In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL)in vitro. After incubation with apoA-I for 24 h, RAW264. 7 cells effluxed 37.65% cholesterol loaded by acetyl LDL (ac-LDL), and 9.78% cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with 100 μg/mL ac-LDL or with 100 μg/mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.

The expression of synaptotagmin II (Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was conselected with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank: NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8% and 10% of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.

In order to investigate the effect of tumor necrosis factor-α (TNFα) on resistin expression in 3T3-L1 adipocytes, and further explore its mechanisms, the differentiated 3T3-L1 adipocytes were incubated with 0, 1, 10, 100 ng/mL TNFα respectively for 24 h, and then the expression of resistin was determined. The differentiated 3T3-L1 adipocytes were incubated with 100 ng/mL TNFα for 3, 6, 24 h respectively, and then the expression of resistin mRNA was analyzed. 3T3-L1 adipocytes were induced to differentiate into mature adipocytes. The cells were randomly divided into 4 groups for culture. In the control group, no drugs were added. Cells of TNFα group were treated with 100 ng/mL TNFα. In Ro-31-8220 group, 5 μmol/L protein kinase C inhibitor Ro-31-8220 was added. With TNFα+Ro-31-8220 group, 100 ng/mL TNFα were added 1 h after the addition of 5 μmol/L Ro-31-8220. All adipocytes were cultured for 24 h. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were employed to detect the expression of resistin gene. Our results showed that resistin protein and mRNA in 3T3-L1 adipocytes were inhibited by TNFα at different concentrations (P<0.01), and the inhibitory effect increased with the concentration (P<0.01). At the same concentrations, the inhibitory effect increased with time (P<0.01). Ro-31-8220 could inhibit its expression and the inhibitive effect remained unchanged with addition of TNFα(P>0.05). It was concluded that TNFα could inhibit the expression of resistin in 3T3-L1 adipocytes. The mechanism may be that the expression of resistin is partly controlled by protein kinase C signal conduction pathway.

To investigate the effects of WIN 55, 212-2 onI_K in cultured rat trigeminal ganglion (TG) neurons, whole-cell patch clamp techniques were used to record theI_K before and after WIN 55,212-2 perfusion at different concentrations. 30 μmol/L WIN 55,212-2 markedly (35.7%±7.3%P<0.01,n=8) inhibitedI_K currents, and the currents were partially recovered after washing. 30 μmol/L WIN 55, 212-2 also induced a significant depolarizing shift in conductance-voltage parameters (control: V_0.5=10.43±4.25 mV, k=16.27±3.86; WIN 55,212-2: V_0.5=24.71±3.91 mV, k=16.69±2.75;n=8,P<0.01 for V_0.5). 0.01 μmol/L WIN 55,212-2 slightly (27.0%±7.9%,P<0.05,n=7) increasedI_K currents, but had no significant change in conductance-voltage parameters (control: V_0.5=10.74±5.27 mV, k=17. 33±2.96; WIN 55, 212-2: V_0.5=11.06±2.05 mV, k=19.69±6.60;n=7,P>0.05 for V_0.5 and k). These results suggested that WIN 55, 212-2 has dual action, which might be through different receptors.

In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expression in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P>0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal thansduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.

To explore the pharmacological effect of 3,4-dihydroxyacetophenone (DHAP) on the apoptosis of RAW264. 7 macrophage cells and the mechanism, RAW264. 7 macrophage cells were treated with 100 or 500 mg/L lipopolysaccharide (LPS), with or without 10⁻⁵ mol/LDHAP for 24 h. Trypan blue dye exclusion assay was used to assess cell viability. Cell apoptosis was morphological studied and flow cytometric assay was used. Tumor necrosis factor-α (TNF-α) level was measured by ELISA methods. IϰB protein was determined by Western blotting. Our results showed that in 100 mg/L LPS-stimulated macrophages, DHAP enhanced the cell apoptosis while in 500 mg/L LPS-stimulated macrophages, DHAP significantly inhibited the cell apoptosis. In both groups, DHAP increased the level of IκB but decreased the level of TNF-α. It is concluded that DHAP has dual effect on the apoptosis of RAW 264. 7 cells treated with different concentrations of LPS. This effect may be due to the inhibition of activation of NF-κB and autocrine production of TNFα. Our study suggests that DHAP may have anti-inflammatory effect on LPS-activated macrophages.

