RESUMO
OBJECTIVE@#The aim of this study is to investigate the effects of inhibitor HNP1 transfection on proliferation and tumor growth of human nasopharyngeal carcinoma cell line HNE1.@*METHOD@#HNE1 cells were transfected with HNP1 by liposome, and the cytotoxic effect was detected by MTT, In vivo tumor growth test was performed in nude mice inoculated with transfected HNE1 cells. The therapeutic effect of HNP1 was evaluated in the inoculated tumors, alpha-defensin 1 expression was detected in implanted tumor tissues by immunohistochemical stain.@*RESULT@#HNP1 transfection significantly inhibited the proliferation of HNE1 cells. MTT assay confirmed cytotoxic effect. In vivo study showed tumor volume was significantly smaller in HNP1 transfection group than that in control group (P < 0.01). Immunohistochemical stain showed alpha-defensin expression was increased in HNP1 transfection group.@*CONCLUSION@#HNP1 transfection can inhibit the proliferation of HNE1 cells, as well as tumor growth.
Assuntos
Animais , Feminino , Humanos , Camundongos , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas , Genética , Patologia , Transfecção , Carga Tumoral , alfa-Defensinas , GenéticaRESUMO
The extracellular domain 2-4 loop cDNA of quail vascular endothelial growth factor receptor quek1 (qVEGFR2) was obtained from plasmid carrying qVEGFR2 by PCR. Then it was cloned into expression vector pPICZalphaA of Pichia pastoris. To construct recombinant expression plasmid pPICZalphaA-qVEGFR2, linearized pPICZalphaA-qVEGFR2 with SacI was transformed to electroporated Pichia pastoris GS115. Subsequently, positive clone was selected by PCR and its phenotype was determined. SDSPAGE and Western blot assays of culture medium from a methanol-induced expression strain demonstrated that recombinant qVEGFR2 proteins were expressed and secreted into the culture medium. These results could provide a basis for further researches on tumor protein vaccine as well as for the preparation of tumor protein vaccine with Pichia pastoris.
Assuntos
Animais , Vacinas Anticâncer , DNA Complementar , Genética , Vetores Genéticos , Genética , Pichia , Genética , Metabolismo , Reação em Cadeia da Polimerase , Codorniz , Receptores de Neurotransmissores , Genética , Proteínas Recombinantes , Genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , GenéticaRESUMO
OBJECTIVE@#To investigate apoptosis induced by vesicular stomatitis virus (VSV) in HNE-1 cancer cells of human nasopharyngeal carcinoma by models of nude mice-BALB/c in vivo.@*METHOD@#HNE-1 cells are collected from culture bottle and infected infra right-side post-back epithelium of nude mice-BALB/c (6 x 10(6) cells/0.1 ml/each mice) to create HNE-1 tumor models of nude mice-BALB/c. When the diameter of HNE-1 tumors is 5 to 8 millimeter, HNE-1 tumor models are treated with VSV (1 x 10(8) pfu /ml) or Saline. By Hoechst 33258-staining under fluorescence microscope, induction of apoptosis by VSV in HNE-1 tumor models are recorded and studied, compared with that by Saline in HNE-1 tumor models in vivo.@*RESULT@#Compared with control group of saline, apoptosis of HNE-1 cancer cells of human nasopharyngeal carcinoma increase apparently in the remaining tumor cells of nude mice treated by VSV (P < 0.01).@*CONCLUSION@#The present study suggests that the treatment with VSV could augment the apoptosis cells of human nasopharyngeal carcinoma in vivo.
Assuntos
Animais , Humanos , Camundongos , Apoptose , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas , Transplante de Neoplasias , Terapia Viral Oncolítica , Vesiculovirus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A DNA fragment encoding human interleukin 4 was obtained by PCR from pORF-hIL4 plasmid. The amplified fragment was inserted into prokaryotic expression vector PQE60 and recombinant protein was expressed in E. Coli M15 by adding isopropyl-beta-D-thiogalactoside (IPTG). The hIL-4 protein was present as insoluble inclusion bodies in the bacterial extract. After denaturation of inclusion bodies with 5 mol/L guanidine hydrochloride, the supernate was diluted to get renaturized. Then dialysis and Ni chelating chromatography were used for purification. TF-1 proliferation assay of recombinant human interleukin 4 was performed, and then rhIL-4 was fit to be used for proliferation of human dendritic cells from monocyte in vitro.
