Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.

Objective To explore the damage effect of sonoporation on the prostate of rabbit,while opening up the blood-prostate barrier by microbubble mediated sonoporation.Methods Fifteen male New Zealand white rabbits were randomly divided into 3 groups:ultrasound (US) group,microbubble (MB) group,ultrasound and microbubble (US+MB) group.Ultrasound was insonated directly on the prostate.Optical microscope,electron microscope and apoptosis index (AI) with TUNEL method were applied to trace the changes of the prostate of rabbit under different conditions.Results There was no significant change in prostatic tissues of group US and MB under the optical microscope.Cytoplasm and nucleoli were stained equally,cells of glandular epithelium were intact and formed orderly.Glandular cavities in these two groups were change very slightly.Glandular epithelium cells of Group US+MB were organized optical under the optical microscope,and there was a mass of eosinophilic liquid in the glandular cavities.Vascular endothelial cell were intact and formed orderly and swollen mitochondria were observed under the electron microscope in MB group and US group.Swollen mitochondria,tight junctions among gland cells were opened,and infiltrated erythrocyte could be found under the eletron microscope in US+MB.AI of group US+MB was markedly higher than that of group US and group MB (P<0.01),and AI of group US was higher than that of group MB (P<0.01).Conclusions Microbubble mediated sonoporation causes damage in the prostate tissue of rabbit,while opening up the blood-prostate barrier with an increased permeability of the prostate.

Objective To compare the in vitro antitumor immune responses induced by bivalent bispecific anti-idiotype antibody G22-I50 and monovalent anti-idiotype antibody G22 and I50, and explore its possible mechanism. Methods Proteins G22-I50, G22, and I50 were induced and identified by Western blot and ELISA. Peripheral blood monoclear cells (PBMC) were isolated and stimulated with G22-I50, G22, and I50 anti-idiotype antibodies, respectively. MTT assay and LDH release test were employed to examine the proliferation and cytotoxicity of the PBMC. The levels of IFN-γ, IL-2, and IL-4 in the supernatant were detected by ELISA and changes of T lymphocyte subsets were determined by flow cytometry. Results Western blot showed that G22-I50, G22, and I50 had specific binding capabilities to FC2 (Ab1). The activities of G22-I50, G22, and I50 had recovered and these proteins could be used in the in vitro study. The proliferation and cytotoxicity of the PBMC stimulated with G22-I50 were significantly higher than those stimulated with G22 or I50, The level of IFN-γ and IL-2 in the culture supernatant of the PBMC stimulated with G22-I50 was higher than that in the G22 or I50 group, but the level of IL-4 did not increase.Compared with the G22 or I50 group, the proportion of CD4+ and CD8+ T cells and CD4+/CD8+ ratio significantly increased, and the proportion of CD4+CD25+ T cells significantly decreased in the PBMC stimulated with G22-I50. Conclusion G22-I50 has more potent immunogenicity and would enhance specific antitumor effect which might relate to improving PBMC proliferation, inducing the secretion of Th1 type cytokines, activating CD8+T cells, and suppressing the expression of CD4+CD25+ T cells.

OBJECTIVE To investigate the antitumor effect of cadmium (Ⅱ) complex of pyrazolone derivatives 1-phenyl-3-methyl-4-propionyl-5-pyrazolone salicyloyl hydrazide-cadmium (Ⅱ) (Cd-PMPP-SAL) on the murine melanoma B16 cells in vitro and in vivo and its mechanisms. METHODS B16 cells were incubated with Cd-PMPP-SAL at 1.0, 1.5, 3.0, 5.0 and 10.0 mg·L-1 for 24, 48 or 72 h. The prolifera? tion rate of B16 cel s was evaluated by MTT assay. B16 cel s were incubated with Cd-PMPP-SAL at 6.25, 12.50 and 25.00 mg·L-1 for 24 h, while cell morphology was observed by Hoechst33258 staining. Apop?tosis of B16 cells was detected by Annexin Ⅴ-FITC/PI staining. The activity of caspases in B16 cells was detected by caspase activity assay. C57BL/6J mice were inoculated subcutaneously with B16 cells to establish a tumor-bearing model. Five days later, Cd-PMPP-SAL at 6.25, 12.50 and 25.00 mg·kg-1 was injected into tumors of C57BL/6J mice once a day for 12 d. The body mass was recorded daily. One day after the last administration, all the mice were killed and the tumor was harvested. Tumor volume and mass were measured, and the tumor inhibitory rates were calculated. Pathological changes of the tumor, liver and lung were observed under a microscope. The expressions of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) in tumor tissues were detected by immuno?histochemistry. The apoptotic cells in transplanted tumor tissues were detected by TUNEL. RESULTS Cd-PMPP-SAL inhibited the proliferation of B16 cells. The IC50 was 4.946 mg · L-1, and 95% confidence interval was 4.24-5.65 mg · L-1. The apoptosis rates(12.8 ± 1.4)% and (18.4 ± 0.4)% of Cd-PMPP-SAL 12.50 and 25.00 mg · L-1 groups were significantly higher than those of control group (1.7 ± 0.1)% (P<0.01). The activity of caspase 3 and 9 of Cd-PMPP-SAL 25.00 mg · L-1 group was significantly higher than that of control group (P<0.01), but there was no significant difference in caspases 3/7. The relative tumor volumes of Cd-PMPP-SAL 6.25, 12.50 and 25.00 mg · kg-1 treated groups from the 8th day of treatment were significantly decreased compared with the model group (P<0.01). The result of paraffin sections showed that the transplanted tumor tissues in Cd-PMPP-SAL 12.50 and 25.00 mg · kg- 1 groups exhibited different degrees of necrosis, but there was no significant pathological damage to the liver or lung tissues of mice. Compared with model group, expressions of VEGF and FGF2 in Cd-PMPP-SAL 12.50 and 25.00 mg · kg-1 treated groups were significantly inhibited (P<0.05), and apoptotic cell rates were significantly higher (P<0.05). CONCLUSION Cd-PMPP-SAL can inhibit growth of B16 cells in vivo and in vitro, which may be associated with induction of tumor cell apoptosis and inhibition of tumor angiogenesis.

