion STIM1 gene correlated significantly with FaDu cell apoptosis .It inhibits apoptosis of FaDu cells ,and it may be a potential diagnostic and therapeutic target for the hypopharyngeal carcinoma .
ABSTRACT
Objective: To investigate the effects of p21/Waf1 gene silencing on replicative potential of bladder cancer-specific oncolytic adenovirus and the proliferation inhibition of EJ cells. Methods: The shRNA targeting p21/Waf1 gene (p21/Waf1-shNA) was designed and synthesized, then the annealing oligonucleotide fragments were subcloned into pMagic 7.1 vector containing coding gene of green fluorescent protein (GFP) to construct recombinant lentiviral plasmid pLVT1051, which was confirmed by PCR and DNA sequencing. The 293T cells were co-transfected with three plasmids including pLVT1051, pCMV-VSV-G and pCMV-dR8.91 to produce the recombinant lentivirus. The EJ cells were infected with recombinant lentivirus carrying p21/Waf1-shRNA, and the puromycin was used to screen out the stably infected EJ cells. The expression levels of p21/Waf1 mRNA and protein in EJ cells after infection with recombinant lentivirus carrying p21/Waf1-shRNA were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. The p21/Waf1 gene-silenced EJ cells were infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, then the expression levels of virus replication-related proteins E1A and Hexon were detected by Western blotting, and the cytotoxic effect on EJ cells was examined by MTT method. Results: Recombinant lentiviral vector carrying p21/Waf1-shRNA targeting p21/Waf1 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus was harvested. The stably infected EJ cells were successfully screened out by using puromycin forr two weeks. The expression levels of p21/Waf1 mRNA and protein in EJ cells infected with recombinant lentivirus carrying p21/Waf1-shRNA were significantly reduced (both P < 0.05). The expression levels of E1A and Hexon proteins in p21/Waf1 gene-silenced EJ cells infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A were up-regulated, and the proliferation inhibition of EJ cells was enhanced (P < 0.01). Conclusion: The p21/Waf1 gene-silenced EJ cells are successfully constructed, and p21/Waf1 gene silencing can enhance the replicative ability of bladder cancer-specific oncolytic adenovirus and significantly inhibit the proliferation of EJ cells.
ABSTRACT
Objective: To investigate the effect of nuclear factor with BRCT domains protein 1 (NFBD1) gene silencing by short hairpin RNA (shRNA) interference on the radiosensitivity of human nasopharyngeal carcinoma CNE-1 cells. Methods: The CNE-1 cells were transfected with NFBD1-shRNA by method of lentiviral infection, the stably transfected cells were selected by puromycin, and the mRNA and protein levels of NFBD1 in CNE-1 cells were detected by real-time fluorogenic quantitative-PCR (RFQ-PCR) and Western blotting, respectively. For the examination of the effect of NFBD1 depletion on the radiosensitivity of CNE-1 cells, the flow cytometry was used to calculate the apoptotic rate and cell cycle distribution, and the survival fraction of the cells was measured via colony formation assay. Results: After transfection with NFBD1-shRNA, the mRNA and protein levels of NFBD1 in CNE-1 cells were decreased obviously. The percentage of the cells in G2/M phase and the apoptotic rate of CNE-1 cells in NFBD1-shRNA group were increased after ionizing radiation (6 MeV) as compared with those of the negative control (NC)-shRNA group (P < 0.05). The survival fraction of CNE-1 cells in NFBD1-shRNA group was significantly reduced after radiation as compared with that of the NC-shRNA group (P < 0.05). Conclusion: Lentivirus-mediated shRNA targeting NFBD1 can steadily silence the expression of NFBD1 gene and enhance the radiosensitivity of CNE-1 cells. Copyright © 2014 by TUMOR.