RESUMO
<p><b>OBJECTIVE</b>To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.</p><p><b>METHODS</b>First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM.</p><p><b>RESULTS</b>It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05).</p><p><b>CONCLUSION</b>Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.</p>
Assuntos
Animais , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas , Genética , Linhagem Celular Tumoral , Ciclofilina A , Genética , Inativação Gênica , Vetores Genéticos , Lentivirus , Genética , Neoplasias Pulmonares , Genética , RNA Interferente PequenoRESUMO
<p><b>OBJECTIVE</b>To study the preventive and therapeutic effects of recombinant IFN-alpha2b for nasal spray on SARS-CoV infection in Macaca mulata (rhesus monkey).</p><p><b>METHODS</b>Ten rhesus monkeys were divided into two groups, 5 in interferon group, and 5 in control group. Before and after SARS-CoV attack, the virus was detected in samples such as pharyngeal swab in all the two groups by Real-time PCR (RT-PCR) and virus isolation was performed.</p><p><b>RESULTS</b>After virus attack, the level of SARS-CoV-specific IgG and neutralizing antibody were induced by SARS-CoV in the interferon group was weaker than in control group. Hematology items showed no apparent changes after virus attack in treated group. Through pathological examination, the morphology of the lung tissues of two Macaques in the treated group was normal, while the other three displayed the interstitial pneumonia with the thickened septum and infiltration with mononuclear cells. Among which, one monkey showed part of thickened septum fused with each other. These lesions in the interferon treated animals were similar to those seen in the animals in control group, but with smaller scope of pathological changes. No significant abnormity was detected in other organs.</p><p><b>CONCLUSION</b>Recombinant IFN-alpha2b could effectively interdict or weaken SARS-CoV injury in monkeys.</p>
Assuntos
Animais , Feminino , Masculino , Antivirais , Usos Terapêuticos , Chlorocebus aethiops , Modelos Animais de Doenças , Interferon-alfa , Usos Terapêuticos , Pulmão , Patologia , Virologia , Macaca mulatta , Doenças dos Macacos , Tratamento Farmacológico , Virologia , Distribuição Aleatória , Proteínas Recombinantes , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Síndrome Respiratória Aguda Grave , Tratamento Farmacológico , Virologia , Células VeroRESUMO
<p><b>OBJECTIVE</b>To study the antigenicity of SARS associated coronavirus (CoV) spike S1 (12-672Aa) domain.</p><p><b>METHODS</b>BALB/c mice were immunized with a plasmid bearing codon-optimized SARS-CoV (Tor2 strain) S1 domain and then boosted with purified S1 protein; the SARS-CoV specific IgG antibody was tested by ELISA and neutralization antibody was determined by in vitro microneutralization assay.</p><p><b>RESULTS</b>S1 domain of SARS-CoV spike, which has been demonstrated harboring the receptor binding domain, successfully elicited SARS-CoV specific IgG antibody in mouse after combined immunization with DNA and purified S1 protein; the antibody elicited solely by S1 could potently neutralize SARS-CoV (HKU-39849) in vitro, 50% of 1 000 TCID50 SARS-CoV challenged cells were protected from viral infection by a 1:1499.68 dilution of mice sera immunized with S1 protein, but negative control sera showed no protection.</p><p><b>CONCLUSION</b>S1 domain of SARS-CoV spike protein, which is responsible for receptor binding, can efficiently and sufficiently induce highly potent neutralizing antibody in mice. This result suggested that S1 domain could be an effective subunit vaccines against SARS-CoV.</p>