Abstract:Objective：To elucidate the distribution of HBV genotypes and subgenotypes in patients with chronic hepatitis B(CHB), hepatocellular carcinoma(HCC) and asymptomatic HBV carriers(ASC) in Shanghai and areas around Shanghai, and to analyze the role of HBV genotypes and subgenotypes in the carcinogenesis and progress of HBV-related diseases. Methods: The HBV genotypes and subgenotypes were determined in 462 HCC patients, 234 CHB patients and 110 ASCs from Shanghai and areas around Shanghai by a multiplex PCR assay. Results: Genotypes A, B, C and D and subgenotypes B2, C1 and C2 were detected. Genotype C(mainly C2, 98.5%) and B(B2, 100%) were more prevalent than other genotypes in our group. Compared with CHB group, HCC group had higher proportion of genotype C(P=0.009) and lower proportion of genotype B(P=0.045). In the patients infected with HBV subgenotypes B2 or C2, the expression of HBeAg in CHB group was significantly higher than that in HCC group(P=0.005; P=0.008), and the expression of anti- HBe was lower in CHB group(P=0.003,P=0.001).In HCC patients, expression of HBeAg in patients infected with mixture genotype was higher than that in those infected with other genotypes(P=0.016 for B2). HCC patients (aged from 40 to 60) with HBV B2 infection had lower viral load than those with C2 and genotype mixture(P=0.029, P=0.021); and patients with HBV C2 infection had lower viral load than those with genotype mixture(P=0.041). Conclusion: Subgenotype C2 is more prevalent than B2 in people living in Shanghai and areas around Shanghai. The compositions of HBV genotypes and subgenotypes are different in patients with CHB, HCC and ASCs. Co-infection with different HBV-genotypes is associated with higher viral load, expression of HBeAg and easier carcinogenesis of HCC.

Abstract:Objective：To detect and identify differentially expressed proteins in the sera of patients with HBV-related primary hepatic carcinoma and discuss their possible role in early diagnosis of primary hepatic carcinoma.Methods: The surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) was used to screen for the differentially expressed serum proteins in patients with HBV-related primary hepatic carcinoma,liver cirrhosis,chronic hepatitis,and healthy adults.Then the most differential protein sample was isolated and purified by ionic exchange chromatography and was subjected to mass spectrometry analysis.The screening results were used to establish a diagnosis model for hepatic carcinoma and the accuracy and sensitivity of the model were assessed.Results: CM10（weak cation exchange）chip found 44 differentially expressed protein peaks (P<0.05) in hepatic carcinoma group compared with those in the healthy adult group,with 21 up-regulated and 23 down-regulated.Fifty-one differential peaks were identified in hepatic carcinoma group compared with those in the liver cirrhosis group (P<0.05), with 47 up-regulated and 4 down-regulated.The expression of protein with a mass-to-charge ratio (m/z) of 5 805 gradually increased in order in the healthy adult, chronic hepatitis, liver cirrhosis and hepatic carcinoma patients; and the expression in hepatic carcinoma group was significantly higher than that in the healthy adult group (P＜0.01); peptide mass fingerprint (PMF) after enzyme hydrolysis showed that 2 of the peptides were partially identical to chondroitin sulfate synthase 2.A diagnosis model of hepatic carcinoma was successfully established based on the differentially expressed proteins,with an accuracy of 94.82％( \[23+32\]/58 ),a sensitivity of 88.46%（23/26），and a specificity of 100%（32/32）.Conclusion: We have successfully screened out the differentially expressed proteins in the sera of patients with HBV-related primary hepatic carcinoma; the most differentially expressed protein (5 805) has 2 peptides partially identical to chondroitin sulfate synthase 2 in sequence.The established model may help to diagnose HBV-related primary hepatic carcinoma.

