The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni²⁺ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni²⁺-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase (CaMK), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was detected in neurons. The results showed that the expression of CaMK, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (P<0.05), and that of iNOS and AC peaked at 8 h and 12 h respectively. It was suggested that there might be some epileptogenic factors in the ACM and such signal pathways as NOS-NO-cGMP, Ca²⁺/CaM-CaMK II and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ_25–35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca²⁺]_i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against A-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca²⁺]i was maintained in the AAv-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

In order to investigate the impairment of mitochondrial membrane phospholipid localization and DNA³⁸⁶⁷ (mtDNA³⁸⁶⁷) deletion and the correlation between cardiac and skeletal muscle cells in mice with viral myocarditis, 50 BALB/c mice were divided into two groups randomly. In experimental group (n=40), the mice were intraperitoneally injected with 0.1 mL Eagle liquid with CVB₃(TCID₅₀=10⁸), while in the control group (n=10), the mice were subjected to equal volume of Eagle liquid. The impairment of mitochondrial membrane phospholipid localization and mtDNA³⁸⁶⁷ deletion rate of cardiac and skeletal muscle were detected separately at day 3, 11 and 24 after injection. The correlation of mitochondrial membrane phospholipid localization and mtDNA³⁸⁶⁷ deletion rate between cardiac and skeletal muscle cells cells was analyzed using Spearman method. At the day 3 after injection, in both cardiac and skeletal muscle cells, mtDNA³⁸⁶⁷ deletion rate was significantly higher in experimental group than in control group (P<0.05), but the localization of mitochondrial membrane phospholipid showed no difference between two groups (P>0.05). At day 11 after injection, the mtDNA³⁸⁶⁷ deletion rate of both cells in experimental group was increased to the peak level (P<0.05), and the impairment of mitochondrial membrane phospholipid localization of both cells also increased markedly in experimental group as compared with control group (P>0.05). At the day 24 after injection, the impairment of mitochondrial membrane phospholipid localization and mtDNA³⁸⁶⁷ deletion of both cells showed a recovery tendency, but still severer than those at the day 3 after injection (P<0.05). The impairment of mitochondrial membrane phospholipid localization and mtDNA³⁸⁶⁷ deletion were consistent and synchronistic between cardiac and skeletal muscle cells, and showed good correlations (P<0.05). The impairment of mitochondria plays an important role in the pathogenesis of viral myocarditis, and the skeletal muscle cells might act as a peripheral “window” to reflect the mitochondrial damage of cardiac myocytes.

In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 (n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribution of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis revealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia, while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercelluar communication by enhancing phosphorylation of Cx43.

The effects of salvia miltiorrhiza Bge (SMB) on left ventricular hypertrophy (LVH) and the expression of tumor necrosis factor-α (TNF-α) in the left ventricle of spontaneously hypertensive rats and the action mechanism were investigated. Normal Wistar-kyoto (WKY) rats were used as negative control, and spontaneously hypertensive rats (SHR) were randomly assigned to receive placebo or SMB. SMB (1 g/kg·d) was injected intraperitoneally for 12 weeks. Systolic blood pressure (SBP) and left ventricular mass index (LVMI) were measured. HE, VG and immunohistochemical staining combined with computed morphometry were employed to evaluate the cardiomyocyte size, diameter, the collagen volume fraction (CVF), perivascular circumferential area (PVCA), and tumor necrosis factor-α (TNF-α) expression in the left ventricular tissue. The results showed, as compared with WKY rats, the SBP, LVMI, cardiomyocyte size, diameter, CVF, PCVA, and TNF-α expression were increased markedly in the 20-week-old spontaneously hypertensive rats. SMB decreased LVMI (P<0.01), size of cardiomyocytes (P<0.01), collagen volume fraction (P<0.01), perivascular circumferential area (P<0.01), and TNF-α expression (P<0.01), but had no effect on SBP (P>0.05). It was suggested that chronic administration of SMB could inhibit and reverse the development of LVH in spontaneously hypertensive rats independent of BP. TNF-α may be involved in the reversal mechanism of LVH by SMB.

In order to investigate the effects of simvastatin on secretion and mRNA expression of interleukin-6 (IL-6) and adiponectin in 3T3-L1 adipocytes, mouse 3T3-L1 adipocytes were stimulated with lipopolysaccharide (LPS). Production and mRNA expression of IL-6 and adiponectin in 3T3-L1 adipocytes were measured using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. The results showed that simvastatin could significantly suppress LPS-induced IL-6 production and mRNA expression in adipocytes (P<0.05), but increase the LPS-induced adiponectin secretion and mRNA expression in a dose-dependent manner (P<0.05). It was suggested that simvastatin could exert beneficial effects on prevention of obesity-induced metabolic changes in adipocytes.

The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor β1 (TGF-β1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was applied for measurement of PIR-A and CD80, CD86, MHC-II mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using ³H-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was analyzed by ELISA. The results showed that PIR positive rate was (28.65±8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21±6.34)%, (58.78±4.70)%, (48.24±6.75)% respectively for IL-10, TGF-β1 and LPS induction (P<0.01), but there was no significantly different among the three groups (P>0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-β1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80, CD86 and MHC-II were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactived T cell proliferation and down-regulated the IFN-γ secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and enhanced the IFN-γ secretion (P<0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC-II expression, which might be the molecular mechanism for the T-DC.

In order to investigate the expression and functional role of HERG1 K⁺ channels in leukemic cells and leukemic stem cells (LSCs), RT-PCR was used to detect the HERG1 K⁻ channels expression in leukemic cells and LSCs. The functional role of HERG1 K⁺ channels in leukemic cell proliferation was measured by MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry. The results showed that herg mRNA was expressed in CD34⁺/CD38⁻, CD123⁻ LSCs but not in circulating CD34⁺ cells. Herg mRNA was also up-regulated in leukemia cell lines K562 and HL60 as well as almost all the primary leukemic cells while not in normal peripheral blood mononuclear cells (PBMNCs) and the expression of herg mRNA was not associated with the clinical and cytogenetic features of leukemia. In addition, leukemic cell proliferation was dramatically inhibited by HERG K⁺ channel special inhibitor E-4031. Moreover, E-4031 suppressed the cell growth by inducing a specific block at the G1/S transition phase of the cell cycle but had no effect on apoptosis in leukemic cells. The results suggested that HERG1 K⁺ channels could regulate leukemic cells proliferation and were necessary for leukemic cells to proceed with the cell cycle. HERG1 K⁺ channels may also have oncogenic potential and may be a biomarker for diagnosis of leukemia and a novel potential pharmacological target for leukemia therapy.

An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction. HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs). The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48−h, HA-1 protein was detected by using Western blot. The cytotoxicty of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.

In order to develop a protocol for clinical grade generation of dendritic cells (DCs) for cancer immumotherapy, aphereses were performed with the continuous flow cell separator and materials were derived from 10 leukemia patients that had achieved complete remission. Peripheral blood monocytes were cultured in vitro with GM-CSF, IL-4 for 6 days, then TNF-(the TNF-group) or TNF-, IL-1, IL-6, PGE₂ (the cytokine mixture group) were added to promote maturation. Cell number was counted by hematology analyzer, and phenotype study (CD1a, CD14, CD83) was carried out by flow cytometry, and the function of DCs was examined by mixed lymphocyte reaction. The results showed that (0.70±0.13)×10⁷/mL (the TNF-α group) and (0.79±0.04)×10⁷/mL (the cytokine mixture group) DCs were generated respectively in peripheral blood obtained by leucapheresis. The phenotypes were as follows: CD1a⁺ (74.65±4.45)%, CD83⁺ (39.50±4.16)%, CD14⁺ (2.90±1.76)% in TNF-α group, and CD1a⁺ (81.86±5.87)%, CD83⁺ (81.65±6.36)%, CD14⁺ (2.46±1.68)% in the cytokine mixture group. It was concluded that leucapheresis may be a feasible way to provide large number of peripheral blood monocytes for DC generation, and combined administration of TNF-, IL-1, IL-6, and PGE₂ may greatly promote maturity.

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

The distribution of hepatitis B virus genotype in Hubei province and its clinical significance were investigated. HBV genotypes of 276 patients were detected by PCR-microplate sandwich hybrization-ELISA technique. The level of HBV DNA was detected by using PCR-fluorescence quantification test. Among 276 patients, there were 78 cases of HBV asymptomatic carriers, 110 cases of chronic hepatitis B (CHB), 62 cases of severe hepatitis (SH) or liver cirrhosis (LC) and 26 cases of hepatocellular carcinoma (HCC). The genotypes of HBV included C, B, mixtures (B+C, B+D, C+D) and D, accounting for 55.8%, 25.4%, 16.7% and 2.1% respectively. The average level of HBV DNA in genotypes C, B, mixtures and D was 1.20×10⁶, 7.81×10⁴, 3.26×10⁵ and 5.01×10⁴ copies/mL respectively. The ratio of SH, LC and HCC in genotype B, C and mixtures was 20%, 30% and 48% respectively. Statistical analysis revealed the percentage of genotype mixtures infection was significantly higher than that of genotype B infection. There was no significant difference in the percentage between genotype B and genotype C or between genotype C and mixtures. The distribution of genotype B, C and mixtures in SH, LC and HCC was significantly different. The frequency of HCC was zero in patients with co-infection. Genotype D was only related with SH and LC. The increased ALT could be converted to categorical grades of severity. From mild, moderate to severity, the prevalence of genotype C showed an opposite trend, although no statistically significant difference was observed. The HBeAg positive rate was higher in patients with genotype C infection than in those with genotype B, especially in the patients whose ages were from 31 to 40 years old. Compared with genotype B, genotype C showed a higher HBeAg positive rate in patients with SH and LC. The percentage of SH, LC and HCC was higher in patients with genotype C and mixtures infection. On the contrary, the percentage of genotype B was lower. The HBeAg positive rate in patients with genotype C infection was higher than those with genotype B infection. Genotype C and mixtures may be associated with development of severe liver disease.

CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine^™2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.

Hypermethylation of the promoter region is an important mean for the transoriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2′-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 μmol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemotherapeutic sensitivity of T24 cells.

In order to study bladder intravesical instillation methods in pure line LEW rats and nude mice, female LEW rats and nude mice aged 2 to 4 weeks were sacrificed. Their urethra and bladder were observed under anatomical microscopy. A trochar was prepared according to the outline and angle of the urethra. Ink was poured into female rats and nude mice bladder though urethra. Filling and staining of bladder were observed and evaluated under anatomical microscopy. Status and methral injury of rats and mice were observed. The results showed that urethra anatomic structure of rats and nude mice was different from that of human urethra. When bladder was filled with ink and became blue, liquid was not seen to leak out. The success rate of intubation was high (100%). Living activities of animals weren’t influenced by intravesical instillation. It was concluded that bladder irrigation might be a kind of valid and utilizable method in pure line rat and nude mouse empirical study. The model may be a more effective tool for study of bladder tumor.

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called “bystander effect” mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ²=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (−). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of “bystander effect”, but also to initiation and progression of prostatic neoplasm.

In order to evaluate the neuroprotective effect of Rosiglitazone Maleate (RSG) against brain ischemic injury, the effects of Rosiglitazone Maleate on the inflammation following cerebral ischemia/reperfusion were investigated. Focal cerebral ischemia was induced by the intraluminal thread for cerebral middle artery (MCA) occlusion. Rosiglitazone Maleate at concentrations of 0.5, 2 and 5 mg/kg was infused by intragastric gavage twice immediately and 2 h after MCA occlusion, respectively. The effects of Rosiglitazone Maleate on brain swelling, myeloperoxidase and interleukin-6 mRNA level in brain tissue after MCA occlusion and reperfusion were evaluated. The results showed that as compared with the model control group, RSG (0.5 mg/kg) had no significant influence on brain swelling (P>0.05), but 2 mg/kg and 5 mg/kg RSG could significantly alleviate brain swelling (P<0.05). All different doses of RSG could obviously reduce MPO activity in brain tissue after MCA occlusion and reperfusion in a dose-dependent manner. RSG (0.5 and 2 mg/kg) could decrease the expression levels of IL-6 mRNA in brain tissue after MCA occlusion and reperfusion to varying degrees (P<0.05) with the difference being significant between them. It was concluded that RSG could effectively ameliorate brain ischemic injury after 24 h MCA occlusion and inhibit the inflammatory response after ischemia-reperfusion in this model.

In order to observe the nutrition state in the severe multiple trauma patients undergoing adjuvant recombinant human growth hormone (rhGH) nutritional support therapy, 45 patients with severe multiple traumas (ISS>25) were randomly divided into 3 groups. All the 3 groups had been supplied with nitrogen and caloricity according to the need of patients for 16 days. The rhGH therapy started 48 h after surgery and lasted for 14 days in two rhGH-treated groups in which rhGH was 0.2 and 0.4 U/(kg · d) respectively, and the resting group served as control one. The levels of nitrogen balance, prealbumin and safety variables (blood sugar, Na⁺, TT₃ and TT₄) were observed and compared among the three groups. The levels of nitrogen balance on the postoperative day (POD) 3 and 5 in the rhGH-treated groups were −1.28±3.19, 5.45±2.00 and −0.18±2.55, 6.11±1.60, respectively, which were significantly higher than those in the control group (−5.17±1.68 and −1.08±3.31, P<0.01). The values of prealbumin on the POD 3 and 5 in the rhGH-treated groups were 180.19±27.15, 194.44±50.82 and 194.94±29.65, 194.11±16.17, respectively, which were significantly higher than those in the control group (117.42±19.10 and 135.63±28.31, P<0.01). There was no significant difference between the rhGH 0.2 U/(kg · d) group and rhGH 0.4 U/(kg · d) group in both of the levels of nitrogen balance and prealbumin. It is concluded that the nutritional support therapy with adjuvant rhGH which starts 48 h after surgery improves the nutrition state of the patients with severe multiple trauma. It is safe for severe multiple trauma patients who accept rhGH at the dose of 0.2 and 0.4 U/(kg · d).

In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.

The model of vaginal candidiasis in Kunming mice was constructed in order to search for the optima construction conditions and provide an economic animal model of Candida albicans (C. albicans) vaginitis. Estrogen benzoate (E₂) was given to mice at different concentrations ranging from 0.0 to 0.05 mg/mouse (4 levels) beginning 72 h prior to vaginal inoculation, then mice were inoculated intravaginally with various concentrations of stationary-phase C. albicans blastoconidia (ATCC90028) (5 levels) in 20 μL of phosphate-buffered saline (PBS) in each E₂ level. General state, scores of genital pathology, the hyphae and vaginal fungal burden (CFU) in vaginal lavage fluid, the hydrops rate of uterus and vaginal tissues for pathological section in mice were observed and obtained at day 2, 4, 7, 14 and 21 after inoculation. The results showed the infection rate in mice was related to the dosage of E₂ and concentration of C. albicans blastoconidia. Additionally there was better cross-effect between the two treated factors. The infection rate was about 80% on the day 4, and could reach 100% on the day 7 until the end of experiment after inoculated intravaginally in groups of E2I3, E₂ 0.025 mg/mouse injected hypodermically and inoculated intravaginally with 5 × 10⁴C. albicans blastoconidia, and large amount of hyphae and blastoconidia could be observe in superficial layer tissue and canal of vaginal by PAS. From the results in our experiment it was concluded that E2I3 was the optima construction condition in kunming mice.

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the control rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regulating the apoptosis of granulosa cells in PCOS rats.

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further development capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cytoplasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hybridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chromosomal diseases.

To explore the dynamic expression and role of Aquaporin5 (AQP5) in lung development and hyperoxia lung injury, gestation 21-day Sprague-Dawley (SD) rats (term=22 days) were randomly assigned to air group and hyperoxia group within 12–24 h after birth. The rats in hypreoxia group were continuously exposed to about 85% oxygen and those in air group to room air. After 1 to 14 days of exposure, total lung RNA was extracted and the expression of AQP5 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry and western-blot were used to detect the expression of AQP5 protein. The results showed that the expression of AQP5 in premature rats lung could be detected at various time points after birth, and the positive staining was restricted to the type I alveolar epithelial cells. In air group, the AQP5 expression was detected in a very low level at day 1, but exhibited a persistent increase after birth. Compared with the air group, the expression of AQP5 in hyperoxia group was increased at day 1, and had significant difference in mRNA level (P<0.05), but decreased significantly in mRNA and protein levels after 4 to 14 days (P<0.01 or P<0.05 respectively). It was concluded that AQP5 might play a key role in the alveolar period of premature rats by regulating the lung water balance. Hyperoxia exposure leads to a down-regulation of the AQP5 expression, which may be an important factor for the development of hyperoxia lung injury.

In order to investigate the clinicopathological characteristics of aortic valve disease in children, all the native surgically excised aortic valves obtained between January 2003 and December 2005 were studied macroscopically and microscopically. The patients’ medical records were reviewed and the clinical information was extracted. According to preoperative echocardiography, intraoperative assessment, and postoperative pathology, combined with clinical symptoms and signs, aortic valve diseases were divided into three categories: aortic stenosis (AS), aortic insufficiency (AI), and aortic stenosis with insufficiency (AS-AI). The etiology was determined according to the macroscopic, microscopic and clinical findings. The results showed that among 70 aortic valves, patient age ranged from 6 to 18 years, with a mean of 15.4 years, and there were 56 boys and 14 girls (male: female=4:1). Forty-four children only had pure aortic valve disease, and the other 26 children had aortic valve disease associated with other heart valve diseases. There were 5 cases of AS (7.14%), 60 cases of AI (85.71%) and 5 cases of AS-AI (7.14%). The causes were congenital aortic valve malformation (32 cases, 45.71%), rheumatic disease (28 cases, 40%), infective endocarditis (7 cases, 10%), Marfan syndrome (2 cases, 2.86%), and undetermined (1 case, 1.43%). It was concluded that the common causes of aortic valve disease in order of frequency in children were congenital aortic valve malformation, rheumatic disease, infective endocarditis, and Marfan syndrome. AI was more common in children with aortic valve disease. Compared with adult patients, congenital bicuspid aortic valve in children was often AI. Histologically, the leaflets of congenital bicuspid aortic valve were mainly myxomatous, fibrosis and calcification less seen. AI was frequently found in rheumatic disease, mostly associated with other heart valve diseases. Macroscopic and microscopic examinations together with clinical information, echocardiographic findings and operative details were important in evaluating the etiology of aortic valve disease.

The expression of silience of death domains (SODD) and its clinical significance and relationship with phospho-NF-κB-p65 proteins in bone marrow cells of childhood acute lymphoblastic leukaemia (ALL) were explored, and the expression of SODD and phospho-NF-κB-p65 in Jurkat cells treated with chemotherapeutic drugs was detected in order to find a new chemotherapeutic target. The expression of SODD and phospho-NF-κB-p65 proteins in bone marrow cells was detected by immunohistochemistry in 25 children with ALL. The apoptosis rate was measured by Annexin-V-Fluorescence/PI double-labeling flow cytometry and the expression of SODD and phospho-NF-κB-p65 proteins determined by Western blotting in the Jurkat cells. It was found that the expression of SODD and active P65 in ALL was significantly higher than that in normal control group (P<0.05). The expression of the SODD and phospho-NF-κB-p65 proteins in the high-risk (HR) group was significantly higher than that in the standard-risk (SR) group (P<0.05). The Pearson rank correlation analysis revealed that there was a positive correlation between SODD and phospho-NF-κB-p65 expression (P<0.01, r=0.69). VCR could effectively induce the apoptosis of Jurkat cells, and down-regulate the expression of SODD and phospho-NF-κB-p65 proteins in a time-dependent manner, but DNR could not down-regulate the expression of SODD effectively. It was concluded that SODD may be closely related to the clinical classification and prognosis of ALL in children. The expression of SODD and phospho-NF-κB-p65 had a definite synergistic relationship with the onset and development of ALL. VCR could down-regulate the expression of SODD and inhibit the NF-κB activation, which could recover the sensibility of apoptosis in leukemic cells.

In order to investigate the role of Th17 cytokines in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-17, IL-23 (p19/p40), and IL-6 in skin lesions and non-lesions of the patients with psoriasis and skin tissues of normal subjects. The results showed that the mRNA expression levels of IL-17, IL-23p19, IL-23p40 and IL-6 in psoriasis leision were significantly higher than those of non-leisions (1.231 ± 0.843 vs 1.003 ± 0.044, 1.166 ± 0.142 vs 0.765 ± 0.133, 1.125 ± 0.104 vs 0.730 ± 0.103, 1.186 ± 0.222 vs 0.976 ± 0.122, respectively, all P < 0.05). Meanwhile, The expression levels of IL-17 mRNA, IL-23p19 mRNA, IL-23p40 mRNA and IL-6 mRNA were higher in non-leisions than those in normal skin tissues (1.003 ± 0.044 vs 0.620 ± 0.104, 0.765 ± 0.133 vs 0.584 ± 0.078, 0.730 ± 0.103 vs 0.000 ± 0.000, 0.976 ± 0.122 vs 0.656 ± 0.121, respectively, all P < 0.05). The overexpression of Th17 cytokines in the skin lesions of patients with psoriasis may indicate that Th17 cytokines play a very important role in the immunopathogenesis of psoriasis.

In order to analyze the in vivo expression of Candida albicans secreted aspartyl proteinases (SAP) in human vaginal infection, the vaginal secretion from 29 human subjects was collected by vaginal swab, and the expression of SAP1-SAP6 was detected by reverse-transcriptase polymerase chain reaction using specific primer sets. It was found that Sap2 and Sap5 were the most common genes expressed during infection; Sap3 and Sap4 were detected in all subjects and all 6 SAP genes were simultaneously expressed in some patients with vaginal candidiasis. It was suggested that the SAP family is expressed by Candida albicans during infection in human and that Candida albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of vaginal candidasis.

In order to compare the clinical characteristics of familial and non-familial bullous lichen planus (BLP), the archival data of 36 BLP patients with positive family history and 21 BLP patients with negative family history diagnosed according to the clinical features and histopathology were collected in our department from 1956 to 2003. The clinical features were analyzed and compared. There were significant differences between familial and non-familial BLP in age of onset, duration of disease and extension of eruption (P<0.01). It was concluded that familial BLP appeared to differ from the non-familial form in its earlier age of onset, longer duration of the disease, more extensive eruption and more tendency to involve nails. Hereditary factors may play a role in the pathogenesis of familial BLP.

In order to investigate the effect of curcumin on proliferation and apoptosis of human pterygium fibroblasts (HPF) in culture and search for a new method to prevent the recurrence after pterygium surgery, HPF was incubated with 0–160 μmol/L curcumin for 24–96 h. The MTT method was used to assay the biologic activities of curcumin at different time points and different doses. The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by immunohistochemistry. The cell cycle distribution was detected by flow cytometry (FCM). Administration of 20–80 μmol/L curcumin for 24–72 h could significantly inhibit HPF proliferation in a dose-and time-dependent manner (P<0.05). After treatment with curcumin at different concentrations of 20, 40, 80 and 160 μmol/L for 24 h, FCM revealed there was a significant sub-G1 peak at each concentration. The number of HPF in G₀/G₁ phase was increased, while in S phase, it was decreased (P<0.05). At the concentration of 20–80 μmol/L, curcumin, in a dose-dependent manner (P<0.05), could inhibit the expression of PCNA in HPF. It was suggesterd that curcumin could significantly inhibit the proliferation of HPF, make HPF arrest in G₀/G₁ phase and induce the apoptosis of HPF in a dose-and time-dependent manner.

In order to study the association of each item of a health examination and lifestyles with health-related quality of life (HRQoL), a cross-sectional survey by a self-administered questionnaire using the 12-Item Short Form Health Survey (SF-12, version 1) was conducted on 145 government managers that took health examinations in Tongji Hospital, Wuhan, China during 2005. The results showed that the determinations of organic functions including BMI, hemoglobin, blood platelet count, urine protein, urine WBC and lipid metabolism (LDL) were associated with HRQoL. The lifestyle items including age, sex, life satisfaction drinking alcohol and kind of alcohol drunk, drinking tea, sleep quality and adhering to bedtime, working overtime, work stress were associated with HRQoL in the multiple regression models. Sex, age and life satisfaction were the lifestyle items most closely associated with HRQoL, while LDL, hemoglobin and urine protein were the health examination items most closely associated HRQoL. This study revealed that both lifestyle and health examination items were associated with HRQoL.