RESUMO
BACKGROUND & OBJECTIVE: Notch signal transduction pathway mediates cell differentiation and proliferation. Its dysfunction is supposed to be involved in tumorigenesis and development. This study was to construct a course recombination enzyme(CRE)-dependent short hairpin RNA (shRNA) expression plasmid targeting Notch1, and investigate its effect on proliferation of cervical cancer cell line HeLa. METHODS: RNA interfering vectors pSico and pSicoR were used to construct CRE-dependent shRNA expression plasmids targeting GAPDH and Notch1; pBS185-CRE was used as an expression vector of CRE. HeLa cells were divided into 4 groups and transfected with pSico, pSico/CRE, pSicoR, and pSicoR/CRE, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were carried out to assess the efficiency of RNA interference (RNAi), and intracellular Notch signal level was tested by CBF-1 reporter plasmid. The proliferation of HeLa cells after CRE-dependent Notch1 RNAi was detected by MTS assay. RESULTS: After transfection of pSico (R)-GAPDH and pSico (R)-Notch1, CRE-dependent green fluorescent cells were detected; Notch1 expression was inhibited; intracellular Notch1 signal level was decreased; the proliferation of HeLa cells was suppressed. CONCLUSION: RNAi of Notch1 mediated by CRE-dependent shRNA expression plasmid can down-regulate intracellular Notch1 signal level and suppress the proliferation of HeLa cells.
Assuntos
Proliferação de Células , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor Notch1/metabolismo , Feminino , Vetores Genéticos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Células HeLa , Humanos , Integrases/genética , Plasmídeos , Receptor Notch1/genética , TransfecçãoRESUMO
AIM: To clone the human beta2-microglobulin(beta2m) gene promoter and study its activity in P815 cells. METHODS: PCR amplification was performed using primers based on human beta2m gene sequence from GenBank and human genomic DNA as a template. The PCR product was directedly ligated into pBluescript II vector for sequencing. The promoter fragment was subcloned into a pcDNA3-EGFP plasmid after it had been identified correctly. A mouse mastocytoma cell line P815 was transiently and stably transfected with the plasmid containing human beta2m gene promoter and enhanced green fluorescence protein(EGFP) gene. The mRNA expression of EGFP in transiently transfected cells was quantified by RT-PCR and that in stably transfected cells was detected by fluorescence microscope and flow cytometry(FCM). RESULTS: A 302 bp DNA fragment was amplified and cloned into the pcDNA3-EGFP vector. RT-PCR analysis showed that EGFP mRNA expression was induced by IFN-gamma in a dose-dependent manner. There was no difference in the fluorescence positive cell rate between the IFN-gamma-treated group and the control group. But the fluorescence intensity of the 5 x 10(5) U/L IFN-gamma-treated group increased about 2 folds compared with that of the control group. CONCLUSION: Human beta2m gene promoter is active in mouse mastocytoma P815 cells. It can regulate the expression of reporter gene under the control of IFN-gamma.
Assuntos
Regiões Promotoras Genéticas/genética , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interferon gama/farmacologia , Mastocitoma/metabolismo , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
EGR-1 gene promoter and CDglyTK gene were used to construct the pcDNA3-EGR-CDglyTK recombinant vector, in which CDglyTK gene expression was under the control of EGR-1 gene promoter. Cationic liposome LipofectAMINE was used to transfect plasmids into human hepatoma 7402 cell line. Subsequently, the transfected cells were treated with different doses of gamma-ray. Northern blot and Western blot showed that ionizing radiation can induce CDglyTK gene expression drived by EGR-1 promoter,in a dose-dependant manner. There was no ionizing radiation-inducible effect in pcDNA3-CMV-CDglyTK control group. Ionizing radiation also markedly enhanced the sensitivity of tumor cells transfected with pcDNA3-EGR-CDglyTK to prodrugs (GCV/5-FC) in tumor cell killing. The data indicated that EGR-1 promoter was inducible by ionizing radiation, whereas the CMV promoter was not. There was a synergetic effect between GCV and 5-FC. The cytotoxic effect of the suicide gene-ionizing radiation combination was stronger than suicide gene alone or ionizing radiation alone.
Assuntos
Proteínas de Ligação a DNA/genética , Raios gama , Proteínas Imediatamente Precoces , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citomegalovirus/genética , Citosina Desaminase , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Proteína 1 de Resposta de Crescimento Precoce , Flucitosina/farmacologia , Ganciclovir/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Lipossomos , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , Plasmídeos/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
rVV (recombinant vaccinia virus) expressing minigene products and rVV expressing full length OVA protein were used to infect several eukaryotic cells. Monoclonal antibody 25.D-1.16 (anti-OVA(257-264)/K(b)) was used to detect the conformation of H-2K(b)-OVA complex on the surface of these cells. The tetramer technique was used to determine their ability to induce antigen specific T cells. The results showed that the tetramer had been generated successfully. Moreover, L929-K(b) cells infected by rVV-OVA can be used as APC to induce CTL response. Specific CTL response was demonstrated by H-2K(b-OVA tetramer staining and cytotoxicity assay. Extracelluar IFN-gamma was also quantitatively analyzed. The results from H-2Kb)-OVA tetramer staining were consistent with cytotoxicity assay. It can be concluded that rVV expressing minigene product is a more efficient source of peptides for class I molecules, and can be more immunogenic than rVV expressing full length protein.
Assuntos
Apresentação de Antígeno/imunologia , Ovalbumina/imunologia , Vaccinia virus/genética , Animais , Linhagem Celular , Testes Imunológicos de Citotoxicidade , DNA Recombinante/genética , DNA Recombinante/imunologia , Feminino , Expressão Gênica , Células HeLa , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/genéticaRESUMO
To study the effect of OVA-linker-beta2m fusion protein as an immunogen to induce CTL mediated immune response in mice. The beta2m gene was amplified by PCR. OVA(257-264) peptide sequence and an 8-amino acid linker were added to the COOH-terminus of beta2m gene, and then cloned into pE42b(+) vector. After that, gene expression, protein purification and refolding were conducted. Then the protein product was used as an immunogen to induce an efficient CTL mediated immune response in vivo. Specific CTL responses were demonstrated by H-2K(b)-OVA tetramer staining and cytotoxicity assay. Extracelluar IFN-gamma was also quantitatively analyzed. The results showed that specific CTL response could be induced in vivo by OVA-linker-beta2m fusion protein. Generation of terameric peptide-MHC complexes in vitro is a powerful tool to stain specific CTL.