Assuntos
Humanos , Escherichia coli , Genética , Metabolismo , Vetores Genéticos , Corpos de Inclusão , Metabolismo , Interleucina-4 , Genética , Dobramento de Proteína , Proteínas Recombinantes , GenéticaRESUMO
OBJECTIVE@#To study the result of HNE-1 cancer cells lesion and HNE-1 cancer cells apoptosis caused by vesicular stomatitis virus in vitro.@*METHOD@#Firstly, HNE-1 cancer cells of human nasopharyngeal carcinoma were cultured in vitro. Secondly, tumor cells were treated with vesicular stomatitis virus (VSV) in different concentration (0.1 moi, 1.0 moi, 10.0 moi and 100.0 moi), while cells without treatment were used as blank assay. Finally, we observed the lesion of HNE-1 cells treated with VSV for 24 to 48 hours under invert microscope, compared with HNE-1 cells of blank assay. By measurement of MTT reduction assay and Hoechst 33258- staining under fluorescence microscope, the effect of VSV killing HNE-1 cancer cells and the induction of apoptosis by VSV in HNE-1 cancer cells were investigated in vitro.@*RESULT@#Compared with blank assay, HNE-1 cancer cells of human nasopharyngeal carcinoma treated with VSV showed phenomenon of lesion under invert microscope. Followed the increase of concentration of VSV, cell survival rate of HNE-1 cancer cells decreased. The research methods of MTT reduction assay and Hoechst 33258 staining under fluorescence microscope defined the effect of VSV killing HNE-1 cancer cells and confirmed that VSV inducing apoptosis of HNE-1 cancer cells in vitro.@*CONCLUSION@#VSV not only exhibited the anti-tumor activity in HNE-1 cancer cells of human nasopharyngeal carcinoma but also showed the potential power of inducing apoptosis of HNE-1 cancer cells in vitro.
Assuntos
Humanos , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Nasofaríngeas , Patologia , Terapia Viral Oncolítica , VesiculovirusRESUMO
Interferon gamma-inducible protein 10, a member of the family of CXC chemokines, is secreted by interferon gamma-stimulated, monocytes, endothelial cells and keratinocytes. Interferon gamma-inducible protein 10 plays an important role in recruiting activated T cells into sites of tissue inflammation. In this experiment, PCR products of Interferon gamma-inducible protein 10 were cloned into prokaryote expression vector pET 32(a) to generate recombinant pET-IP10 with S-Tag at the N-terminus, and expressed successfully in E. coli BL21 (DE3). The total expressed products amounted to 25.3% in all bacterion proteins. pET-IP10 mainly formed inclusion body in E. coli. Soluble recombinant protein accounted for 20% among IP-10 fusion protein. The soluble recombinant proteins were purified by using S-Tag affinity chromatography effectively with purity of over 90%. The chemotaxis biological activity of purified Interferon gamma-inducible protein 10 could specifically exhibit the directional migration of stimulated T cells at concentration of 100 ng/ml. The results indicated that the strategy we used in this experiment was effective for recombinant Interferon gamma-inducible protein 10 production with biological activity.
Assuntos
Humanos , Quimiocina CXCL10 , Escherichia coli , Metabolismo , Vetores Genéticos , Proteínas Recombinantes , Linfócitos T , Biologia Celular , TiorredoxinasRESUMO
Mouse colon cancer cells CT26 were transfected with constructed plasmid expressing mouse soluble B lymphocyte stimulator (msBlyS). Single cell clones were selected with 100 microg/ml Zeosin and subcloned by serial limiting dilution. Eight resistant transfectants were isolated and expanded, and five of them displayed the desirable msBlyS cDNA band amplified by semi-quantitative RT-PCR assay. Western blot analysis showed that only msBlyS molecules of the expected size were detected in the cell lysates from transfectants. The supernatant of transfectants could costimulate B cell proliferation in standard costimulation assay. Thus we have successfully screened the stable transfectants expressing high levels of msBlyS in CT26 cells, which could be used as cancer vaccines for further anti-tumor immunotherapy.
Assuntos
Animais , Camundongos , Anticorpos Monoclonais , Fator Ativador de Células B , Linfócitos B , Alergia e Imunologia , Vacinas Anticâncer , Genética , Clonagem Molecular , Neoplasias do Colo , Patologia , DNA Complementar , Genética , Epitopos de Linfócito B , Genética , Proteínas de Membrana , Genética , Proteínas Recombinantes , Genética , Recombinação Genética , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa , GenéticaRESUMO
A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR. The amplified fragment was inserted into prokaryotic expression vector PQE30. Recombinant protein was expressed in E. Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix. Then it was identified by sequence analysis and Western blot analysis. The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis. And a specfic band was shown by Western blot analysis. These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant.