OBJECTIVETo study the effect of histone deacetylation 6 (HDAC6) siRNA on the growth of xenografted human laryngeal squamous cell carcinoma cell line Hep-2 in nude mice and underlying mechanism.

METHODSLaryngeal squamous cell carcinoma cell line Hep-2 cells were subcutaneously injected to the back of nude mice and transplanted tumor model was established after one week. Nude mice was divided into three groups including blank control group, empty vector group and HDAC6 siRNA group, and the tumor growth was observed. Ki-67 proliferation index was detected by immunohistochemistry. Western blot, in situ hybridization and immunohistochemistry were used to detect the mRNA and protein expressions of HDAC6 in xenograft. The expressions of Bcl-2 and Bax proteins were examined by Western blotting. Cell apoptosis was detected by TUNEL.

RESULTSThe mean volume of xenograft transfected with HDAC6 siRNA was less than that of xenograft transfected with empty vector or that of xenograft with blank control treatment (P < 0.05). HDAC6 siRNA effectively down-regulated the expressions of HDAC6 mRNA and the expressions of HDAC6 and Bcl-2 proteins, but up-regulated the expression of Bcl-2 protein in xenografts, with significant differences (all P < 0.05). The proliferation index of Ki-67 in HDAC6 siRNA transfection group was significantly lower than that in blank control group or empty vector group (P < 0.05). TUNEL assay demonstrated that HDAC6 evidently evoked cell apoptosis (P < 0.05).

CONCLUSIONHDAC6 siRNA could effectively inhibited the growth of xenografted human laryngeal carcinoma cell line Hep-2 in nude mice, down-regulate the expressions of HDAC6 and bcl-2, and up-regulate the expression of bax.

Animals ; Cell Line, Tumor ; Down-Regulation ; Female ; Histone Deacetylase 6 ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

Objective To assess the sensitivity of indices of hip development in children with spastic diple-gia resulting from cerebral palsy. Methods X-ray images of the hips of 57 children with cerebral palsy ( the cere-bral palsy group) were checked, and the acetabular index ( AI), femur head migration percentage ( MP), center-edge angle and neck-shaft angle (NSA) were compared with those of normal children ( the control group, n = 30).Results The differences in MP and NSA between the two groups were significant. The prevalence of hip subluxation was 20.45% among the children with spastic diplegia who could not walk independently, and the prevalence was sig-nificantly greater in children 3 to 5 years old than among those under 3. Conclusion The MP can be used as a sen-sitive index to evaluate hip development. Age is a relevant factor affecting the hip development of children with cere-bral palsy.

OBJECTIVETo investigate the effects of histone deacetylase 2 (HDAC2) expression on cell proliferation, apoptosis and migration of laryngeal squamous cell carcinoma (LSCC) Hep-2 cells.

METHODSHDAC2 siRNA and control siRNA were transfected into LSCC Hep-2 cells by lipofectamine 2000, and cells were divided into three experimental groups: untreated group, control siRNA group and HDAC2 siRNA transfection group. Western blotting was utilized to detect the expression of HDAC2 protein in Hep-2 cells. Cell proliferation and apoptosis were investigated by CCK-8 kit and flow cytometry, respectively. Boyden chamber was used to study cell migration. Expressions of cell apoptosis and cell migration related proteins were detected by Western blotting.

RESULTSHDAC2 siRNA significantly down-regulated the expression of HDAC2 protein in LSCC Hep-2 cells. Down-regulation of HDAC2 expression coincided with an inhibition of cell proliferation and migration along with an induced cell apoptosis of Hep-2 cells. Moreover, down-regulation of HDAC2 expression significantly increased the expressions of caspase-3 and caspase-9 proteins but decreased the expressions of matrix metalloproteinases (MMP)-2 and MMP-9 proteins.

CONCLUSIONSHDAC2 may play a pivotal role in the initiation and development of LSCC. Down-regulation of HDAC2 expression mediates cell apoptosis. Cell migration inhibition may be tightly associated with overexpression of caspase-3 and caspase-9 along with down-regulation of MMP-2 and MMP-9 expressions.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Down-Regulation ; Histone Deacetylase 2 ; genetics ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo observe the effects of small interfere RNA (siRNA) targeting the c-myc in combination with 5-fluorouracil (5-Fu) on the growth of Hep-2 cells in vitro and in vivo.

METHODSHep-2 cells transfected with or without c-myc siRNA were treated with 5-Fu for 48 h. C-myc protein levels in Hep-2 cells were detected using the Western blot. The cell cycle was analyzed by flow cytometry. Hep-2 cells were subcutaneously inoculated into the back of BALB/c nude mice to establish the implanted laryngeal squamous carcinoma model. PBS, c-myc siRNA, and 5-Fu, alone or in combinations were administered i.p. The mice were sacrificed after the treatments and the tumor masses were removed to determine the tumor volume and weight. The inhibitory rate was calculated. Expression of c-myc in tumor tissue was detected by immunocytochemistry and cell apoptosis was analyzed by terminal transferase dUTP nick end labeling (TUNEL).

RESULTSThe protein levels of c-myc decreased after transfected with c-myc siRNA. C-myc siRNA-transfected cells showed an increase in the percentage of cells in the GO-G1 phase and a decrease in the percentage of cells in the S phase. When combined with 5-Fu, the results were improved. The tumor growth was faster in the control group and was significantly slower in the c-myc siRNA plus 5-Fu group than that in the c-myc siRNA group or 5-Fu group (P < 0.05). The tumor weight in the c-myc siRNA plus 5-Fu group was significantly smaller than that in the c-myc siRNA or 5-Fu group (P < 0.05). Immunohistochemistry showed that c-myc siRNA inhibited the expression of c-myc in tumor tissues in the c-myc siRNA group and c-myc siRNA plus 5-Fu group (P < 0.05). The number of apoptotic cells in the c-myc siRNA plus 5-Fu group was higher than those in the c-myc siRNA groups (P < 0.05).

CONCLUSIONSC-myc siRNA inhibits the expression of c-myc in Hep-2 cells and in the tumor tissues of nude mice. C-myc siRNA combined with 5-Fu inhibits the growth of implanted laryngeal squamous carcinoma and promotes cell apoptosis. C-myc could become a novel target for the treatment of laryngeal squamous carcinoma.

Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Fluorouracil ; pharmacology ; Gene Silencing ; Humans ; Laryngeal Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Small Interfering ; genetics ; Transfection

AIM:To investigate the effects of thalidomide ( THD) on the activation of connective tissue growth factor ( CTGF) gene promoter induced by transforming growth factor β1 ( TGF-β1 ) in human embryonic lung fibroblasts ( HELF) .METHODS:DNA sequence of CTGF gene promoter was cloned into luciferase reporter gene vector to construct the recombinant eukaryotic expression vector pGL 3-CTGFP, and the recombinant vector was transfected into HELF cell line.The effects of TGF-β1 and THD on the activation of CTGF gene promoter were detected by dual-luciferase analysis . RESULTS:TGF-β1 increased the reporter gene activity dose-dependently (P<0.05), with a plateau at 5 μg/L being 2.16 folds as high as the control .TGF-β1-induced increase in the reporter gene activity was also time-dependent ( P<0.05).After exposure to TGF-β1(5 μg/L), the level of luciferase activity reached its peak at 12 h and was 2.52 folds as high as the control .THD significantly inhibited TGF-β1-induced increase in the reporter gene activity in a dose-dependent manner , but its basal activity was not changed .CONCLUSION: TGF-β1 stimulates the transcriptional activity of CTGF gene promoter in HELF cells in a dose-and time-dependent manner , while THD may inhibit the effects dose-dependently .

OBJECTIVETo explore the silencing effect of short hairpin RNA (shRNA) on the CD28 of mice T lymphocytes by CD28-shRNA expressing plasmid evaluate the interfering effects (chronology and stability) mediated by shRNA and select out the most efficient CD28 shRNA sequence.

METHODSThree CD28 specific and one non-specific shRNA expressing plasmids were constructed and then transfected separately into mice spleen T lymphocytes. Non-transfected cells and non-specific shRNA were taken as controls. Inhibitory effect of CD28 shRNA was demonstrated by real-time quantitative PCR and Western blots. The sequence of the highest RNA interference (RNAi) efficacy was screened.

RESULTS(1) CD28 shRNA expressing plasmids were successfully constructed; (2) Three CD28 specific shRNAs effectively inhibited the expression of CD28 at the mRNA and protein levels, and there was a statistically significant difference comparing with the controls (P < 0.01): The copies of CD28 in mice spleen cells at the mRNA levels were persistently decreased by 99.62%, 99.89% and 99.80% respectively after 20 days, and so did at the protein level [(84.90 +/- 0.65)%, (96.49 +/- 0.03)%, (91.76 +/- 0.32)% respectively]. The highest inhibitory rate was in CD28 shRNA-2 group.

CONCLUSIONS(1) Specific shRNA can mediate long-term and stable silencing effects on CD28 gene; (2) shRNAs matching different sites of CD28 gene exert differential inhibitory effects.

Animals ; CD28 Antigens ; genetics ; Cells, Cultured ; Gene Expression Regulation ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; RNA Interference ; T-Lymphocytes ; metabolism ; Transfection

OBJECTIVETo investigate the failure of internal fixation on displaced femoral neck fractures in adults under fifty-five years old retrospectively inorder to pay more attention to the treatment of these fractures.

METHODSFrom Junary 2007 to June 2010,18 failed cases of internal fixation on displaced femoral neck fractures in adults under fifty-five years old were treated,there were 13 males and 5 females with an average age of (48.0 +/- 6.0) years old ranging from 27 to 55. Among them, 17 patients were treated with cannulated screws and 1 patient was treated with intramedullary nail; 16 patients were diagnosed as osteonecrosis and 2 patients as osteonecrosis associated with nonunion.

RESULTSThe average time from internal fixation to failure was 23 months (ranged, 8 to 32 months). The quality of fracture reduction in Garden index was poor. The Harris Hip Score was (56.0 +/- 12.5) (ranged,33 to 80). Eight cases of osteonecrosis and 2 cases of nonunion combinated osteonecrosis were received total hip arthroplasty. Hip resurfacing arthroplasty were performed for other 5 osteonecrosis. Because of no evident clinical symptoms,the other 3 cases received conservative treatment. The patients with total hip arthroplasty and hip resurfacing arthroplasty were followed-up for 34 months ranging from 12 to 53 months. After operation,the Harris score was (94.0 +/- 3.0) ranged 89 to 96.

CONCLUSIONOsteonecrosis is a common complication after internal fixation on displaced femoral neck fracture in adults under fifty-five years old. More attention should be paid to the treatment of displaced femoral neck fracture in those patients.

Adult ; Female ; Femoral Neck Fractures ; diagnostic imaging ; physiopathology ; surgery ; Fracture Fixation, Internal ; adverse effects ; Humans ; Male ; Middle Aged ; Recovery of Function ; Retrospective Studies ; Tomography, X-Ray Computed ; Treatment Failure

OBJECTIVEHaemophilus (H.) influenzae is a gram-negative bacillus that is a common commensal organism of the human upper respiratory tract and an important cause of human diseases such as pneumonia, meningitis, septicemia, epiglottitis and cellulitis. Strains of H. influenzae are classified according to their capsular polysaccharide. There are six serotypes, designated as a through f. In addition, there are nonencapsulated strains. Although the type of infectious diseases caused by H. influenzae has changed considerably in recent years because of the widespread and routine immunization of children against type b H. influenzae (Hib), Hib remains an important pathogen. Ampicillin is the drug of choice for treating many infections caused by H. influenzae, but its usefulness has been compromised by the increasing prevalence of ampicillin-resistant strains. The continued monitoring of resistant strains by using genotyping methods may provide insights into the epidemiology of transmission. A molecular epidemiological study of ampicillin-resistant H. influenzae derived from nasopharyngeal swabs specimens of children less than 5 years of age with respiratory tract infection were investigated in this study.

METHODSA total of 899 isolates were collected from Beijing, Shanghai, and Guangzhou during 2000-2003. Susceptibility to ampicillin was determined by using E-test. Ampicillin-resistant H. influenzae strains were selected according to National Committee for Clinical Laboratory Standards (NCCLS) 2002 breakpoints. Nested PCR method with primers specific for bexA gene and b capsulate type-specific gene was established. Genotyping by pulsed-field gel electrophoresis (PFGE) and multiplex PCR assay was performed for all ampicillin-resistant H. influenzae strains.

RESULTSSeventy-four ampicillin-resistant H. influenzae strains were obtained. Two strains were positive by nested PCR, characterized as b genotype. The incidence of Hib in ampicillin-resistant H. influenzae strains was 2.7%; 38 genotypes were detected by PFGE. Detection of five types strains of clonal dissemination by PFGE accounted for 55.4% in all ampicillin-resistant H. influenzae strains. Among them eighteen H. influenzae strains belonged to one type, accounted for 24.3% in all ampicillin-resistant H. influenzae strains. Thirty one genotypes were identified by multiplex PCR assay for ampicillin-resistant H. influenzae. The identity ratio of PFGE and multiplex PCR was 63.5%.

CONCLUSIONIn Beijing, Shanghai and Guangzhou areas 55.4% of ampicillin-resistant H. influenzae strains had clonal dissemination during the 4 years.

Ampicillin Resistance ; genetics ; Anti-Bacterial Agents ; pharmacology ; Child, Preschool ; China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Haemophilus Infections ; epidemiology ; microbiology ; Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Nasopharynx ; microbiology ; Polymerase Chain Reaction ; Respiratory Tract Infections ; microbiology

This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.
ADAM Proteins ; genetics ; ADAM10 Protein ; Amyloid Precursor Protein Secretases ; genetics ; Animals ; Cell Line ; Cloning, Organism ; Genes, Reporter ; Genetic Vectors ; Luciferases ; genetics ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Plasmids ; Promoter Regions, Genetic