Objective To screen the anti-nasopharyngeal carcinoma scFv from a human anti-nasopharyngeal carcinoma single-chain phage antibody library, and identify its characteristics. Methods The single-chain phage antibody library was subjected to three rounds of positive and negative cell panning and enrichment, and then it was selected by ELISA. The binding specificity of phage antibodies with naso-pharyngeal carcinoma cells was confirmed by immunohistochemistry. Results After panning, enrichment and testing by ELISA, 3 phage an-tibody clones reacting with CNE2 more strongly than HUVEC and NP69 were picked out from 4212 clones. One clone, HNSAO33, was fur-ther analyzed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. HNSAO33 spe-cifically reacted to nasopharyngeal carcinoma cells in most human nasopharyngeal carcinoma tissue sections except a few human normal naso-pharyngeal tissue sections. The distinction of positive rates was of a great statistical significance. Conclusion ELISA and immunohisto-chemistry results confirmed HNSAO33 specifically bind with nasopharyngeal carcinoma cells. The seFv fragment against nasopharyngeal carci-noma may be further developed and applied in clinical diagnosis and therapy of nasopharyngeal carcinoma.

Objective To explore the safety and clinical efficacy of central venous infusions of concentrated potassium chloride corrects hypokalemia by micro-pump in intensive care unit(ICU). Methods The data was analyzed retrospectively on 78 patients with hypokahmia in ICU. They were randomly divided into 2 groups: general group (39 cases) with potassium concentration 40 mmol/L and the rate 10-20 mmol/h,concentrated potassium group(39 cases) with potassium concentration 100-300 mmol/L and the rate 40-200 retool/h, calculating the whole potassium dosage respectively, examining the initial potassium concentration, urine volume per hour, the mean time to aimed potassium concentration and side effect.Results The initial potassium concentration and the whole potassium dosage were no significant difference between the two groups [(2.9 ± 0.2), (3.0 ± 0.2) mmol/L and (85.2 ± 8.7), (92.3 ± 7.6) mmol, respectively](P >0.05). It took longer time reaching the aimed potassium concentration in general group than that in concentrated potassium group [(17.25 ± 4.49) hours and (5.67 ± 0.75) hours, respectively] (P < 0.01).There were no comphcations such as hyperkalemia, fatal arrhythmia and phlebitis. Five patients were bloating in general group. Condusions Under meticulous monitoring, it is effective and relative safely to correct hypokahmia by central venous infusions of concentrated potassium chloride using micro- pump in ICU. The therapy is of clinical value in treating hypokalemia patients.

Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.

Objective To evaluate perioperative nutritional support for patients with acute abdomen in enhanced recovery after surgery (ERAS) programme.Methods A total of 490 patients with acute abdominal disease were collected,287 in ERAS group and 203 in conservative perioperative management (CPM) group.Biochemical and clinical markers of the 2 groups were compared.Results ERAS group had higher plasma albumin level,quicker bowel function recovery,lower postoperative complications,shorter hospital stay,and a lower WHO pain rating scale (all P ＜ 0.05).Conclusion With perioperative nutritional support,ERAS programme can accelerate recovery after emergency surgery,reduce the rate of overall complications,promote bowel function recovery,and decrease morbidity in the perioperative period for patients with acute abdominal disease.

OBJECTIVE: To determine the effect of gamma-aminobutyric acid(GABA) on proliferation and malignant phenotypes of hepatocellular carcinoma cell line HepG-2. METHODS: HepG-2 cells were cultured by routine method, and then treated with different concentrations of GABA. The proliferation of HepG-2 cells was measured through MTT, doubling time and cell cycles by flow cytometry. The malignant phenotypes were investigated by soft agar colony formation assay. RESULTS: Compared with the control group, GABA efficiently stimulated the proliferation of HepG-2 cells in a dose-dependent manner and affected the distribution of cell cycles of HepG-2 cells. The doubling time of the control group and the GABA-treated group were 39.0, 30.6, 30.0, 27.3, 26.6, and 38.2 h, respectively. The colony formation rates were 3.2%, 4.2%, 5.4%, 6.6%,6.5%, and 3.5%, respectively. Tumorigenicity testing showed that the average weights of tumor was 1.382 g, and 0.285 g for the 2 groups. The difference between the control group and the GABA-treated groups was significant (P<0.01). CONCLUSION GABA can enhance the proliferation and malignant phenotypes of HepG-2 cells.
Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Hep G2 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Phenotype ; gamma-Aminobutyric Acid ; pharmacology