ABSTRACT
OBJECTIVE@#To study the effect on the expression of protein kinase CK2alpha and growth of human laryngeal carcinoma xenograft in nude mice by applying small interfering RNA (siRNA) specific to protein kinase CK2alpha.@*METHOD@#Human laryngeal carcinoma Hep-2 cells were implanted under the skin of nude mice. After the tumors grew to a definite size, the tumors were injected with siRNA expression plasmid specific to protein kinase CK2alpha. The weight and volume of subcutaneous tumors were measured. The expression level of protein kinase CK2alpha mRNA and protein of tumors were measured with reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical technique, respectively.@*RESULT@#Protein kinase CK2alpha mRNA and protein expressions were significantly decreased in tumors transfected with siRNA expression plasmid specific to protein kinase CK2alpha (P<0.05). The tumor grew slowly after transfected with siRNA expression plasmid specific to protein kinase CK2alpha (P<0.01).@*CONCLUSION@#The siRNA expression plasmid specific to protein kinase CK2alpha may suppress the growth and the protein kinase CK2alpha expression of subcutaneous tumors. RNA interfering technology may be a new strategy for the treatment laryngeal cancer.
Subject(s)
Animals , Female , Humans , Mice , Casein Kinase II , Genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Mice, Inbred BALB C , Mice, Nude , Plasmids , RNA, Small Interfering , Genetics , Transfection , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE@#To explore the methods of culture, identification and label of embryonic rat neural stem cells.@*METHOD@#The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of BrDU. The neural stem cells (NSCs) were marked with fluorescent dye, bisbenzimide (Hoechest33342) and induced to differentiate. The differentiated cells were detected with Neuron Specific Enolase (NSE) and Glial Fibrillary Acidic Protein (GFAP) immunocytochemical fluorescent staining respectively.@*RESULT@#Nest-like clusters of neural stem cells were obtained in suspension and the cells could be differentiated into neurons and astrocytes which maintaining the main characteristics of NSCs after 8 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 8 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells.@*CONCLUSION@#The cells from embryonic rat hippocampus possessed the abilities of division, multiplication and self-renew, which were believed to be the main characteristics of NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye and could be used as donor cells in experimental research on NSCs transplantation.
Subject(s)
Animals , Female , Pregnancy , Rats , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Cells, Cultured , Embryonic Stem Cells , Cell Biology , Multipotent Stem Cells , Neural Stem Cells , Cell Biology , Neurons , Cell Biology , Rats, Sprague-DawleyABSTRACT
OBJECTIVE@#To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and study the possible role it may play in the etiology of autoimmune inner ear disease.@*METHOD@#A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30kd was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freund's adjuvant, another 10 guinea pigs were immunized with complete Freund 's adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique.@*RESULT@#The purity of the protein was demonstrated by a single band at the 30 kD site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F =6.48, P <0. 01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only.@*CONCLUSION@#P0 protein from guinea pig's inner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein is successfully established.
Subject(s)
Animals , Adenoviridae , Genetics , Cochlea , Metabolism , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel , Gene Expression , Gene Transfer Techniques , Genes, Homeobox , Genes, Reporter , Genetic Vectors , Guinea Pigs , Myelin P0 ProteinABSTRACT
In order to study the effect of 5, 6-Dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose-and time-dependent manner. After being treated with 0, 10, 20, 40, 80 microm mol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68+/-0.19)%, (1.95+/-0.12)%, (8.51+/-0.26)%, (11.26+/-0.17)% and (14.99+/-0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 microm mol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.
ABSTRACT
In order to study the effect of 5, 6-Dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose- and time-dependent manner. After being treated with 0, 10, 20, 40, 80 μmmol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68±0.19) %, (1.95±0.12)%, (8.51 ±0.26)%, (11.26±0.17) % and (14.99±0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 μmmol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.
ABSTRACT
OBJECTIVE To construct siRNA eukaryotic expression vector targeting protein kinase CK2?and to investigate its inhibitory effect on invasion of the HEp-2 cell line in human laryngeal carcinoma. METHODS siRNA expression vector psiRNA-hH1neo-CK2 targeting protein kinase CK2?was constructed by gene recombination,and then was transfected into the HEp-2 cells by lipofectamine methods. Protein kinase CK2?mRNA and protein of the transfected cells were detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blot,respectively. The invasion of the transfected cells was measured by Boyden chamber.SP method was used to examine the expressions of MMP2 and TIMP2 protein of the transfected HEp-2 cells. RESULTS Protein kinase CK2?siRNA expression vector was successfully constructed by gene recombination. Compared with non-specific interfering groups and blank groups, protein kinase CK2?mRNA and protein were significantly decreased respectively in the psiRNA-hHIneo-CK2 groups(P
ABSTRACT
In order to study the expressions of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in human laryngeal squamous cell carcinoma (LSCC) and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues (both P < 0.01), and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ill + IV tissues of LSCC as compared with the stage I + II tissues of LSCC (P < 0.01). There was a high positive correlation between VEGF and COX-2 expression in LSCC (r = 0.756, P < 0.01). These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/genetics , Laryngeal Neoplasms/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Biomarkers, Tumor , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/geneticsABSTRACT
In order to study the expressions of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in human laryngeal squamous cell carcinoma (LSCC) and its significance,the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues (both P＜0.01), and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ⅲ + Ⅳ tissues of LSCC as compared with the stage Ⅰ + Ⅱ tissues of LSCC (P ＜0. 01). There was a high positive correlation between VEGF and COX-2 expression in LSCC (r=0. 756,P＜0.01). These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.