This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles ans apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded. The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated. DNA replication was evaluated by [³H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [³H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G₀/G₁ by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G₀/G₁ phase.

To investigate the effect of curcumin on immune function of mice, the effect of curcumin was examined on the proliferation of spleen lymphocytes of mice and the function of phagocytosis of peritoneal macrophage by using MTT test and the expression of the nucleoprotein of NFκB p65 was determined in spleen lymphocytes by employing Western Blot. Our results showed that curcumin could enhance the phagocytosis of peritoneal macrophages. Lowdose curcumin could upregulate the proliferation of spleen lymphocytes of mice, and highdose curcumin could suppress the proliferation of spleen lymphocytes. Curcumin could suppress the expression of NFκB p65. Our study suggested that curcumin can regulate immune function of mice in a dosedependent manner. The possible underlying mechanism might be its ability to suppress the activity of NFκB p65.

To examine the relationship between host survival and the type of immune response in different organs during disseminated candidiasis, the murine model of disseminated candidiasis was established by injection withCandida albicans via tail vein. The survival time was observed for up to 60 days. And the expression levels of cytokines in the spleen and kidney, including IFN-γ and IL-4, were determined with RT-PCR. Our results showed that in the spleen, both non-fatal and fatal inoculum caused a type II immune response with steady expression levels of IFN-γ and the obviously increased levels of IL-4. While in the kidney, non-fatal inoculum induced a type I immune response with the obviously increased levels of IFN-γ and the steady expression levels of IL-4. However, fatal inoculum induced a type II immune response with a constant expression of IFN-γ and the evidently increased levels of IL-4. It is concluded that in disseminated candidiasis, host survival is associated with the type of immune responses in the kidney, but not in the spleen.

To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3. 1 (+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1(+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 μg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P<0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in culture IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.

In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003. 58±1 626. 57) and the expression of IL-5 mRNA (0. 8300±0. 0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888. 47±1103. 56 and 0. 3050±0. 0208, respectively,P<0.01), while the indexes (23 219. 83±1 024. 86 and 0. 3425±0. 0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P<0.01). The indexes (87 107.41±1 342. 92 and 0. 8225±0 0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P<0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.

To examine the mRNA expression of matrix metalloproteinase 9 (MMP-9) and the gelatinase activity of its inhibitor, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) in the primary epithelial cells of patients with COPD, airway epithelial cells were taken from 15 COPD patients and culturedin vitro. The patients were divided into three groups, COPD group, normal smoking control group and non-smoking control group, with 5 subjects in each group, on basis of the smoking history and lung function. The semi-qualitative RT-PCR was employed to determine the mRNA levels of MMP-9 and TIMP-1 and SDS PAGE was used for the determination of the gelatinase activity of MMP-9 and TIMP-1. Our result showed that the mRNA of MMP-9 and TIMP-1 in epithelial cells of the non-smoking subjects was at a low level. The mRNA of MMP-9 and TIMP-1 in COPD patients and smokers was significantly higher than that in non-smokers (P<0.05). No significant difference was found in the levels of MMP-9 and TIMP-1 in epithelial cells between the COPD patients and smokers. The MMP-9/TIMP-1 ratios in COPD patients and smokers were significantly lower than that of non-smokers (P<0.05). The gelatinase activity in the epithelial cells of both COPD patients and normal smokers was increased (P<0.05), but no difference existed in the gelatinase activity in the epithelial cells between COPD patients and normal smokers. It is concluded that the transcription of MMP-9 and TIMP-1 and the gelatinase activity of MMP-9 and MMP-2 in the epithelial cells in COPD patients were increased, which resulted in an imbalance of MMP-9/TIMP-1, thereby causing pulmonary fibrosis. These factors play important roles in the pathogenesis of COPD.

To investigate role of Notch1–3 in hyperoxia-induced lung injury in newborn rat exposed to 85% O₂, SD rat litters born on the 22th day were randomly divided into two groups: room air group and hyperoxia group. The animals were sacrificed 1, 4, 7, 10, 14 and 21 days after continued exposure to oxygen (n=40, oxygen>0.85) or room air (n=40). 6 rats each group were used to assess lung histological changes by HE staining and expression of Notch in lungs by immunohistochemistry. Total RNA was extracted by Trizol reagent from frozen lung tissues. Notch mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that 7, 14 and 21 days after O₂ exposure, hyperoxia group showed lung injury characterized by pulmonary edema, hemorrhage and lung development arrest. Positive staining for Notch1, Notch 2 in hyperoxia group was much lower than those in room air group at all time points (P<0. 01,P<0.05), but compared with the controls, the hyperoxia group showed higher expression of Notch3 (P>0.05). Immunostained cells were typically airways epithelia, alveolar epithelial and inflammatory cells, and fibroblasts in hyperoxia group (P<0.01). Notch mRNA levels showed similar change as protein level (P<0.01). It is concluded that the prolonged exposure to 85% O₂ resulted in abnormal expression of Notch receptors, which might contribute to the pathogenesis of hyperoxia-induced lung injury in newborn rats. The decreased inhibition of Notch1 might be one of the protective reaction and major mechanisms for proliferation/differentiation of type II alveolar epithelial cells. The up-regulation of Notch3 activity might result in the lung development arrest of the newborn rats.

To explore the mechanism of Notch in hyperoxia-induced preterm rat lung injury, 2-days-old preterm SD rats were randomized into control and hyperoxia group (FiO₂≥0.85). On day 1, 7, 14 and 21, 8 rat pups of each time point were used to assess histopathological changes of lung with HE staining and to evaluate the expression of Notch1 and Notch3 with immunohistochemistry. Notch1, Notch3, Aquaprin5 (AQP5) and surfactant protein C (SP-C) mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). The results showed that the lung injury in the hyperoxia group was characterized by retarded lung alveolization and differentiation of alveolar epithelial type II cells (AEC II). Positive staining of Notch1 in hyperoxia group was weaker than controls at every time point (except for day 7), while positive staining of Notch3 was much stronger (P,0.05,P<0.01). Notch1, Notch3 mRNA level showed similar change as protein level. AQP5, SP-C mRNA decreased significantly as compared with that of the controls (P<0.01). We are led to conclude that hyperoxia results in abnormal expression of Notch, which is likely to contribute to the pathogenesis of lung injury through regulating proliferation and transdifferentiation of alveolar epithelial cells.

To examine the deletion and point mutation of WWOX (WW domain containing oxidoreductase) exons 6–8 in human non-small cell lung cancer and their possible relationship with pathological stages, tumor tissues and the corresponding normal tissues were obtained from 44 Chinese patients who had undergone surgery for non-small cell lung cancer. RNA was extracted from each sample and deletion and mutation of WWOX exons 6–8 were analyzed by RT-PCR and DNA sequencing. Our results showed that 28 of 44 (63.6%) lung cancer samples showed loss of WWOX exons 6–8 transcript and the deletion was detected in only 3 of 44 (6.8%) corresponding adjacent normal tissues (P<0.05). The transcript sequencing analyses of the 16 lung cancer samples without transcript loss of WWOX exons 6–8 revealed no difference from the sequence of Gen-Bank. Moreover, the deletion of WWOX exons 6–8 was significantly higher in the smokers when compared with the non-smokers. It is also higher in the men and squamous carcinomas than in women and adenocarcinomas (P<0.05). The deletion, however, was not found to be associated with pathological stages of the tumors. Our study documented a high incidence of deletion of WWOX exons 6–8 in non-small cell lung cancer in Chinese patients and suggested that the frequent loss of WWOX exons 6–8 might play an important role in the tumorigenesis of non-small cell lung cancer in Chinese. WWOX exons 6–8 may serves as a candidate molecular target of smoking carcinogenesis, and point mutation is not a predominant way of alteration of WWOX exons 6–8.

The expression of basic fibroblast growth factor (bFGF) in rat liver fibrosis and hepatic stellate cells (HSCs) and the relationship between the expression of bFGF and rat liver fibrogenesis were studied. Sixty male SD rats (230–260 g) were divided into 4 groups randomly (the 0 week group, 1 week group, 4 week group and 8 week group). Liver fibrosis was induced by subcutaneous injection of carbon tetrachloride. The sections of rats’ liver in each group were tested by Van-Gieson (V-G) staining and immunohistochemistry. The expression of bFGF mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). HSCs were isolated by the combined methods of collagenase IV perfusion and density gradient centrifugation. The expression of bFGF protein in cultured HSCs was detected by Western blot. Images of immunohistochemistry detection, agarose gel electrophoresis of RT-PCR and SDS-polyacrylamide gel electrophoresis of Western blot were analyzed semiquantitatively by image-analyzing system. The results were analyzed by statistics. The results showed that the fibers were gradually increased in the sections of rat liver with the prolongation of the model induction. At the end of the 8th weeks, liver fibrosis was formed. The expression of bFGf detected by immunohistochemistry showed a similar tendency of gradual increase. At the end of the 8th weeks, the bFGF expression could be observed in many regions in sections and the strongest expression was in interstitial cells including HSCs and some hepatocytes in regions around the portal area and central veins. Also there was moderate expression widely in extracellular matrix (ECM). In RT-PCR detection and Western blot detection of HSCs cultured in vitro, the similar tendency of gradual increase was evident either. It is suggested that bFGF is related with liver fibrosis of rats closely and may be a fibrogenesis factor of liver. bFGF possibly regulates liver fibrogenesis through regulating metabolism of extracellular matrix (ECM) by autocrine and paracrine stimulation.

To investigate the expression and the role of three isoforms of Serum and Glucocorticoid-inducible Kinase (SGK) in experimental diabetic nephropathy (DN), 12 male C57BL/6 mice of 8-weeks-old were divided into two groups. Streptozotocin (STZ)-induced diabetic nephropathy and normal controls were analyzed at the end of the 4th week after the induction of diabetes. Renal hemodynamics and histological studies were performed. The expression of SGK1 mRNA, SGK2 mRNA and SGK3 mRNA of kidney cortex were measured by RT-PCR, and the cortical SGK1 protein was detected with Western blotting. Our results showed that the blood glucose, blood HbA1c, 24-h urinary protein, creatinine clearance and the renal index were all increased in DN group. More extracellular matrix (ECM) accumulation was observed. The level of cortical SGK1 mRNA and protein were up-regulated in DN group in comparison with control group. SGK2 and SGK3 mRNA were elevated in DN mice. In DN, mRNA level of three SGK isoforms and SGK1 protein were increased significantly. It is concluded that SGKs may contribute to the early renal injury of DN.

To investigate the role of NF-κB in endotoxic shock in rats, the model of endotoxin-shock rats was induced by intravenous infusion of lipopolysaccharide (LPS). 1 h, 2 h, 4 h and 6 h after LPS injection, the activation of NF-κB in blood mononuclear cells and the content of TNF-α and IL-6 in plasma was detected by enzyme-linked immunoadsordent assay (ELISA). The level of mean arterial pressure (MAP) and the histopathological changes of lung and liver were also observed. The activation of NF-κB in mononuclear cells increased 1 h after LPS injection and reached its peak 2 h after the injection, and its level was higher than that of normal group. The level of TNF-α was increased 1 h after the infusion and peaked 2 h after the injection, and its level was higher than that of normal group after LPS infusion. The content of IL-6 increased gradually with time, the IL-6 level was higher than that of normal group after LPS injection. MAP was decreased gradually with time and its level was lower than that of normal group after LPS injection. Pathological examination showed that endotoxic shock could cause pulmonary alveolar hemorrhage, edema and infiltration of inflammatory cell in lung tissue and congestion, edema, capillary dilation and inflammatory cell infiltration in liver tissue. It is concluded that NF-κB can up-regulate the expression of TNF-α and IL-6 in plasma and play an important role in endotoxin-induced shock in rats.

To investigate the killing effect ofPNP/MeP-dR suicide gene system on hepatoma cells, pcDNA3. 0/PNP, an eukaryotic expression vector harboringE. coli PNP gene, was transfected into human hepatoma HepG2 cells by liposome-mediated method. A HepG2 cell line with stablePNP gene expression, HepG2/PNP, was established with presence of G418 selection. The cell growth curves were determined with trypan blue staining. The sensitivity of HepG2/PNP to MeP-dR and bystander effects were assayed by MTT and FCM methods. The enzymatic activity of the product ofPNP gene was determined by HPLC method. The cytotoxic effects of MeP-dR on HepG2/PNP cells were obvious (IC₅₀=4.5 μmol/L) and all HepG2/PNP cells were killed 4 days after the treatment with 100 μmol/L MeP-dR. In mixed cultures containing increasing percentages of HepG2/PNP cells, total population killing was demonstrated when HepG2/PNP cells accounted for as few as 5% of all HepG2 cells 8 days after the treatment with 100μmol MeP-dR. High-pressure liquid chromatography (HPLC) demonstrated that thePNP enzyme could convert MeP-dR into 6-MP.PNP/MeP-dR suicide gene system had an advantage over traditional suicide gene systems for hepatoma gene therapy. Our e results suggest that high-level bystander effects of this system result in significant anti-tumor responses to hepatoma gene therapy, especiallyin vivo.

The experience in diagnosis and treatment of bleeding complications in severe acute pancreatitis (SAP) by transcatheter arterial embolization was summarized. The clinical data of 19 SAP patients complicated with intra-abdominal bleeding in our hospital from Jan. 2000 to Jan. 2003 were analyzed retrospectively and the therapeutic outcome, of TAE was evaluated statistically. The results showed that the short-term successful rate of hemostasis by TAE was 89.5% (17/19), the incidence of re-bleeding after TAE was 36.8% (7/19) and the successful rate of hemostatis by second TAE was 71.4% (5/7). It was concluded that the intra-abdominal bleeding in SAP was mainly caused by the rupture of erosive/infected pseudoaneurysm. Mostly, the broken vessels were splenic artery and gastroduodenal artery. In terms of emergence hemostatis, TAE is the most effective method. Surgical hemostasis is necessary if hemostasis by TAE is failed or re-bleeding occurs after TAE.

In order to study the effects of electromagnetic fields (EMFs) on proliferation, differentiation and intercellular cyclic AMP (cAMP) in mouse bone marrow mesenchymal stem cells (MSCs)in vitro, the mouse bone MSCs were isolated and culturedin vitro. The third passage MSCs were divided into 4 groups and stimulated with EMFs. The cellular proliferation (MTT), the cellular differentiation (alkaline phosphatase activity, ALP), and the intercellular cAMP level were investigated at different time points. The results showed that EMF (50Hz pulse burst 2 mT peak) inhibited the cellular proliferation (P<0.05), enhanced the cellular differentiation (P<0.05), and increased the intercellular cAMP level (P<0.01) in the early time of the stimulation (1–3 days), but the intercellular cAMP level did not increased further in the later days. We are led to conclude that the cAMP may be involved in the mediation of the growth inhibitory and differentiation-inducing signals of specific EMFsin vitro.

To clone Uroplakin II gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GIII/T₃N₀M₀ tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0, software. Full length cDNA of Uroplakin II gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted betweenXba I andHindIII restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin II negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin II were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin II cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100% homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP II for Uroplakin II, was successfully constructed. After being transferred with pcDNA-UP II for 72 h, cellular Uroplakin II mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin II gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin II gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

To investigate the impact of exercise on the expression of adiponectin and GLUT4 mRNA in type 2 diabetic rats, type 2 diabetic rat model was made. The diabetic rats were treated with swimming training for 8 weeks. The expression of adiponectin mRNA in perirenal fat and GLUT4 mRNA in skeletal muscles were assessed by reverse transcription polymerase chain reaction (RT-PCR) and the levels of blood glucose, serum insulin, and blood lipid were measured. Our results showed that the expression of adiponectin mRNA and GLUT4 mRNA in diabetic model group was decreased by 45% (P<0.01), 43% (P<0.01) respectively. The gene expression of adiponectin and GLUT4 was increased significantly in swimming group (P<0.05 andP<0.01, respectively). Compared with the model group, fasting insulin, TG, TC and FFA were decreased significantly in the training group (P<0.05 orP<0.01) as compared with model group. It is concluded that exercise can promote the expression of adiponectin mRNA and GLUT4 mRNA in type 2 diabetic rats, which may be one of the mechanisms responsible for the amelioration of insulin resistance in the rats.

In order to explore the effects of metformin combined with cyproterone acetate (CPA) on the clinical features, endocrine and metabolism of the patients with polycystic ovarian syndrome (P-COS), 50 cases of non-obese PCOS were randomly subjected to CPA (CPA treatment group,n=25) and CPA+metformin (n=25) treatment for 6 months. Before and after treatment the body mass index (BMI), waist: hip ratio (WHR), ovarian volume, serum gonadotrophin, androgen and sex hormone-binding globulin (SHBG) levels, and fasting lipid, glucose and insulin levels were measured. The results showed that all of the parameters in two groups were similar before treatment. After treatment for 6 months in the CPA+metformin group, BMI and WHR were significantly decreased, while insulin sensitivity was significantly decreased as compared with those before treatment. In CPA group, no significant changes were found before and after treatment. Combined use of CPA and metformin could result in the reduction of serum androstenedione and increases of serum SHBG levels as compared with the CPA treatment alone. It was concluded that combined use of CPA and metformin could improve the insulin sensitivity, and further suppress the hyperandrogenism in non-obese women with PCOS.

The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03%. The S phase cells accounted for 0 to 54% in the cell cycle and the positive expression of Ki67 ranged from 0 to 52%. There were 40 cases of LPI (64.5%) including 15 negative cases and 22 cases of HPI (35.5%) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

The inhibitory effects of two kinds of selective cyclooxygenase-2 inhibitors on the proliferation of human carcinoma of larynx Hep-2in vitro and their corresponding mechanisms were investigated. Hep-2 cells were cultured with two kinds of selective cyclooxygenase-2 inhibitors (Sc-58125 and Celecoxib) at various concentration for 24 h. Morphological changes were observed under the phase microscopy and the growth suppression was detected by using MTT colorimetric assay. Apoptotic DNA fragments were observed by agarose gel electrophoresis and the cell cycle and apoptotic rate were detected by flow cytometry (FCM) respectively. Hep-2 cells became rounded and detached from the culture dish after being treated with Celecoxib for 24 h, however, they remained morphologically unchanged with Sc-58125. Sc-58125 could increase G₂ phase cells, whereas, Clecoxib rose G₁ phase cells. Both of the two effects were dose-dependent. Moreover, the Hep-2 cells cultured with 50μmol/L and 100μmol/L Celecoxib showed obvious apoptosis, with the nuclear DNA of cells exhibiting characteristic DNA ladder. So Sc-58125 could inhibit the proliferation of Hep-2 cells by altering the G₂ phase cells. However, Celecoxib had the same effect by changing the G₁ phase cells and inducing apoptosis at higher concentration.

To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied bothin vitro andin vivo. With thein vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy. And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With thein vivo examination, 20 SD rats were randomized into control group (n=10) and electrical acupuncture group (EA group,n=10). EA (0.5–1.5 V, 4–16 Hz, 30 min) was applied to “Zusanli” acupoint for 30 min at rat’s hind paw, the localization of Cx43 was immunohistochemically detected. The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43 was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43 on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91% and 29.53% respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

To evaluate the normal appearance of epiphyseal and physeal cartilage on Gadolinium (Gd)-enhanced MR imaging. The appearance and enhancement ratios of 20 proximal and distal femoral epiphyses in 10 normal piglets were analyzed on Gd-enhanced MR images. The correlation of the MR imaging appearance with corresponding histological findings of immature epiphyses was examined. Our results showed that Gd-enhanced MRI could differentiate the differences in enhancement between physeal and epiphyseal cartilage and show vascular canals within the epiphyseal cartilage. Enhanced ratios in the physeal were greater than those in the epiphyseal cartilage (P<0.005). It is concluded that Gd-enhanced MR imaging reveals epiphyseal vascular canals and shows difference in enhancement of physeal and epiphyseal cartilage.

To determine the (tttta)_n repeat polymorphisms at the promoter region ofCYP11α gene, and study its linkage to hyperandrogenism of polycystic ovary syndrome (PCOS) in Chinese women, a case-control study was conducted in the Reproductive Medical Center of the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). 96 PCOS patients and 78 healthy control women were included.CYP11α (tttta)_n repeat-polymorphism genotyping analysis was performed by using polymerase chain reaction (PCR). Serum pituitary hormone and total testosterone levels were measured by ELISA. 4 differentCYP11α (tttta)_n allelles were identified, corresponding to 4-, 6-, 8-, and 9-repeat-unit alleles. The frequency and distribution of these alleles are 0.16, 0.33, 0.38, and 0.13 respectively in PCOS patients, as compared with 0.20, 0.34, 0.35, and 0.11 respectively in healthy controls. There were no significant differences between these two groups. Moreover, no correlation between the polymorphism ofCYP11α gene and serum testosterone level of patients with PCOS and controls was observed. It is concluded that microsatellite polymorphism (tttta)_n of geneCYP11α exists in Chinese women and the polymorphism ofCYP11α gene does not play an important role in the pathogenesis of Chinese patients with PCOS, especially in patients with hyperandrogenism.

To investigate the effect of immature dendritic cells (iDCs) on experimental autoimmune myasthenia gravis (MG), iDCs were generated in low dose of GM-CSF, and then they were pulsed with acetylcholine receptor (AchR) and transferred to allogeneic rats. After 3 weeks, all rats were immunized with AchR and complete Freund’s adjuvant (CFA) and observed for the corresponding indices of MG for 7 weeks. Our results showed that compared with mature DCs (mDCs) generated at high dose of GM-CSF plus additional stimulation by lipopolysaccharide, iDCs expressed significantly lower levels of MHC-II, CD80 and CD86, and their ability to uptake FITC-Dextran was stronger but the ability of stimulating proliferation of allogeneic T cells were weaker. Like controls, after immunization, all rats transferred with iDCs, mDCs and AchR-pulsed mDCs showed typical symptoms in 4 to 7 weeks. The amplitude of electromyogram wave dropped obviously, the level of serum AchRab increased and neuromuscular junction showed typical damage of MG. In contrast, no conspicuous changes were noted in rats transferred with AchR-pulsed iDCs. The results suggest that iDCs could be generated by inducing bone marrow precursors in low dose of GM-CSF, AchR-pulsed iDCs could induce tolerance of EAMG. The dysfunction of DCs may play an important role in the initiation and maintenance of normal immune response in MG.

To study the mechanism of the innoxious touch-stimulus on the modulation of hyperalgesia and the expression of the C-fos and the nerve growth factor (NGF) receptor-TrkA in the spinal dorsal horn neurons following the chronic constriction injury (CCI) of the sciatic nerve in rats, 60 female Sprague-Dawley rats were randomly divided into sham-operation group and CCI group, with each group being further divided into 3 subgroups on the 7th, 14th and 28th day after operation (n=10). The mechanical and the thermal withdrawal threshold were assessed following the touch stiumulation after the CCI, immunohistochemical methods were employed to observe the expression of the C-fos and TrkA in spinal dorsal horn. Our results showed that the hyperalgesia appeared on the 4th day and reached the maximal level on the 14th day after operation. The expression of the C-fos also increased significantly and reached its maximal level on the 14th day after the touch-stimulus. Meanwhile, the TrkA expression was elevated significantly in both groups, as compared with basic data, and the difference was statistically significant (P<0.05). It is concluded that the level of the C-fos expression changed with the paw withdrawal threshold variation and increased markedly following the innoxious touch-stimulus. The expression of the TrkA receptors also increased gradually following the development of the neuropathic pain. The results suggest that C-fos may play a crucial role in the development of the hyperalgesia in the earlier-time of the neuropathic pain, but TrkA receptors may be involved in the long-lasting adaptive changes of the central pathway in neuropathic pain.

In order to study the relationship between serum specific IgE (sIgE) and allergen skin test, allergen skin tests and detections of sIgE in 220 allergic patients of Wuhan area were analyzed. The coherent rate of the two methods was beyond 70% (P<0.01). It was concluded that thein vitro andin vivo detection methods of allergens have a high coherence and can be used as the effective ways to diagnose the allergic diseases in clinical practice.

Spermicidal effect of Jieze No. 1 (JZ1) in combination with nonoxynol-9 (N-9) was examinedin vitro. The minimum spermicidal concentration of JZ1 decoction, N-9 and their mixture solution in 20 s and 3 min were examined by improved spermicidal test of Sander-cramerin vitro. The percentages of progressively moving spermatozoa, moving spermatozoa and viable spermatozoa were also observed 20 s, 3 min and 30 min after the addition of the liquid medicine. Our results showed that sperms did not recover their activities in a revival test when the minimum spermicidal concentration of either JZ1 decoction, or N-9, or the mixed solution of the two agents, was used. N-9 (JZ1 in the mixed group) showed significant differences in the percentages of progressively moving spermatozoa, moving spermatozoa, and visible spermatozoa in 20 s, 3 min, and 30 min, when compared with N-9 alone (P<0.01). We are led to conclude that JZ1 decoction can improve N-9 spermicidal actionin vitro, and when used in combination with N-9, it has synergic effect.