Abstract:Objective：To investigate the influence of troglitazone, a potent peroxisome proliferator-activated receptor (PPAR) gamma agonist, on proliferation and β-catenin signaling pathway of human liver cancer cell line HepG2 in vitro, and to discuss its possible anti-cancer mechanism. Methods: HepG2 cells were cultured in vitro and the cell growth was assessed by MTT assay after exposure to different concentrations of troglitazone (5, 10, 20, 40, 80 and 100 μmol/L) for 120 h, and the results were compared with that of the control cells (cultured normally). Flow cytometry was used to assess cell cycle of HepG2 cells treated with troglitazone at 10 μmol/L and of normal control cells. The subcellular location of β-catenin was investigated by immunocytochemistry in troglitazone（10 μmol/L）-treated and control cells. Expression of cyclin D1 and c-myc proteins was examined by Western blotting assay. Results: MTT assay demonstrated that, after treatment with 5, 10, 20, 40, 80 and 100 μmol/L of troglitazone, the cell survival rates were (96.8±1.2）%,（53.4±1.2）%,（42.3±1.2）%, （31.4±1.0）%,（13.6±0.8）% and （9.6±0.7）%, respectively. Compared with control cells, cells treated with 10 μmol/L troglitazone showed an increased proportion of cells at the G0/G1 phase（\[67.6±0.5\]% vs \[56.3±1.5\]%，P＜0.01) and decreased proportion of cells at the S phase（\[20.6±0.5\]% vs \[25±1.0\]%，P＜0.01）. β-catenin was located in the nucleus of the control cells and in the cytoplasm of the troglitazone-treated cells. Western blotting analysis showed that the expression of c-myc and cyclin D1 proteins in troglitazone-treated cells was lower than that in the control cells. Conclusion: Troglitazone can inhibit the proliferation of HepG2 cells in a dose-dependent manner, which may be associated with regulation of the β-catenin signaling pathway and inhibition of target protein expression.

Abstract:Objective：To investigate the mechanism for decrease of glucocorticoid receptor mRNA (GR mRNA) during heat shock response. Methods: The changes of GR mRNA level in SMMC-7721 cells were examined by semi-quantitative RT-PCR during heat shock response. We designed 2 RNA fragments paired with GR gene intron E and cross exon 3, 4, and 5, which were used as probes for in situ hybridization with the sequences in SMMC-7721 cells at different periods after heat shock. The result was subjected to confocol microscope observation and computer image analysis. The transcription, degradation, and splicing of GR mRNA were investigated. Results: RT-PCR showed that the GR mRNA level was decreased during heat shock response. In situ hybridization revealed that both GR mRNA and GR pre-mRNA levels were lower in the heat shock group than in the non-heat shock group(P<0.05). GR pre-mRNA level was higher in cells treated with actinomycine D before heat shock than in cells only treated with actinomycine D, while the GR mRNA level was lower in cells treated with actinomycine D before heat shock(P<0.05). Conclusion: During heat shock response, GR mRNA transcription is suppressed, the splicing of GR pre-mRNA is suppressed, and GR mRNA degradation is accelerated.

Abstract:Objective：To investigate the expression changes and the subcellular localization of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in hepatocellular carcinoma (HCC) and cell line of different cell densities. Methods: The expression of hnRNP K in HCC was detected by immunohistochemical staining. The differential expression of hnRNP K in the HCC tissue (n=30) and paired paracancerous tissue was identified by Western blotting assay. The expression and sub-cellular location of hnRNP K in HCC cell line of different cell densities were determined by immunocytochemical staining and Western blotting assay. Results: Expression of hnRNP K was mainly detected in the nucleus of HCC cells. Twenty-one of the 30 hepatic cancer tissues had higher hnRNP K expression than the paired paracancerous tissues. Sixteen of the 20 HCC samples with HBV infection had an increased expression of hnRNP K; in the 10 HCC samples without HBV infection,5 had an increased expression of hnRNP K. Immunocytochemical staining and Western blotting assay both showed that the expression of hnRNP K was increased in the nucleus of HCC cells with the increase of cell densities （30%, 50% and 90% confluence）; the result of Western blotting assay also showed that there was no obvious change in hnRNP K expression in the cytoplasm of HCC cells of different cell densities（30%, 50%, 70% and 90% confluence）. Conclusion: The expression of hnRNP K is elevated in HCC tissues and has an increasing tendency in the nucleus with the increase of cell density. Infection of HBV may be associated with the up-regulation of hnRNP K in HCC cells.

Abstract:Objective：To investigate the inhibitory effect of baicalin on the fluorouracil (Fu) resistant hepatocarcinoma cell(HCC) BEL-7402/5-Fu and its possible mechanism. Methods:Hepatocarcinoma cell line BEL-7402 and Fu-resistant hepatocarcinoma cell line BEL-7402/5-Fu were cultured in vitro. The inhibitory effect of baicalin on the BEL-7402/5-Fu cells was assessed by MTT assay; the intracellular rhodamine fluorescence intensity was observed by flow cytometry; the expression of MDR1 gene was detected by RT-PCR; and the expression of protein P-glycoprotein (P-gp) was analyzed by Western blotting assay. Adhesion assay was conducted using Matrigel model. Expression of beta 1-integrin and E-CD protein was detected by immunofluorescence technique. Results:Baicalin inhibited the proliferation of both BEL-7402 and BEL-7402/5-Fu cells, with IC50 of baicalin being 34.2 mg/L and 36.6 mg/L, respectively. Baicalin (5 mg/L and 10 mg/L) partially reversed the resistance of BEL-7402/5-Fu to Fu, with the reversal folds being 28.6 and 46.7, respectively. Baicalin (5 mg/L and 10 mg/L) increased the sensitivity of BEL-7402 cells to Fu, with the sensitivity-enhancing folds being 1.4 and 2.1, respectively. Baicalin also increased the concentration of rhodamine and expression of integrin β1, inhibited the expression of MDR1 gene and P-gp, E-CD protein, and reduced the adhesion capacity, with the effect of 10 mg/L baicalin significantly effective than that of 5 mg/L baicalin (all P＜0.05).Conclusion:Baicalin can inhibit the proliferation of BEL-7402/5-Fu in vitro, and partially reverse the resistance to Fu, which is attributable to the increased accumulation of intracellular drug concentration, inhibited expression of MDR1 gene.

Abstract:Objective：To screen for the differentially expressed proteins during rat liver regeneration after partial hepatectomy(PH) by proteomics technique. Methods: Healthy male SD rats were randomly divided into 2 groups: hepatectomy group and sham operated group. The hepatectomy model was produced by 70% PH(n=35) and the sham operated rats（n=5）underwent the same surgical protocol without hepatectomy. Rats were executed at 2, 12, 24, 36, 48, 72 and 168 h after partial hepatectomy (each time 5 rats) and the right lobes were harvested. The total protein was extracted and analyzed by two-dimensional gel electrophoresis and mass spectrometric analysis. The differential proteins were then analyzed by Western blotting. Results: The spots of differential protein began to rise at 2 h after PH and peaked at 36 h after PH. A total of 78 protein spots were identified and 35 significant protein spots were found by mass spectrometric analysis. The 35 protein sports fell into 5 types according to their dynamic changes: 3 up-regulated and 2 down-regulated (with different regulation time periods and amplitudes); and their functions involved oxidative stress response, acute reaction, lipid and energy metabolism, intracellular signaling transduction, cell proliferation, etc., with some having unknown functions. Western blotting analysis showed that the prohibitin protein began to increase 2 h after PH and decreased to the normal level after 48 h. Conclusion: It is indicated that many proteins and signal transduction pathways participate in the liver regeneration after partial hepatectomy.

Abstract:

Abstract:Objective：To observe the therapeutic effect of major histocompatibility complex (MHC) class Ⅱ transactivator mutant (CⅡTAm) for gene therapy of mouse experimental autoimmune thyroiditis (EAT) and explore the possible mechanisms.Methods: Thirty-one healthy female CBA/J mice were randomly divided into 4 groups，namely EAT model group(n=8)，CⅡTAm therapy group(n=9)，GFP control group (n=9)，and normal control group(n=5). Animals in the first 3 groups were immunized with porcine thyroglobulin (pTg) and complete or incomplete Freud’s adjuvant (CFA/IFA) to establish EAT model; mice in the CⅡTAm therapy group and GFP control group were also treated by intravenous recombinant adenovirus Ad-CMV-CⅡTAm and Ad-GFP, respectively, while those in the EAT model group were injected with equal volume of normal saline. Mice in the normal control group received no special treatment. All mice were sacrificed on the 29th day after the first immunization. The thyroid pathological changes were examined using H-E staining; the expression of MHC Ⅱ molecules in the thyroid was examined using immunohistochemical staining; the spleen lymphocyte proliferation and IFN-γ secretion stimulated by pTg were examined in their culture supernatant; the titer of plasma anti-pTg autoantibody was assayed by ELISA; and the CD4+ T cells in both peripheral blood and spleen was analyzed by flow cytometry. Results: H-E staining showed that the infiltration index of thyroid lymphocyte in the CⅡTAm therapy group (0.3±0.5) was significantly lower than that in the EAT model group (1.4±0.4) and the GFP control group (1.5±0.2, both P<0.01). Immunohistochemical staining showed diffused expression of MHC Ⅱ molecules in the thyroid of the EAT model group and GFP control group, compared to very weak expression in the CⅡTAm therapy group and the negative expression in the normal control group. The lymphocyte stimulation index (SI) against 80 μg/ml pTg in the CⅡTAm therapy group was significantly lower than that in the EAT model group and the GFP control group（P<0.05）. The IFN-γ secretion in the culture supernatants showed a similar difference as SI in all the groups (P<0.01). The titer of plasma anti-pTg autoantibody in the CⅡTAm therapy group was significantly lower than those in the EAT model group and the GFP control group (both P<0.01). The positive rate of ICOS on CD4+ T cells in the CⅡTAm therapy group was significantly lower than that in the EAT model group and the GFP control group（both P<0.01）. Conclusion: Ad-CMV-CⅡTAm recombinant adenovirus can inhibit the MHC Ⅱ molecule expression in the thyroid of EAT mouse and the proliferation of self-reactive T cells, attenuate the inflammatory cells infiltration in the thyroid, and decrease the titer of plasma anti-pTg autoantibody, indicating that CⅡTA mutants might have therapeutic effect for EAT.

Abstract:Objective：To screen for an optimized siRNA sequence targeting rat Toll-like receptor 4 (TLR4) in vitro. Methods: The full length gene of rat TLR4 was cloned and inserted into pEGFP-C1 plasmid to construct pEGFP-rTLR4. Three pairs of siRNAs targeting rTLR4 were chemically synthesized and were co-transfected with pEGFP-rTLR4 into HEK-293 cells via Lipofectamine2000. Cells were also co-transfected with siRNA targeting EGFP and negative control siRNA. The expression of EGFP was observed under inverted fluorescene microscope and flow cytometry. Results: Compared with the negative control group, 3 pairs of siRNAs targeting TLR4 and one pair of siRNA targeting EGFP significantly suppressed the EGFP expression (P<0.05); the inhibitory effect of siRNA2（gene sequence:5′-GTC TCA GAT ATC TAG ATC T-3′,19 bp, 1 352-1 370）was the strongest one, with an interference efficiency over 75%.Conclusion: We have successfully obtained the siRNA sequence targeting TLR4 mRNA, which can efficiently suppress the expression of rat TLR4 mRNA in vitro.

Abstract:Objective：To observe the influence of lentiviral vector-mediated RNA interference on expression of human APRIL (a proliferation-inducing ligand) gene in human pancreatic cancer cell line CFPAC-1, so as to pave a way for APRIL gene-targeted gene therapy of pancreatic cancer. Methods: Gene engineering technique was used to screen 3 RNA interference sequences targeting APRIL gene; the sequences were separately cloned into the pGCL-GFP vector to construct LV-APRIL shRNA1, LV-APRIL shRNA2 and LV-APRIL shRNA3, which were subsequently confirmed by PCR and DNA sequencing analysis. The titer of lentivirus was determined after 293T cells were cotransfected with LV-APRIL shRNA, pHelper 1.0 and pHelper 2.0. The 3 kinds of recombinant lentiviruses were injected into CFPAC-1 cells and the APRIL mRNA and protein expression were examined by real-time RT-PCR and Western blotting, respectively,and the result was compared with those of the non-transfected and blank vector transfected CFPAC-1 cells. Results: PCR analysis and DNA sequencing confirmed that the 3 APRILshRNA sequences were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5×107 TU/ml,6×107 TU/ml and 4×107 TU/ml, respectively. APRIL expression in CFPAC-1 cells was significantly inhibited at both mRNA and protein levels compared with the non-transfected and empty vector transfected CFPAC-1 cells (P<0.05). After transfection with LV-APRIL shRNA1 and LV-APRIL shRNA2, APRIL mRNA expression decreased by 73％ and 68％, APRIL protein expression decreased by 66％ and 59％ (P<0.05), respectively; there was no significantly difference between the non-transfected and empty vector transfected CFPAC-1 cells.Conclusion: Three lentiviral RNAi vectors of APRIL gene have been successfully constructed, and they can effectively inhibit the expression of APRIL gene in CFPAC-1 cells in vitro.

Abstract:Objective：To establish an androgen-independent human prostate cancer cell line model LNCaP-AI. Methods: LNCaP cells were cultured in absence of hormone for a long-term to establish LNCaP cell line LNCaP-AI, which can live without hormone. MTT, immunofluorescence and RT-PCR techniques were used to study the proliferation activity of LNCaP-AI cells and expression and secretion level of PSA by LNCaP-AI cells in absence of hormone.Results: After cultured for 3 months, LNCaP cells gradually became accustomed to the non-hormone condition, showing the characteristics of androgen-independent LNCaP-AI cell line. LNCaP-AI cells rapidly proliferated under the non-hormone condition and secreted PSA.However, PSA mRNA expression level in LNCaP-AI cells was 44% that of the LNCaP cells under hormone condition.Conclusion: Androgen-independent LNCaP-AI cell line may simulate the development of androgen-independence in prostate cancer cells and is an ideal model of androgen-independent prostate cancer cell line.

Abstract:Objective：To investigate the relationship of mitochondrial DNA 4977 bp (mtDNA 4977) deletion in the peripheral blood with severity and stability of coronary atherosclerosis. Methods：We selected 90 unrelated patients with coronary atherosclerotic heart disease (CAD) who were diagnosed by coronary angiography (CAG). The severity of pathological changes of the coronary artery was assessed by the number of diseased coronary branches and Gensini score. The CAD patients were further divided into subgroups according to the clinical types, the number of diseased coronary branches and Gensini score. Control group included 60 healthy age-matched subjects. The relative amount of mtDNA 4977 deletion was determined using a nested polymerase chain reaction (PCR) protocol. White blood cell (WBC) count, high sensitive C reactive protein (hsCRP), lipids (TC, TG, LDL-C, HDL-C), plasma glucose (FPG, 2hPG), blood pressure (SBP, DBP) and body mass index (BMI) were all measured. The information on age, sex and medical histories, including smoking status, hypertension and diabetes mellitus,were obtained in all subjects. Clinical parameters, biochemical indicators, the incidence and relative amount of mtDNA 4977 deletion were compared between the subgroups; the correlation coefficients of mtDNA 4977 relative amount with WBC count, hsCRP and other conventional risk factors for CAD were calculated. Results：The incidence and relative amount of mtDNA 4977 deletion in the peripheral blood in CAD patients were significantly higher than those in the controls (P＜0.01). No significant differences were found in the incidence and relative amount of mtDNA 4977 deletion between patients with angina pectoris and acute myocardial infarction (P＞0.05). MtDNA 4977 deletion incidence and relative amount increased with the increase of diseased coronary branches and Gensini score.In CAD patients mtDNA 4977 deletion relative amount was positively correlated with the number of diseased coronary branches and Gensini score(P＜0.01), not correlated with WBC count and hsCRP. Conclusion: Peripheral blood mtDNA 4977 deletion can be used to predict the severity of coronary atherosclerosis, though it is not associated with the stability of pathological changes of the coronary artery.

Abstract:Objective：To investigate the differential expression of serum soluble TNFrelated apoptosisinducing ligand (sTRAIL) between ankylosing spondylitis (AS) and rheumatoid arthritis patients (RA) and to discuss its clinical significance. Methods: Sixty AS patients, including 38 HLAB27positive ones and 22 HLAB27negative ones, 20 rheumatoid arthritis (RA) patients and 30 healthy individuals were included in the present study. The AS patients were divided into active group and inactive group based on bath ankylosing spondylitis disease activity index (BASDAI). The concentrations of serum sTRAIL were measured by ELISA in all groups. Erythrocyte sedimentation rate (ESR) and Creactive protein (CRP) were detected automatically by ESR automatic analyzer and specific protein analyzer. Results: The serum sTRAIL concentration was significantly higher in AS patients (both HLAB27positive and negative AS patients) than in RA patients and healthy controls (P<0.01); no significant difference was found between HLAB27positive and negative AS patients. Serum sTRAIL concentration was significantly higher in active AS group than in inactive AS group（P<0.01）. Serum sTRAIL and CRP concentrations were correlated with each other in HLAB27positive AS patients（r=0.609，P=0.000）, but not in HLAB27negative ones. Serum sTRAIL concentration was not correlated with ESR in AS patients. Conclusion: Serum sTRAIL is obviously upregulated in AS patients, which is not associated with the status of HLAB27. However, the association between sTRAIL and CRP is influenced by the status of HLAB27.

Abstract:

Abstract:Objective：To observe the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in non-small cell lung cancer(NSCLC), and to discuss its clinical significance. Methods: The expression of EGFR and VEGF was detected in 82 NSCLC and 20 non-malignant pulmonary samples by immunohistochemical method. The expression of EGFR and VEGF in NSCLC patients with various pathological characteristics was observed and the correlation between them was analyzed. Results: The positive rates of EGFR and VEGF in 82 NSCLC samples were obviously higher than those in the 20 non-malignant samples（53.66% vs 0 for EGFR, 62.20% vs 25％ for VEGF, both P<0.05）. The expression of EGFR in NSCLC samples was significantly correlated with the sex of patients, pathological types of cancer (squamous-cell carcinoma vs adenocarcinoma), presence of lymph node metastasis, and TNM stages（P<0.05）. The expression of VEGF in NSCLC was correlated with lymph node metastasis and TNM stages (P<0.05). It was also noticed that higher expression of EGFR or VEGF was associated with poor prognosis of patients (P<0.05). The expression of EGFR was correlated with VEGF expression in pulmonary tissues of NSCLC patients（rs=0.314,P<0.05）. Conclusion: EGFR and VEGF are over-expressed in NSCLC tissues and the 2 are correlated with each other, which may serve as predicator for prognosis and a therapeutic target of NSCLC patients.

Abstract:Objective：To observe the changes of mu-opioid receptors (MOR) expression in acute inflamed knee joint synovium tissue of dogs,so as to discuss the feasibility of using peripheral local opioid for analgesia in acute inflammation.Methods: Knee joint synovium tissues were taken from 9 dogs with acute arthritis and 8 dogs with normal knee joints.The expression of MOR protein and mRNA was examined by immunohistochemistry and real-time quantitative PCR.Results: The expression of MOR mRNA in the acute inflamed group was significantly higher than that in the normal control group （\[34.40±5.48\] % vs\[16.54±8.03\] %,P<0.05).Immunohistochemical result showed more positive staining of MOR particles and stronger signal in the acute arthritis group than in normal control group.The immunohistochemical index of MOR positive cells in the acute arthritis was significantly higher than that in normal control group (\[323 175.00±92 614.94\] vs \[175 444.10±75 149.06\],P<0.05).Conclusion: MOR exists in the knee joint synovium tissue of dogs,and acute inflammation can enhance the expression of MOR.

Abstract:Objective：To study the changes and rules of the reference point potentials of electrocardiograms (ECGs) before and after pacing. Methods: The head-chest lead and routine lead ECGs of the patients with permanent pacemaker implantation were recorded with and without right ventricular pacing; the Q, R and S wave amplitudes were compared between the 2 ECGs.Results: Without right ventricular pacing, the amplitudes of Q and S wave of routine lead were higher than those of the head-chest lead; the amplitude of R wave was lower than that of the head-chest lead. With right ventricle pacing, the amplitudes of Q, R, and S waves of routine lead were lower than those of head-chest lead(all P<0.01).Conclusion: The transduction direction of the cardiac excitement has great influence on the potentials of the reference point.

Abstract:Objective：To separate the nematocyst venom (NV) and tentacle-only extract (TOE) from the jellyfish Cyanea capillata and to analyze the difference between their haemolytic activities and the influencing factors.Methods: NV and TOE were separated by autolysis and centrifugation.The influence of concentration, temperature and pH value on the haemolytic activities of NV and TOE were observed.Results: NV and TOE were successfully separated.The concentration-associated haemolytic curves were “S” shaped for both NV and TOE.The HU50 of NV and TOE were 8 μg/ml and 67 μg/ml, respectively; and the haemolysis of NV was about 8.4 times of that of TOE.Temperature had great influence on haemolysis of both and the highest haemolysis were both at 40℃.pH value also had influence on haemolysis.The strongest haemolysis activity was found both at pH 8 and TOE was more sensitive.Conclusion: NV and TOE both have haemolysis activity,with the activity of NV stronger than that of the latter.The haemolysis activity is influenced by concentration, temperature and pH value.

Abstract:Objective：To prepare humanized antinuclear antibody Fab fragment. Methods: The reconstructed humanized antinuclear antibody (ANA) Fab phage display library was enriched by 4 rounds of panning and was identified by indirect ELISA method.Phasmid DNA isolated from positive clones was deprived of gⅢ gene. After self-ligation the recombinant plasmid was used to transform E.coli. XL1-Blue, then XL1-Blue was induced by IPTG to product soluble human antinuclear antibody Fab fragment. Finally, soluble human antinuclear antibody Fab in the supernatant was identified by indirect ELISA method and immunofluorescence. Results: The eluted phages were enriched by more than 200 folds after 4 rounds of panning. Two positive clones were isolated from the ANA Fab library. Electrophoresis after XhoⅠdigestion proved that the self-ligation was successful after deletion of gⅢ gene. The results of indirect ELISA indicated that the 2 positive clones of Fab had specific anti-dsDNA activity. Indirect immunofluorescence showed homogeneous fluorescence within nuclei of Hep2 and monkey hepatic cells and in the Crithidia kinetoplast. Conclusion: We have successfully prepared soluble, specific human antinuclear antibody Fab fragment, which paves a way for preparation of high affinity antinuclear antibody Fab fragment.

Abstract:Objective：To analyze the polymorphism in human cDNA sequence of prothymosin-α (ProTα) by sequencing analysis. Methods: The cDNA of human ProTα was amplified from cells of peripheral blood and cord blood by RT-PCR．The product of RT-PCR was purified and linked with vector pMD18-T. After cloning and sequencing, the sequence of ProTα cDNA was compared with the standard sequence to analyze the polymorphism in the ProTα cDNA sequence. Results: The cloned ProTα cDNA sequence was different from that of the standard. We found 2 kinds of variations: (1) The nucleotide in 107 position was varied and the nucleotides in 110-121 and 191-205 positions were deleted; (2) The nucleotide in 306 position was deleted, mainly in the 60-80 years old group. Conclusion: We have identified 2 kinds of variations in human ProTα cDNA, but the first 28 amino acid in the N-terminal of cDNA of human ProTα are not involved therefore the variations do not affect the function of human ProTα.

Abstract:Enteric nervous system consists of myenteric and submucous plexuses. Myenteric plexus mainly innervates the enteric motion; submucous plexus modulates the gastrointestinal secretion and local blood flow. P2 (ATP) receptors exist in both of the above plexuses and play an important role under physiological and pathophysiological conditions. This article reviews the distribution, function and pharmacological characteristics of different P2 receptors, in an effort to provide new evidence for studying the role of P2 receptors in the diagnosis and therapy of enteric diseases.

Abstract:Objective：To apply chromogenic in situ hybridization (CISH) for detection of HER2 gene amplification in breast cancer tissues and to discuss some modifications of the CISH method. Methods: HER2 gene amplification was detected by CISH in 60 breast cancer specimens with an immunohistochemical score over 2+. The correlation between the results of IHC and CISH was analyzed.Our experience in CISH manipulation was summarized and optimization to CISH was discussed. Results: CISH identified gene amplification in 91％(40/44) specimens with an IHC score of 3+ and in 50％(8/16) specimens with an IHC score of 2+．The total concordance rate between IHC and CISH was 80％(48/60, P<0.01)．The thickness of sections should be controlled within 4-5 μm; the denaturation should be complete; and the post-hybridization washing temperature and time were also very important and the temperature should be controlled at 70-75℃. The dyeing time of hematoxylin should also be restrictedly controlled. Positive control should be set up in the experiment for high quality of the experiment. Conclusion: CISH has high concordance rate with IHC in examining HER2 amplification and it may be a new method for detection of HER2 gene. The thickness of the sections, the post-hybridization washing temperature and time, and the time of hematoxylin dyeing should be strictly controlled.

Abstract:目的：回顾性分析115例再次肾移植患者的临床资料，观察再次肾移植的效果并分析其影响因素。方法:回顾性分析我院自1978年7月至2006年10月间115例再次肾移植术后人/肾1年存活率，并与同期首次移植患者作对比；分析首次移植失败原因、血透过渡时间、术前补体依赖性细胞毒性试验（CDC）、群体反应性抗体（PRA)水平、免疫抑制方案等因素对移植术后效果的影响。结果:再次移植组术后移植肾1年存活率明显低于首次移植组（69.6% vs 88.7%，P<0.05）。血透过渡时间<6个月组患者移植肾功能恢复正常的比率明显高于6~12个月、12~24个月、>24个月各组（P<0.05）；CDC<5%组移植肾存活率明显高于5％~10％及>10%组（72％ vs 31％ vs 0，P<0.05）；再次肾移植受者PRA<10%组术后急性排斥反应发生率明显低于PRA>10%组（30.2％ vs 75.0％），而移植肾1年存活率明显高于后者（74.4% vs 60.0%，P<0.05）；免疫抑制方案中应用抗体诱导组术后移植肾1年存活率明显高于未用抗体诱导组（81.0% vs 63.4%，P<0.05）。结论:再次移植肾存活率低于首次移植；首次肾移植失败原因、血透过渡时间、术前PRA水平、术前淋巴细胞毒性反应水平及免疫抑制方案均影响再次肾移植效果。

Abstract:目的：观察盐酸甲氯芬酯对脑梗死患者心率变异性(heart rate variability,HRV）的影响，探讨其对脑梗死患者心脏自主神经功能损伤的疗效。方法:选择91例脑梗死患者，随机分为2组（甲氯芬酯治疗组46例、吡拉西坦对照组45例），选取同期50例健康体检者作为正常对照；甲氯芬酯治疗组静滴盐酸甲氯芬酯，吡拉西坦对照组静滴吡拉西坦，共15 d；比较各组HRV各项参数的变化。结果:与健康人群相比，脑梗死患者SDNN、SDANN、SDNN Index、RMSSD、PNN50、HRVTI各项指标均显著降低（P＜0.05）。甲氯芬酯治疗后HRV各项指标均明显高于治疗前（P＜0.05）；吡拉西坦治疗后SDNN、SDANN、SDNN Index较治疗前增高（P＜0.05），而RMSSD、PNN50、HRVTI无显著变化。治疗前2组各项指标无统计学差异，治疗后甲氯芬酯治疗组各项指标高于吡拉西坦对照组（P＜0.05）。结论:脑梗死后心脏自主神经活动受到抑制，甲氯芬酯能改善脑梗死后心脏的自主神经功能，对预防心血管事件有一定价值。

Abstract:目的：建立优化的高效毛细管电泳法,并将其应用于测定肝力保胶囊中苦参碱和氧化苦参碱的含量。方法：采用Agilent3DCE高效毛细管电泳仪、未涂渍熔融石英毛细管（68.5 cm×75 μm,有效长度60 cm）,缓冲液50 mmol/L Na2HPO4（水甲醇＝41,pH＝5.5）,运行电压25 kV,温度25℃,检测波长214 nm,以氨茶碱为内标,建立优化的高效毛细管电泳法,并测定3批次肝力保胶囊中苦参碱和氧化苦参碱的含量。结果：苦参碱和氧化苦参碱均能够基线分离,符合含量测定要求；回归方程分别为Y=222.80X+3.635 3（r=0.999 9）和Y=213.02X+34.268 6（r=0.999 9）；线性范围分别为33.98～339.8 μg/ml和47.35～473.5 μg/ml。苦参碱和氧化苦参碱的日内和日间精密度（RSD）均<3.0%,最低检测限分别为13.60 μg/ml和18.90 μg/ml,加样回收率结果分别为102.3%(RSD=0.57%,n=3)和101.1%(RSD=0. 41%,n=3)。3批样品中苦参碱的含量（%）分别为2.78±0.043、2.50±0.069、2.56±0.040；没有检测到氧化苦参碱的峰。结论：优化的高效毛细管电泳法稳定简便,结果可靠,能够用于测定苦参碱和氧化苦参碱含量；优化的高效毛细管电泳法测定的苦参碱含量可作为肝力保胶囊质量控制指标。

Abstract:目的：评价氪黄激光早期治疗视网膜中央静脉阻塞黄斑水肿的临床疗效。方法：用氪黄激光治疗视网膜中央静脉阻塞黄斑水肿共52例（52眼），氪黄激光波长568 nm，对黄斑区水肿采用格栅样光凝，光凝距黄斑中心小凹300 μm以上，避开视盘黄斑束，功率60~200 mW，光斑直径100 μm，曝光时间0.1 s，光凝168~287点，光斑反应Ⅰ~Ⅱ级。术后随访观察疗效。结果：术前视力为0.193±0.051，术后视力为0.221±0.045，两者比较无统计学差异；术后视力提高11眼（21.2%），不变29眼（55.8%），下降12眼（23.1%）。光凝后黄斑水肿完全消退41眼（78.8%），部分消退9眼（17.3%），不变2眼（3.8%）。结论：早期采用氪黄激光治疗视网膜中央静脉阻塞黄斑水肿有效，但不能提高视力。

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