Objective:To construct eukaryotic expressing vector of the full length coding sequence of Bad gene and to express the gene in the basal cell carcinima A431 cells.Methods:Bad gene was amplified from Hela cell line by RT-PCR and the fragment of the cDNA was cloned into eukaryotic expressing vector pcDNA3.1-myc by ligating the fragment into XhoI and EcoRI site.The recombinant plasmid pcDNA3.1-myc-Bad was identified by DNA sequencing and restriction enzyme analysis.The gene transfection mediated by lipofectin was used to introduce the eukaryotic expressing vector of pcDNA3.1-myc-Bad into human basal cell carcinima A431 cells. After selection with G418, resistant colonies were obtained.Trasfection efficiency was identified by Western blot and SABC-FITC assay.Cell proliferation was examined by cell counting and colonogenic assay after transfection.Results:A 500 bp DNA fragment was amplified with RT-PCR.Sequence and restriction enzyme analysis showed that the recombinant plasmid pcDNA3.1-myc-Bad was constructed successfully.In human basal cell carcinima cell line A431 Bad gene was expressed.The cell proliferation was inhibited by 62.6% and colonogenesis by 39.9% by the transfection of the gene.Conclusion: Human Bad gene was successfully cloned.Transfection of basal cell carcinoma cells with the gene may inhibit the cell proliferation and colonogenesis.

Objective To explore effective methods of diagnosis and treatment for delayed rupture of the spleen (DRS) through retrospective analysis of 32 cases. Methods A retrospective study was done in 32 cases with DRS. The effects of CT, ultrasound and diagnostic peritoneal lavage on diagnosis of DRS was observed and compared. Results Splenectomy was performed in 26 cases, among which one died four days after splenectomy because of complicated brain injury and six given conservative treatment under continuous monitoring discharged. The mean hospitalization duration was 18.5 days. Conclusions Diagnostic peritoneal lavage can improve the positive rate of peritoneal puncture. Ultrasound is an important method for diagnosing DRS and closely monitoring the patients receiving conservative treatment.

Objective To observe the effeet of cotransduction of wt-p53,B7-1 and GM-CSF genes mediated by recombinant adenovirus on cell growth, apoptosis and immunogenicity of neuroblastoma cell line SH-SY5Y, Methods We transfected SH-SYSY cells separately with recombinant adenovirus, recombinant adenovirus mediating human wild type p53(wt-p53) gene and BB-102.We separately named them with SH/Ad,SH/p53 and SH/BB-102 group.The protein expression of p53, B7-1 and GM-CSF were measured by Western blot, FCM and enzyme linked immunosorbent assay. Cell number was counted and growth curves were drawed. Cell apoptosis was tested by FCM. Lymphocyte proliferation and cytokine secretory was evaluated by mixed lymphocyte culturing. Results After transection with BB- 102, p53, B7-1, and GM-CSF, high-efficiency expression of target genes were found. Growth of the cells was inhibited, apoptosis was induced in the SH/p53 and SH/BB-102 groups. Proliferation of lymphocytes was stimulated and cytokine increased visorously in the SH/BB-102 group. Condusions After transfection with BB-102, growth of the SH-SY5Y cells was inhibited,apoptosis was induced,and immunogenicity was greatly enhanced.

Objective To investigate the effects of sodium butyrate (SB) on fetal gastric epithelial cells (GECs) in vitro damaged by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Methods The fetal gastric epithelial cells were isolated, cultured and identified. Then the cells of the second generation were cocultured with 10 -5 mol/L, 10 -6 mol/L, 10 -7 mol/L SB for 4 h, then added with 10 -5 mol/L MNNG culturing for another 24 h. After the treated cells were finally cultured in normal culture medium for 40 d, the unscheduled DNA synthesis (UDS), lipid peroxidation (LPO) and ras p21 were detected. The cells were only cultured with 10 -5 mol/L MNNG served as negative control and without MNNG or SB as normal control. Results The level of UDS, the contents of lipid peroxidation products and the concentrations of ras p21 in GECs treated with 10 -5 mol/L and 10 -6 mol/L SB decreased obviously, and had significant differences with that of GECs treated without SB. Conclusion SB can effectively prevent the damage of GECs induced from MNNG.

Adenocarcinoma ; genetics ; metabolism ; Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; Child ; Child, Preschool ; DNA-Binding Proteins ; biosynthesis ; genetics ; Female ; Gastric Mucosa ; metabolism ; Genes, Tumor Suppressor ; Humans ; Male ; Middle Aged ; Nuclear Proteins ; biosynthesis ; genetics ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Diseases ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; Tumor Protein p73 ; Tumor Suppressor Proteins

Objective To construct series of reporter plasmids with truncated and deleted hTERT promoter.Methods Gene fragments of hTERT promoter was amplified by PCR and cloned into pGL3-Basic to construct luciferase reporter vectors.Dual luciferase assays were performed with cell lysates of HepG2 and COS-7 cells cotransfected with hTERT promoter reporter plasmids and pRL-TK.Results Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed and respectively named pGL3B-895,pGL3B-371,pGL3B-DELS2,pGL3B-349,pGL3B-329,pGL3B-318,pGL3B-306.Dual luciferase reporter assays showed that all the reporter vectors have promoter activity both in HepG2 and COS-7.Conclusion Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed,and their promoter activity were verified.These plasmids provide necessary experimental naterials for further investigation of regulation of hTERT during hepatocarcinoma development.

Objective To explore a new peptide-based approach independent of HLA to generate antigen-specific CD+ CD8+T cells. Methods Peripheral blood mononuclear cells(PBMC) were stimula- ted for 6 h with IE-1 peptide pool. Then the activated IFN-γsecreting ceils were tested by immunomagnetic selection. And the selected cells were cultured with radio-inactivated PBMC in medium with 100 IU/ml IL-2 for 4 weeks. Results The generated T cell lines consisted of IE-1 specific CD4+ T (6.88%) and CD8+ T cells 92.99%, which demonstrated antigen-specific killing and cytokine secretion. Conclusion T ceils can be proliferated with this new procedure, and maintain its phenotype and antigen-specific function.

Objective:This paper aimed to investigate the effects of isocorydinone on cell proliferation in SiHa human cervical carcinoma cell lines. Methods:Different concentrations of isocorydione (100, 200, 400, 800, and 1200 μmol/L) were used to treat SiHa human cervical carcinoma cells in vitro for 24, 48, and 72 h. Methyl thiazolyl tetrazolium (MTT) assays were conducted to determine the inhibitory action of isocorydione. Flow cytometry was performed to detect the cell cycle in SiHa human cervical carcinoma cells af-ter treatment with 400 μmol/L isocorydione. Hoechst 33342 staining was used to observe the micro-morphological changes of SiHa cell nucleus after the treatment. The expression of Bcl-2, Bax, and caspase-3 proteins in cervical carcinoma SiHa cell lines was determined using western blot analysis. Results: MTT assays showed that isocorydione inhibits the proliferation of SiHa cells in a dose- and time-dependent manner (P<0.05). The flow cytometry results showed that SiHa cervical carcinoma cells treated with different concen-trations of isocorydione exhibited increased cell cycle. Compared with the control group, Hoechst 33342 staining showed that SiHa cells became narrow, with nuclear pyknosis and fragmentation, and formed an apoptotic body after treatment with 400 μmol/L isocoryd-ione for 48 h. Furthermore, western blot analysis proved that isocorydione significantly inhibited the proliferation of SiHa cell lines, and the expression of Bax protein was increased. By contrast, the expression of Bcl-2 protein decreased gradually. Consequently, the ra-tio of Bax/Bcl-2 increased, as well as the expression of caspase-3 protein. Conclusion:Isocorydione exhibited an overt inhibitory ac-tion on SiHa cells. Isocorydione promoted the occurrence of cell apoptosis, which may be associated with related proteins of mitochon-drial apoptotic pathway.

ObjectiveTo evaluate the disinfective effect of a new peracetic acid solution for digestive endoscope.MethodsForty endoscopes were divided into experimental group and control group,10 gastroscopes and 10 enteroscopes in each group,the experimental group was disinfected with the new peracetic acid solution for 10 min,the control group was disinfected with 2％ glutaral for 10 min,the disinfection effect was compared.Subsequently,80 other endoscopes were divided into 4 groups,10 gastroscopes and 10 enteroscopes in each group,each group was disinfected for 2 min,3 min,4 min and 5 min,the disinfection efficiency was evaluated.ResultsThe disinfection rates of gastroscopes and enteroscopes in the control were 100％ (10/10)and 90％ (9/10)respectively.Bacteria were found in both endoscopes.In the experimental group,disinfection rates of both gastroscopes and enteroscopes were 100％ (10/10),and no bacterium was found,which was superior to the control.disinfection rates of gastroscopes of 3 min,4 min and 5 min were all 100％ (10/10),which were higher than that of 2 min group (30％) (P ＜0.05).Bacteria were found in 3 min group.Disinfection rates of 4 min and 5 min group were 100％ ( 10/10),which were higher than that of 3 min group (80％)(P ＜0.05).Bacteria were found in 4 min group,and 2 min group was not disinfected.ConclusionThe new peracetic acid solution is effective for clinic digestive endoscope disinfection,and is superior to 2％ glutaral.

Peptide self-assembled multilayers ( SAMs ) was coated onto the quartz surface. The assembly conditions, such as the assembly agent concentration and assembly time, were examined. The SAMs was characterized via UV-vis absorption spectrometry and scanning electron microscope. Our results showed that the optimal concentration and assembly time for the 3-aminopropyl-triethoxysilane aqueous were 1% ( V/V ) and 3 h respectively, and those of gold nanoparticles were 2. 4 × 10-4 mol/L and 12 h, respectively, while those of peptide solution were 1 × 10-4 mol/L and 12 h, respectively. A new sensitive method, based on the theory that peptide could be cleaved at the site of Arg-Gly by thrombin, was established to detect thrombin. In addition, a good linear relationship was obtained in the range from 2. 8×10-12 mol/L to 9. 9×10-10 mol/L, and the detection limit was 1. 4×10-12 mol/L. The peptide self-assembled multilayers were also used in the analysis of blood serum samples, and the recovery rate was within the range from 91. 6% to 107. 6%.