Objective:To construct a fusion DNA vaccine pEGFP-C3-B7.2-hTERT and to observe the capability of pEGFP-C3-B7.2-hTERT DNA vaccine to induce specific anti-tumor immune responses and to inhibit growth of the hepatoma transplanted from H22 cells. Methods: The B7.2 and hTERT cDNA (amplified by RT-PCR), together with pEGFP-C3 as the vector were used to construct fusion gene plasmid pEGFP-C3-B7.2-hTERT, which was then used to vaccinated C57BL/6 mice for 3 times at a 7 d interval. Animals vaccinated with pEGFP-C3-B7.2, pEGFP-C3-hTERT, pEGFP-C3 and PBS were taken as controls. Splenocytes CTLs of immunized mice, the levels of IL-2, IFN-? in the culture supernatant, the levels of antibodies against hTERT, and the changes of the T lymphocyte subsets in the peripheral blood were all examined. The mice harboring hepatocarcinoma H22 cells were challenged with pEGFP-C3-B7.2-hTERT and the tumor forming time and the survival periods of mice were observed. Results: Agarose gel electrophoresis, nuclease digestion and sequencing confirmed the successful construction of pEGFP-C3-B7.2-hTERT. The CTLs activity of splenocytes from the mice immunized with pEGFP-C3-B7.2-hTERT fusion gene was significantly higher than those of the other groups (P

Animals ; Cell Survival ; Cells, Cultured ; GTP-Binding Proteins ; metabolism ; Isoproterenol ; adverse effects ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, beta-2 ; metabolism

Objective To study the expression and significance of ?-catenin in keratosic dermatosis and non-melanocytic skin tumors. Methods ?-catenin expression was determined in 10 specimens of normal skin,10 cases of seborrheic keratosis(SK),26 cases of actinic keratosis(AK),17 cases of Bowen's disease(BD) and 35 cases of primary cutaneous squamous cell carcinoma(SCC) by immunohistochemical EnVision two-step staining method. Results The expression of ?-catenin was significantly higher in SK,AK,BD and SCC than in normal skin(P0.05),but that was significantly lower in SK and AK than in BD(P0.05). Conclusion The cytoplasmic and/or nuclear expression of ?-catenin may play a role in the progression from normal skin to keratosic dermatosis and non-melanocytic skin tumors.

Objective To generate of human induced pluripotent stem cells from umbilical cord matrix cells(UMC).Methods Sox2 and Klf4 and Oct4 and c-Myc were transfected into UMC cells with retrovirus,and thcn UMC cells was reprogrammed to iPS cells.Gene expression was confirmed with RT -PCR and the integration was confirmed with cell karyotype.iPS cells were further validatcd with cell alkaline phosphatase detection and immunofluorescence staining,differentiating into teratomas in vivo and embryoid bodies in vitro.Results iPS cells were similar to embryonic stem cells (ES).The expression of Nanog,Oct4,Rex1 and Sox2 in iPS cells were higher than UMC cells,and Sox2,Klf4,Oct4,c-Myc silenced in iPS cells.Exogenous genes were inserted into the nucleus of iPS cells,which was confirmed by 1％ agarose gel electrophoresis.iPS ccll karyotype was normal,alkaline phosphatase activity increased,ES cell-specific proteins,including SSEA3,SSEA4,TRA-1-60,TRA-1-81 and Nanog,were expressed.iPS cells were differentiated into a teratoma in vivo and embryoid bodies in vitro.Conclusions iPS cells were similar to ES cells,which had pluripotency.

ObjectiveGeneration of human induced pluripotent stem cells from human skin fibroblasts.MethodsSox2, Klf4, Oct4, c-Myc were transfected into HSF cells with retrovirus, and then HSF cells was reprogrammed to iPS cells.Detecting cells endogenous and exogenous gene, analyzing karyotype,cells alkaline phosphatase staining and immunofluorescence staining, differentiating into teratomas in vivo and embryoid bodies in vitro were performed.ResultsiPS cell morphology was similar to embryonic stem cells (ES).The expression of Nanog, Oct4, Rex1, Sox2 in iPS cells were higher than HSF cells, and Sox2, Klf4, Oct4, c-Myc were silenced for the iPS cells.Exogenous genes were inserted into the nucleus of iPS cells, which was confirmed by 1％ agarose gel electrophoresis.iPS cell karyotype was normal, alkaline phosphatase activity increased, ES cell-specific protein expressed.iPS cells were differentiated into a teratoma in vivo and embryoid bodies in vitro.ConclusionsiPS cells were similar to ES cells, which have pluripotency.

AIM: To observe the expression of HIF-1? mRNA, HIF-1?, VEGF and iNOS proteins and to investigate their relationship in h ypoxia-treated SW480 cells. METHODS: HIF-1?, VEGF and iNOS proteins were measured by immuno cytochemistry. Western-blot was used to detect HIF-1? protein. HIF-1? mRNA wa s measured by in situ hybridization. RESULTS: Under hypoxic condition, SW480 cells expressed proteins of HIF-1?, VEGF and iNOS more strongly than that under normoxia condition. How e ver, under hypoxia condition, these three proteins expressed weakly or negativel y when the cells treated with genistein, the inhibitor of HIF-1?. Expressions o f HIF-1? and VEGF proteins in cultured SW480 cells under hypoxic condition were completely or partially inhibited by the addition of SNP but the expression of iNOS was unaffected. Another NO donor NOC5, however, induced the expression of t hese three proteins. L-NAME, a non-specific inhibitor of NOS, inhibited the expr ession of HIF-1?, VEGF and iNOS. The levels of HIF-1? mRNA changed slightly i n different oxygen condition or addition of genistein, NO donor or iNOS inhibitor . CONCLUSIONS: Hypoxia induces the expression of HIF-1?, therefor e upregulates the production of VEGF and iNOS. During hypoxia, SNP inhibits but N OC5 promotes HIF-1? expression, indicating that different NO donor acts on the cells through different mechanisms.

Objective To demonstrate the carbonic anhydrase Ⅲ (CAⅢ) for 25 000 protein decreased in skeletal muscle of myasthenia gravis (MG). Methods The protein molecular properties responsible to antibodies against 25 000 protein and CAⅢ were analyzed by a combination method of two-dimensional electrophoresis and immuno-Western blot. Competitive binding reactions of the antibodies to the purified 25 000 protein and muscular homogenate were observed by using immuno-Dot blot and immuno-Western blot, respectively. The expression of CAⅢ from normal and MG muscles was detected by immuno-Western blot. Results Combination analysis of two-dimensional electrophoresis and immuno-Western blot showed that the protein of immunological responsible to antibodies against 25 000 protein and CAⅢ had an identical molecular mass and isoelectric point. Competitive binding reactions proved that 25 000 protein and CAⅢ were the same substance, either by immuno-Dot blot or by immuno-Western blot. In addition, a much similar result was obtained when the levels of 25 000 protein from normal and MG muscles were detected by antibodies against 25 000 protein and (CAⅢ) by immuno-Western blot. Conclusion 25 000 protein decreased in the MG skeletal muscle was proved to be just a known protein CAⅢ, which made a basis for further exploring the relationship of CAⅢ deficiency and MG pathogenesis.

Objective To study the effect of chest wall vibration therapy on bronchiolitis. Methods A total of 64 patients with bronchiolitis were divided into an experimental group and a control group, the former included 34 cases and the latter included 30 cases. The experimental group received both routine treatment and chest wall vi- bration, while the control group only received routine treatment. PaO_2, PaCO_2, SaO_2, Heart Rate (HR) and Respi- ration (R) were observed, respectively, in the experimental group and the control group at the beginning and the end of the third day. Time needed for expectoration and length of hospital stay in the two groups were observed. Results It was shown that PaO_2, PaCO_2, SaO_2 , HR, R were significantly improved at the end of the third day when compared with those at the beginning in both groups(P

AIMTo investigate the role of overexpression of beta2-adrenoceptor on contraction in cardiac myocytes isolated from failure hearts of rats and primarily analyses its mechanisms.

METHODSPrimarily cultured cardiac myocytes were infected with adenovirus containing the sequence for human beta2-adrenoceptors. The expression of beta2-adrenoceptors was tested by Western blot. The contraction amplitudes induced by isoprenaline stimulation were measured.

RESULTSOverexpression of beta2-adrenoceptor increased the content in failure cardiac myocytes. The contraction amplitudes in failure cardiac myocytes were lower than that in the control (P < 0.01). Overexpression of beta2 adrenoceptor improved the contraction of failure cardiac myocytes (P < 0.01, Failure+ Adv.Beta2 group vs. Failure group). Selective beta2-adrenoceptor antagonist ICI 118,551 partially reversed the effects (P < 0.05, Failure+ Adv.beta2 + ICI group vs Failure + Adv.beta2 group), but the contraction amplitudes in this Failure +/- Adv.beta2 + ICI 118,551 group were still higher than that in only heart failure group (P < 0.05). Selective beta1 adrenoceptor antagonist CGP20712A completely inhibited the effects of overexpression of beta2 adrenoceptor on contraction amplitude in failure cardiac myocytes.

CONCLUSIONOverexpression of beta2-adrenoceptors improves the contraction of cardiac myocytes isolated from failure hearts of rats. The effect is related to beta1-adrenoceptor.

Adenoviridae ; genetics ; Adrenergic beta-Antagonists ; pharmacology ; Animals ; Cells, Cultured ; Heart Failure ; metabolism ; physiopathology ; Humans ; Imidazoles ; pharmacology ; Isoproterenol ; pharmacology ; Male ; Myocardial Contraction ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, beta-2 ; genetics ; metabolism

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Humans ; Leptin ; chemistry ; genetics ; metabolism ; Molecular Weight ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism