Abstract:

Abstract:Objective: A computational model of insulin secretion and glucose metabolism for assisting the diagnosis of diabetes mellitus in clinical research is introduced. The proposed method for the estimation of parameters for a system of ordinary differential equations (ODEs) that represent the time course of plasma glucose and insulin concentrations during glucose tolerance test (GTT) in physiological studies is presented. The aim of this study was to explore how to interpret those laboratory glucose and insulin data as well as enhance the Ackerman math-ematical model. Methods: Parameters estimation for a system of ODEs was performed by minimizing the sum of squared residuals (SSR) function, which quantifies the difference between theoretical model predictions and GTT's experimental observations. Our proposed perturbation search and multiple-shooting methods were applied during the estimating process. Results: Based on the Ackerman's published data, we estimated the key param-eters by applying R-based iterative computer programs. As a result, the theoretically simulated curves perfectly matched the experimental data points. Our model showed that the estimated parameters, computed frequency and period values, were proven a good indicator of diabetes. Conclusion: The present paper introduces a computational algorithm to biomedical problems, particularly to endocrinology and metabolism fields, which involves two coupled differential equations with four parameters describing the glucose-insulin regulatory system that Acker-man proposed earlier. The enhanced approach may provide clinicians in endocrinology and metabolism field in-sight into the transition nature of human metabolic mechanism from normal to impaired glucose tolerance.

Abstract:Objective: The association between ribonuclease L (RNASEL) gene polymorphisms and prostate cancer risk has been widely reported, but the results of these studies remained controversial and underpowered. We performed a meta-analysis of 28 studies to evaluate the association between Arg462Gln and Asp541Glu polymorphisms in the RNASEL gene and prostate cancer risk. Methods: Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between RNASEL polymorphisms and prostate cancer risk. Results: A significantly increased prostate cancer risk was found for the Arg462Gln polymorphism in Africans (Gln/Gln vs Arg/Arg: OR = 2.50, 95%CI = 1.28-4.87; Gln/Gln vs Gln/Arg + Arg/Arg: OR = 2.54, 95%CI = 1.30-4.95), but not in Europeans and Asians. Additionally, the Asp541Glu polymorphism was associated with increased total prostate cancer risk (Glu-allele vs Asp-allele: OR = 1.04, 95%CI = 1.01-1.07; Glu/Glu vs Asp/Asp: OR = 1.22, 95%CI = 1.03-1.46; Glu/Glu vs Glu/Asp + Asp/Asp: OR = 1.09, 95%CI = 1.02-1.16). In the stratified analysis for the As-p541Glu polymorphism, there was a significantly increased prostate cancer risk in Africans and Europeans, and in hospital-based prostate cancer cases. Conclusion: The meta-analysis results showed evidence that RNASEL Arg462Gln and Asp541Glu polymorphisms are associated with prostate cancer risk and could be low-penetrance prostate cancer susceptibility biomarkers.

Abstract:Objective: To investigate a possible association of LMP2/LMP7 genes with chronic hepatitis C virus (HCV) infection, and to assess whether LMP2/LMP7 genes could influence the outcomes of HCV infection among drug users. Methods: Genomic DNAs of 362 anti-HCV sero-positive drug users and 225 control drug users were ex-tracted from the peripheral blood leukocytes. The sero-positive patients were divided into those who had persistent infection and those who had spontaneously cleared the infection. Polymorphisms of LMP genes were determined by PCR combined with restriction fragment length polymorphism (RFLP). Results: The distribution of LMP2 genotypes among the control, persistent infection and spontaneous clearance groups were not different. However, the LMP7 codon 145 Gln/Lys, Lys/Lys, and Gln/Lys+Lys/Lys genotypes were found significantly more frequent in the persistent infection group than in control group (OR=1.75, 95%CI=1.06~2.90; OR=3.16, 95%CI=1.23-8.12; OR = 1.94, 95%CI=1.21-3.12, respectively). Similarly, the frequencies of the codon 145 Gln/Lys, Lys/Lys, and Gln/Lys+Lys/Lys genotypes were found significantly more frequent in the persistent infection group than in the spontaneous clearance group (OR=1.64, 95%CI=1.04-2.57; OR=2.40, 95%CI=1.09-5.28; OR=1.76, 95%CI=1.15-2.69, respectively). Stratified analysis indicated that combined genotype Gln/Lys + Lys/Lys of the LMP7 gene was related to an increasing susceptibility to HCV infection (OR=1.91, 95%CI=1.02-3.55; OR=2.19, 95%CI=1.24-3.89; OR=1.91, 95%CI=1.05-3.48, OR=2.86, 95%CI=1.41-5.78, respectively) and the risk of persistent HCV infection (OR=1.94, 95%CI=1.12-3.34; OR=2.02, 95%CI=1.21-3.38; OR=1.78, 95%CI=1.12-2.85, OR=2.23, 95%CI=1.09-4.58, respectively) among > 30-year-old, males, the injection drug user (IDU) subjects and/or the shorter duration drug users (≤5 y). Conclusion: These results suggest that polymorphism of the LMP7 gene may have an influence on the outcomes of HCV infection, and is one of the factors accounting for the genetic sus-ceptibility to HCV infection among drug users.

Abstract:Objective: Interleukin-1β (IL-1β) plays an important role in the development of type 1 and type 2 diabetes mellitus. Resveratrol, a polyphenol, is known to have a wide range of pharmacological properties in vitro. In this research, we examined the effects of resveratrol on IL-1β-induced β-cell dysfunction. Methods: We first evalu-ated the effect of resveratrol on nitric oxide (NO) formation in RINm5F cells stimulated with IL-1β using the Griess method. Next, we performed transient transfection and reporter assays to measure the transcriptional activ-ity of peroxisome proliferator-activated receptor-γ (PPAR-γ). We also used Western blotting analysis to assess the effect of resveratrol on inducible nitric oxide synthase (iNOS) expression and nuclear factor-κB (NF-κB) translo-cation to the nuclei in cells treated with IL-1β. In addition, we assessed the transcriptional activity of NF-κB using an electrophoretic mobility shift assay (EMSA). Finally, we evaluated the effect of resveratrol on IL-1β–induced inhibition of glucose-stimulated insulin secretion in freshly isolated rat pancreatic islets. Results: Resveratrol significantly suppressed IL-1β-induced NO production, a finding that correlated well with reduced levels of iNOS mRNA and protein. The molecular mechanism by which resveratrol inhibited iNOS gene expression appeared to involve increased PPAR-γ activity, which resulted in the inhibition of NF-κB activation. Further analysis showed that resveratrol could prevent IL-1β-induced inhibition of glucose-stimulated insulin secretion in rat islets. Con-clusion: In this study, we demonstrated that resveratrol could protect against pancreatic β-cell dysfunction caused by IL-1β.

Abstract:Objective: It has been reported that the intravenous anesthetic propofol (PPF) has anti-inflammatory effects in vitro and in patients. The purpose of this study was to investigate whether PPF has anti-inflammatory effects in lipopolysaccharide (LPS)-induced septic shock by inhibiting the induction of pro-inflammatory cytokines [inter-leukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)] and high mobility group box 1 (HMGB1) in rats. Methods: Thirty six male Wistar rats were randomly assigned to one of three groups (control group, PPF + LPS group and LPS group; n = 12 per group). Control group rats received a 0.9% NaCl solution (NS) by the tail vein. The PPF + LPS group rats received PPF (10 mg/kg bolus, followed by infusion at 10 mg/(kg·h) through a femoral vein cath-eter) 1 h before LPS (7.5 mg/kg) administration via the tail vein. The LPS group rats received injection of LPS (7.5 mg/kg) via the tail vein. Hemodynamic effects were recorded as well as mortality rates, and plasma cytokine con-centrations (TNF-α, IL-6, HMGB1) were measured for the 24-h observation period. Results: The mean arterial pressure and heart rate of the PPF + LPS group were more stable than those of the LPS group. The mortality at 24 h after the administration of the LPS injection was much higher in the LPS group (58.3%) compared to the PPF + LPS group (25.0%). Plasma concentrations of cytokines (IL-6 and TNF-α) and HMGB1 were significantly reduced in the PPF + LPS group compared to the LPS group (P < 0.05). Conclusion: Pretreatment with PPF reduced the mortality rate of rats and attenuated the pro-inflammatory cytokine responses in an endotoxin shock model through an anti-inflammatory action inhibiting induction of HMGB1.

Abstract:Objective: Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health prob-lem. It is necessary to develop standard rabies virus diagnostic tools, especially for diagnosing the strains preva-lent in China. Methods: Monoclonal antibodies (MAbs) specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay (ELISA), isotyping, affinity assay, immunofluorescence assay (IFA), and immunocytochemistry. The MAb, whose affinity was higher for antigen, was used to establish an antigen capture-ELISA (AC-ELISA) detection system and test the efficiency by using clinical samples. Results: The heavy chain subclasses of two MAbs were all determined to be IgG2a. The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis, whereas the 3C7 MAb showed the highest affinity for anti-gen. IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples. Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb. Conclusion: It was potentially useful for the further devel-opment of highly sensitive, easily handled, and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic, especially in China.

Abstract:Objective: The recent advent of flow cytometry (FCM), coupled with fluorescent dyes, has been success-fully applied to assess mitochondrial function. The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium (Rh123/PI) dual fluorescent staining and FCM in asthenospermia and oligoastheno-zoospermia. Methods: Twenty-five fertile men (with normal sperm parameters) and 230 infertile patients were examined. Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups: asthenospermia (n = 30) and oligoasthenozoospermia (n = 25). Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function. Results: Significant differences were found between the normal and abnormal semen samples (P < 0.05) when Rh123+/PI-, Rh123-/PI+ and Rh123-/PI- sperm were examined by FCM, but there was no significant difference between the asthenospermia (P = 0.469) and oligoasthenozoospermia group (P = 0.950) when Rh123+/PI- and Rh123-/PI+ sperm were then examined; how-ever, a significant difference was found between the 2 groups (P = 0.003) when Rh123-/PI- sperm were examined. There was no correlation between Rh123-/PI- sperm and semen parameters in the normal group, but there was a significant negative correlation between the sperm concentration and Rh123-/PI- sperm in asthenospermia and oligoasthenozoospermia patients (r = -0.509, -0.660; P = 0.018, 0.038). Conclusion: Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mito-chondrial membrane potential in asthenospermia and oligoasthenozoospermia.

Abstract:Objective:To construct human single chain variable fragment (scFv) antibody library using phage display technology,and screen out special antibodies against the glycoprotein of rabies virus(RABVG) . Methods:The total RNA was isolated from the donors vaccinated with rabies vaccines and was used to amplify VH and VL genes by RT-PCR. VH and VL genes were joined together by overlap PCR to form scFv. The purified scFv gene repertoires were cloned into the phage vector pComb3XSS to construct the primary phage library. Panning against RABVG was performed for 5 rounds and the phage library was identified. The positive recombinant phages identified by ELISA were used to infect E.coli TOP10F′ for soluble scFv. Results:A recombinant phage antibody library against RABV was successfully constructed. 43 clones were found to bind to RABVG. 15 clones that had higher A450 values were detected with DNA sequencing and 3 of them were confirmed. The scFv gene was transformed into E.coli TOP10F′ for expression and purification. The purified scFv was proved to specifically bind to RABVG from the ELISA results. Conclusion:A human phage-display antibody library has been successfully constructed, and the selected scFv fragment can specifically bind to RABVG. It could be used in further studies of generation of human anti-RABVG antibodies for clinical application.

Abstract:Objective:To investigate the effect and mechanisms of sunitinib on PDGF-BB-induced proliferation and migration of human airway smooth muscle cells (HASMCs). Methods:HASMCs derived from human bronchus were divided into four groups:normal group,PDGF-BB group,PDGF-BB and sunitinib intervention group,sunitinib group. Proliferation was measured by MTT. Cell cycle progression was analyzed by flow cytometry. Migration was examined using Boyden chamber apparatus. The protein expressions of p-PDGFR-βand p-AKT were determined by western blot analysis. Results:PDGF-BB(20 ng/ml) significantly induced proliferation and migration of HASMCs as compared to controls. Sunitinib(0.3~9 nmol/L) inhibited the HASMCs proliferation and migration in response to PDGF-BB (20 ng/ml). Flow cytometry disclosed that PDGF-BB(20 ng/ml) increased significantly HASMCs ratio in S phase(P < 0.05);Sunitinib decreased HASMCs in S phase stimulated by PDGF-BB(P < 0.05). The phosphorylated protein levels of PDGFR-β and AKT in PDGF-BB group were higher than those of the normal group (P < 0.05),but were decreased in PDGF-BB and sunitinib intervention group compared with control(P < 0.05). Conclusion:It appears that sunitinib can directly inhibit PDGF-BB induced HASMCs proliferation and migration through the modulation of the PI3K/Akt pathway.

Abstract:Objective:Connective tissue growth factor(CTGF) can induce the expression of integrin-linked kinase(ILK)in human renal tubular epithelial cell line(HK-2). In this study,we investigated the modulatory effect of lipoxin A4(LXA4)on action of CTGF-induced ILK expression,and explored the mechanisms of the action of LXA4. Methods:Cultured HK-2 cells were stimulated with CTGF with or without pretreatment of LXA4. The expression of ILK mRNA was detected by RT-PCR. The expressions of ILK protein,p42/P44 mitogen-activated protein kinase(p42/44 MAPK)(also called ERK1/2) and phosphoinositide 3-kinase(PI3-K) were detected by western blot. Results:CTGF up-regulated the mRNA and protein expressions of ILK,and up-regulated the expression of phospho-ERK1/2 and phospho-PI3-K. LXA4 dose-dependently inhibited the above action of CTGF. Conclusion:LXA4 inhibits CTGF-induced expression of ILK via ERK1/2 and PI3-K-dependent signal pathway.

Abstract:Objective:To investigate the mechanism of 1,25(OH)2D3 in preventing skin aging. Methods:The phenotype of skin was compared between wild-type mice and 1α-hydroxylase gene knock-out littermates at 10 weeks of ages by histology,immunohistochemistry,western-blot and biochemical analysises,respectively. Results:1α-hydroxylase gene knock-out mice displayed a premature aging phenotype in skin including thinner skin with increased senescence associated β-galactosidase activity,flattening of the dermal-epidermal junction,reduced subcutaneous fat deposition and hair follicles number. Proliferating cell nuclear antigen(PCNA) immunopositive cells in skin were decreased, but terminal deoxynucleotidyl transferase dUTP nick end labeling positive areas were increased significantly in 1α-hydroxylase gene knock-out mice compared to their wild-type littermates. The expression of p16,p27,p53,NF--资B,active caspase-3 was up-regulated markedly in skin from 1α-hydroxylase gene knock-out mice. Reactive oxygen species and malondialdehyde levels were raised,whereas activities of superoxide dismutase and glutathione peroxidase were reduced in 1α-hydroxylase gene knock-out mice. Conclusion:1,25(OH)2D3 plays an important role in preventing skin aging by stimulating cell proliferation,inhibiting cells apoptosis and reducing oxidative stress.

Abstract:Objective:To construct and express a recombinant expression vector containing a mouse parathyroid hormone related protein (PTHrP) 1~84,and to examine the effect of the purified product on bone formation. Methods:The PTHrP1~84 gene fragment was amplified by RT-PCR and inserted into corresponding sites of expression vector pGEX-2TK,and the fusion protein expressed in E.coli BL21 induced by IPTG. After purification and identification,its roles on the proliferation and differentiation of bone marrow mysenchymal stem cells was evaluated in vitro. Results:The new recombinant expression vector pGEX-2TK/mPTHrP1~84 was constructed successfully. The gene recombinant product was soluble. The treatment with purified mPTHrP1~84 in bone marrow cell cultures not only significantly increased colony-forming unit-fibroblast (CFU-f),but also significantly increased alkaline phosphotase and collagen positive CFU-f and calcified nodules. Conclusion:Results demonstrated that gene recombinant mPTHrP1~84 can stimulate bone marrow mysenchymal stem cell to proliferate and differentiate into osteoblasts.

Abstract:Objective:To construct and identify pEGFP-HDGF over expression plasmid in U87 cells. Methods:HDGF fragment was amplified by RT-PCR method and then inserted into pEGFP-N1 vector. The level of HDGF was identified by RT-PCR and western blot after transfection of positive recombinant plasmid into glioma cell line U87. The biological activity of HDGF-GFP fusion protein was tested by the proliferation assay. Results:Fluorescence microscopy demonstrated that pEGFP-HDGF was successfully transfected into U87 cells. Over expression of HDGF was confirmed by RT-PCR and western blot. The proliferation rate of U87 cell transfected with pEGFP-HDGF plasmid was significantly higher than that of negative control. Conclusion:pEGFP-HDGF over expression plasmid with biological activity was successfully constructed and stablely transfected into U87 cells.

Abstract:Objective:To construct early growth response gene-1(Egr-1) and its shRNA expression vectors,and to assess their function on rat glomerular mesangial cell(GMC). Methods:The CDS area of Egr-1 and three reverse repeated motifs targeting of Egr-1 gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1-myc-his-A and pGCsi.U6.neo.GFP. After screened and confirmed,the recombinant plasmids were transfected into GMC,then the level of Egr-1 protein in rat GMC was measured by western blot to prove its expression and find out the optimal shRNA. Results:It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vectors were correct. The results of western blot showed that the constructed pcDNA3.1/Egr-1 plasmid expressed correctly and the optimal shRNA,which effectively silenced the target gene,was Egr-1 shRNA-2. Conclusion:The pcDNA3.1/ Egr-1 plasmid and its shRNA were constructed successfully. These data provide experimental tools for studying biological functions of Egr-1 gene in the future.

Abstract:Objective:To explore the effects of microRNA143,microRNA145 transfection on biological behaviors of cholangiocarcinoma cell line QBC939,and the relationship between microRNA143,microRNA145 and extrahepatic cholangiocarcinoma. Methods:RNA of 11 pairs of specimens of extrahepatic cholangiocarcinoma and normal bile duct was extracted,and then the expression of microRNA143 and microRNA145 was detected by real-time PCR. Expression plasmid of microRNA143(or microRNA145) was transiently transfected into human cholangiocarcinoma cell line QBC939,and then cell proliferation,cell cycle and cell apoptosis were determined by MTT and flow cytometry assay. Results:Compared with normal bile duct,the relative expression ratio of microRNA143 and microRNA145 in extrahepatic cholangiocarcinoma respectively was 0.048,0.035(P < 0.05). Compared to control group,cholangiocarcinoma cell growth was effectively inhibited in microRNA143 group and microRNA145 group,the percentage of cells in G1 phase was increased[(microRNA143 group and microRNA145 group:66.08%±1.93% and 65.57%±1.85%)vs(control 42.87%±1.37%)],the percentage of cells in S phase was decreased[(19.16%±1.15% and 21.40%±1.65%) vs(control 38.76%±1.83%)]and the ratio of cell apoptosis was increased[(18.49%±2.26% and 16.89%±2.47%)vs 4.68%±1.65%](P < 0.05). Conclusion:The expression of microRNA143 and microRNA145 is down-regulated in extrahepatic cholangiocarcinoma,and they may correlate with tumorigenesis. With up-regulation of microRNA143 or microRNA145 expression in cholangiocarcinoma cell line QBC939,cell proliferation can be inhibited,cell cycle can be arrested and cell apoptosis can be increased.

Abstract:Objective:To study the change of the localization of aldolase A (ALDOA) in Hela cells before and after X ray exposure,and impact of the treatment on cell survival rate. Methods:The whole encoding sequence of ALDOA was cloned into the eukaryotic expressing pECFP-C1-ALDOA plasmid,and then transfected into Hela cells in the experimental group. Meanwhile,the pECFP-C1 vector was transfected into Hela cells as a control. The expression and localization of pECFP-C1-ALDOA and pECFP-C1 were investigated by fluorescent microscopy before and after X ray exposure,and the cell survival rate was detected by XTT analysis. Results:The constructed plasmid was verified by double enzyme digestion and DNA sequencing;the fluorescent microscopy revealed that pECFP-C1-ALDOA located predominantly in cell endochylema while pECFP-C1 dispersed equally in the whole cell;pECFP-C1-ALDOA located into cell nucleus 24 hours after X ray exposure,however,the localization of pECFP-C1 did not change significantly;XTT analysis revealed that the survival rate of the pECFP-C1-ALDOA-transfected Hela cells was much higher than untransfected cells after X ray exposure. Conclusion:Inhabiting the expression of ALDOA may increase Hela cell death induced by X-ray,so ALDOA could be used as a new target for cancer radiation therapy.

Abstract:Objective:To explore the differentiation of rat bone mesenchymal stem cells to dopaminergic neurons by chemically defined sequential culture media in vitro. Methods:The rat bone mesenchymal stem cells were isolated from rat marrow by density gradient centrifugation and the fourth generation was induced into dopaminergic neurons by treated with bFGF,EGF,NCM,SHH and FGF8. Dopamine associated proteins and genes in the treated cells were examined by Western blot,immumofluore-scence and RT-PCR. Results:After 2 weeks of stimulation,the cells expressed the characteristics of dopaminergic neurons. RT-PCR discovered the expression of mRNA of DAT,Nurr1,AADC,VAMT and TH. Immumofluorescence staining indicated that the ratio of TH positive neural cells was significantly increased after induction for 2 weeks(41.18±3.24) %. Conclusion:Chemically defined sequential culture media can induce differentiation of BMSCs into DA neurons in vitro,and the expression of mature neurons characteristics of the transdifferentiated cell. BMSCs might be a suitable and available source for the in vitro derivation of DA neurons and cell transplantation therapy on some central neural system diseases such as Parkinson’s disease.

Abstract:Objective:To improve efficiency of transgenes by albumin microbubble,which is a non-virus gene delivery vector. Method:Polyethylenimine coated albumin microbubble(PAMB) was prepared by sonicating the mixture of human albumin,polyethylenimine and glucose. The transgene efficiency of polyethylenimine,polyethylenimine+albumin,PAMB and Lipofectamine 2000 was compared by detecting the positive cell ratio of green fluorescent protein(GFP) using flow cytometry. Results:The average efficiency of transgene by polyethylenimine,polyethylenimine+albumin,PAMB and Lipofectamine 2000 was (13.75±4.88)%,(5.20±1.15)%,(49.17±6.75)% and (53.72±5.69)%,respectively. The efficiency of PAMB group was higher than that in polyethylenimine group(P < 0.05),however,there was no statistical significance between PAMB group and Lipofectamine 2000 group (P > 0.05). Conclusion:Albumin microbubble modified by polyethylenimine is highly effective on transgene,and it is a useful non-virus gene delivery vector. This study may provide a new method for gene transfection in vivo.

Abstract:Objective:To optimize the culture conditions of glioma U87 cells for proliferation inhibiting assay. Methods:After down-regulation of HDGF expression by RNA interference in U87 cell line,cells were inoculated in 96-well plate. MTS cell proliferation assay was performed under three different conditions including serum free culture condition,containing 10% FBS culture condition and plate precoated with Matrigel. Results:U87 cell proliferation was inhibited after down-regulation of HDGF level. Cell proliferation inhibiting rates in serum free group,10% FBS group and Matrigel precoated group were 18%,9%,28%,respectively. Conclusion:Pre-coating the plate with Matrigel may be optimal for cell proliferation inhibiting detection after RNA interference.

Abstract:Objective:To explore the anti-tumor mechanism of paeoniflorin by human gastric cancer cell line SGC7901/VCR. Methods:After SGC7901/VCR cells were treated with paeoniflorin in different concentration for 48 h,methyl thaizolyl tetrazolium(MTT) method was used to detect the inhibition rates of the cells. Flow cytometry(FCM) was employed to detect SGC7901/VCR cell apoptosis ratios.Western blot was used to test the expression of NF-κB,Bcl-2 and Bax. Activation of NF-κB transcription was detected by ELISA. Results:Paeoniflorin restrained cell proliferation and induced apoptosis of SGC7901/VCR cells in a dose-dependent manner. Paeoniflorin significantly inhibited the activity of NF-κB in nuclear. The inhibition pattern of NF-κB was confirmed by western blot and ELISA. After SGC7901/VCR were treated with 0,40,80,160,320 μg/ml of paeoniflorin for 48 h respectively,the protein levels of Bcl-2 were down-regulated in a dose-dependent manner,while those of Bax were not affected. Conclusion:Paeoniflorin can inhibit the proliferation and induce the apoptosis of SGC7901/VCR cells,which might be partly related to Bcl-2 up-regulation by inhibiting NF-κB activation.

Abstract:Objective:To establish hypoxia model with human umbilical vein endothelial cell line (Eahy926) and investigate the effect of tetramethylpyrazine(TMP) on Eahy926’s fibrinolytic function with hypoxia. Methods:The cells were divided into three groups:control group,CoCl2-treated group (1.6 mmol/L) and CoCl2+TMP-treated groups (0.08 and 0.16 g/L,respectively). Hypoxia was mocked by treatment of CoCl2. Cell viability was evaluated by Hoechst staining and cell counting kit-8 (CCK-8);the expression of t-PA,u-PA and PAI-1 was measured by semi-quantitative RT-PCR;t-PA,u-PA and PAI-1 in the supernatant of culture medium were detected by ELISA. Results:Cell viability was decreased in CoCl2-treated and CoCl2+TMP-treated groups compared with the control group (P < 0.01). Treatment of TMP improved cell viability in a dose-dependent manner (P < 0.01). Moreover,CoCl2 induced a decreasing mRNA expression (P < 0.01) and secretion of t-PA (P < 0.01);TMP treatment elevated t-PA mRNA expression and contents of the cell supernatant (P < 0.01) in a dose-dependent way. Conclusion:TMP can effectively protect Eahy926 cell from hypoxia-induced damage and increase viability of cells in a concentration-dependent manner;TMP might enhance the fibrinolytic function of endothelial cells by regulating the expression and secretion of t-PA.

Abstract:Objective:To observe the relationship between myocardial apoptosis of diabetic mellitus and uncoupling protein 2 (UCP2). Methods:Diabetic animal models were induced by streptozotocinon on UCP2 knock-out mice(UCP2-/-) and C57BL/6 mice,rearing for 7 and 28 days each group. The expressions of myocardial apoptosis relative protein,caspase 3,were investigated using western blot. Hochest fluorescence staining detected morphological changes of myocardial cells in left venticle of diabetic mice. Results:Hochest staining results showed cells nuclear condensation,granular fluorescence and other typical features of apoptosis in DM-28-day group. Myocardium of diabetic mice exhibited significant concentration of chromatin at 7 days and aggravated at 28 days,with high fluorescence intensity. However,slight concentration of chromatin was detected on UCP2-/-+DM mice comparison of DM mice at 28 days. Image analysis showed that the expression of cleaved-caspase 3 protein levels decreased slightly in UCP-/-+DM-7-day group comparing with DM-7-day group(P > 0.05). The expression of cleaved-caspase 3 protein levels decreased significantly(P < 0.05)in UCP-/-+DM-28-day group comparing with DM-28-day group. Conclusion:UCP2 may be one of the most important factors which contribute to myocardial apoptosis of diabetic mellitus.

Abstract:Objective:To examine the changes of IL-6 level in the condition of cultured THP-1 with or without human adipocytes,and to explore the relationship between inflammation and obesity. Methods:Co-culture system for monocytic cell line THP-1 and human adipocytes was established through transwell combined with treatment of JNK inhibitor-SP600125. Meanwhile,cultured THP-1 and human adipocytes respectively acted as controls. The accumulation of IL-6 in cultured media supernatant was assayed by enzyme-linked immunosorbant assays (ELISA) for every group. Results:The results of ELLSA showed that the IL-6 was markedly increased in the co-culture of THP-1 and human adipocytes,and the level of IL-6 was decreased when treated with JNK inhibitor. Conclusion:There might be a paracrine loop between the monocytic cell line THP-1 and adipocytes. The level of IL-6 is increased under co-culture condition,while JNK inhibitor could decrease the IL-6 release,which might become a new therapy target for obesity.

Abstract:Objective:To observe the expression of C telopeptide of type Ⅱcollagen(CTX-Ⅱ)in serum and protein extract from joints of experimental osteoarthritis(OA),and to explore the value of CTX-Ⅱ as a biomarker in early diagnosis and condition assessment. Methods:96 adult SD rats were randomly divided into two groups:Sham-operated group (n=48),OA model group(n=48). OA was induced by unilateral surgical transection of the anterior cruciate ligament. Joints were isolated 0,3days,1,2,4,8weeks after surgery for protein extraction and histology. CTX-Ⅱ in protein extracts and serum was measured by ELISA. Results:Levels of CTX-Ⅱ in serum and protein extract were both significantly increased in OA model group compared with sham-operated group on each time point. The pathological features of OA were observed during 2~8 weeks and became gradually more serious. The changes in level of CTX-Ⅱ in serum and protein extraction showed a close correlation. Conclusion:Serum CTX-Ⅱ level increases before the appearance of characteristic pathology of OA and is in coincidence with the changes of CTX-Ⅱ in the tissue. It can be a sensitive reference indicator for early diagnosis and condition evaluation of OA.

Abstract:Objective:To investigate the effect of methylmethacrylate monomer to New Zealand white rabbit’s cardiopulmonary function by injecting methylmethacrylate monomer into vein. Methods:According to different injecting materials,New Zealand white rabbits were divided into two groups:Experimental group (methylmethacrylate monomer) and control group(physiological saline). According to different doses,experimental group were divided into three sub groups:MAX group (0.35 ml);MID group (0.10 ml);MIN group(0.02 ml). The fluctuations of blood pressure,heart rate and the changes of the oxygen partial pressure of blood were monitored and pathological examinations of the lung and heart were carried out postoperatively. Results:Significant decrease of blood pressure,heart rate and oxygen saturation were detected in 6 cases out of 7 subjects in MAX group,and then died 30 minutes later. Moderately change was observed in MID group,but no one died. Slightly change happened in MIN group. No significant change happened in control group. Pulmonary hemorrhage and embolism were seen in lung preparation of MAX group and MID group. Subendocardial thrombus was observed in heart preparation of MAX group. Conclusion:Methylmethacrylate monomer has cardiac muscle toxicity,and this closely correlates with the dose of monomer. The physical chemical property of cement and toxicity of monomer may be a factor lead to“bone cement response syndrome”,but not the major factor of this pathological process.

Abstract:Objective:To investigate the expression levels of OPG,RANKL,RANK,and the level and activity of NF-κB p65 in patients with non-small cell lung cancer(NSCLC),then to explore their clinical significance. Methods:The gene expression levels of OPG,RANKL,RANKwere assessed by real-time PCR and the level and activity of NF-κB by western blot in cancer and corresponding adjacent lung tissues of 32 NSCLC patients and normal lung tissues of 15 chest trauma or inflammation patients matched. Results:A significant increasing gradient of gene expression of OPG,RANKL,RANK and RANKL/OPG ratio was identified in normal,adjacent lung tissues of NSCLC and cancer tissues,as well as the intranuclear/total ratio of NF-κB p65,whereas the level of total NF-κB p65 was comparable among the three groups. Moreover,the mRNA levels and RANKL/OPG ratio of the axis in cancer tissues of NSCLC with lymph node metastasis was significantly higher than that in NSCLC without lymph node metastasis. The intranuclear/total ratio of NF-κB p65,but not the total level,was also increased notably in cancer tissues of NSCLC with lymph node metastasis. Conclusion:The activation of OPG/RANKL/RANK axis may play an important role in pathogenesis and lymphatic metastasis of human NSCLC through activating the NF-κB signal transduction pathway.

Abstract:Objective:To determine the relationship between expression of nuclear factor kappa B(NF-κB),interleukin-8(IL-8) and cytotoxin-associated protein A(CagA),and evaluate the clinical implication of CagA in tumorigenesis and progression of gastric carcinoma. Methods:Tissue specimens from 54 cases of chronic gastritis and 58 cases of gastric cancer were evalutaed for the expression of CagA,NF-κB and IL-8 by immunohistochemistry and western blot. The relationship between NF-κB,IL-8 and CagA were detected by correlation analysis. Results:There was no difference in positive rate of CagA protein between chronic gastritis and gastric cancer groups(53.7% vs 62.1%,P > 0.05). The positive rate of NF-κB and IL-8 were significant higher in peri-cancer tissues of gastric cancer than that in chronic gastritis(P < 0.05). In the peri-cancer tissues of gastric cancer,the expression of NF-κB and IL-8 were significant higher in CagA positive group than that of CagA negative group(P < 0.05). And there was a positive correlation between the expression of NF-κB,IL-8 and CagA. Conclusion:CagA protein might be important in tumorigenesis and progression of gastric carcinoma by up-regulating the expression of NF-κB and IL-8.

Abstract:Objective:To explore the apoptosis rate of thyroid cells from normal,Graves disease(GD),Hashimoto thyroiditis(HT) and detect the apoptosis related proteins. Methods:The apoptosis rate of thyroid cell from normal GD and HT thyroid gland was measured by the terminal deoxynucleotidyl transferase mediated d-UTP nick labeling(TUNEL),and apoptosis related proteins Fas and FasL were confirmed by immunohistochemical methods,and the expression of Fas mRNA was measured by RT-PCR. Results:Higher apoptosis rate was found in GD and HT TEC,and there was an obvious and almost equal expression of FasL(P > 0.05). The difference in expression of Fas protein and mRNA in three groups thyroid glands was significant (P < 0.01),and the expression of Fas in normal tissue was the lowest and trivial,but the Fas expression in HT was the highest and distinctive. Conclusion:These data indicate that apoptosis plays an important role in the pathogenesis of AITD,and Fas/FasL may be the signal molecule related to apoptosis of TEC.

Abstract:Objective:To analyze the relationship between the cytochrome P450 2A13(CYP2A13) gene and tongue cancer. Methods:CYP2A13 mRNA and protein were detected by real-time fluorescent quantitative PCR and western blot respecytively in 26 cases of oral cancer and their corresponding adjacent tissues. Results:CYP2A13 mRNA and protein were expressed in tongue cancer and their surroundings. And the expression of CYP2A13 mRNA and protein in tongue cancer is much lower than in their surroundings (P < 0.05). Conclusion:Down-regulated expression of CYP2A13 may be involved in the development and progression of tongue cancer due to the increase activity of certain carcinogens,and the exact mechanism needs to be further studied.

Abstract:Objective:To investigate the relationship of the CCND1 G870A polymorphism and the risk of ovarian cancer. Methods:In a case-control study of 107 patients with newly diagnosed ovarian cancer and 114 cancer-free controls frequency-matched by age,the G870A polymorphisms of CCND1 gene were genotyped by the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)method.We further stratified the age,age at menarche,age at first live birth,parity,number of abortion and menopausal status of cases. Results:No significant association was observed between the CCND1 G870A polymorphism and ovarian cancer risk. Conclusion:No obvious association was found between the CCND1 G870A polymorphism and ovarian cancer risk in Chinese population.

Abstract:Objective:To investigate the influence of blood pressure level on large arterial compliance(C1) and small arterial compliance(C2). Methods:Nine hundred and fifty three subjects were divided into optimal,normal,high normal blood pressure and hypertension groups according to their blood pressure levels. C1 and C2 were measured by pulse wave analysis. Statistical methods included analysis of covariance and stepwise regression analysis. Results:C1 and C2 were decreased with increased blood pressure levels(P < 0.000 1). Stepwise regression analysis showed that C1 and C2 were decreased with increased age,systolic blood pressure and heart rate;men had higher C1 and C2 than women;and smokers had a lower C2 than non-smokers. Conclusion:C1 and C2 are decreased with blood pressure levels. Non-hypertensives with blood pressure levels at normal or high-normal ranges have lower arterial compliance than those with optimal blood pressure.

Abstract:Objective:To study the relationship between blood pressure,blood glucose and the different causes of death in elderly,and then provide a guidance for clinical prevention. Methods:883 dead patients with relatively complete information were investigated,to choose cases aged over 60 years as subjects. The 501 patients were divided into two groups:180 diabetics and 321 non-diabetics,and then were subgrouped according to blood pressure:diabetes/hypertension group(DM/HT,108 cases,60.14%),diabetes group(DM,72 cases,39.86%),hypertension group(HT,130 cases,40.50%),non diabetes/hypertension group(non-DM/HT,191 cases,59.50%) respectively. Sex,age,blood pressure,blood glucose and cause of death were observed in each group. Results:① The incidence of hypertension in the elderly were 40% in non-diabetes and 60% in diabetes,and higher in men;②Cerebral hemorrhage was the first cause of death(more than 30%) in DM/HT and HT groups;meanwhile vascular accident,mostly infarction was only 20% in DM and non-DM/HT groups;③Malignant tumors was the first cause of death in DM and non-DM/HT groups,and meanwhile it was not related with hypertension(except for renal spindle cell carcinoma);④There was a high prevalence of infection in the two sub-groups of diabetes;⑤The uremia prevalence was higher in DM/HT group and HT group than in the other two subgroups;⑥Multiple regression analysis showed systolic blood pressure and diastolic blood pressure were associated with cardio-cerebral vascular accident,unrelated to tumors and infection,the systolic blood pressure was also associated with uremia;fasting blood glucose were associated with the four causes of death. Conclusions:There was a relationship between blood pressure and blood glucose with the different causes of death in the elderly,and we should carry into disease prevention accordingly to different groups.

Abstract:Objective:To study the relationships between the resting heart rate(RHR) and the cognitive function of patients with type 2 diabetes mellitus (T2DM). Methods:Cognition was evaluated with questionnaires in 140 T2DM patients. The RHR was divided into three groups: <75 beats/min(bpm) group(65 cases),≥75 bpm and <85 bpm group(44 cases) and ≥85 bpm group(31 cases). The cognitive levels were compared among the three different RHR groups. Results:① There were significantly differences on the immediate memory of mini-mental state examination(MMSE) and naming picture,and the executive function and the linguistic capacity among the three groups(P < 0.05). ②It has no difference among the three groups in sex,age,the educational level,the duration of diabetes,combined hypertension or not,systolic blood pressure(SBP),hemoglobin A1c(HbA1c),cholesterol(TC),triglyeride(TG),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),lipoprotein(a) [LP(a)] and thyrotropic stimulating hormone(TSH) except diastolic blood pressure (DBP),the albumin/creatinine ratio in urine (UAIB/Cr) and the high sensitivity C-reactive protein (HsCRP). ③The RHR has negative corrections with the total score,orientation and linguistic capacity of MMSE,and naming picture,filling the lack of picture among the three groups (P < 0.05),and the executive function,composition of a picture and memory of MMSE(P < 0.01). ④There was a significant difference on RHR between UAIB/Cr <30 mg/g group and ≥30 mg/g,<300 mg/g group. And the RHR has prominent different between HsCRP ≥5 mg/L group and <5 mg/L group. Conclusion:The RHR has obvious correlation with cognitive function of patients with T2DM. RHR higher,damaged domains of cognition more.

Abstract:Objective:To investigate the relationship between obstructive sleep apnea syndrome (OSAS) and serum ferritin (SF). Methods:80 patients with OSAS were enrolled as subjects and 80 normal person as control Based on apnea hypopnea index(AHI),the patients were divided into mild,moderate,and severe subgroups. SF levels were measured by electrogenerated chemiluminescence. Results:The levels of SF in OSAS group were higher than those in the control group;there was a positive correlation between the level of SF and AHI. Conclusion:OSAS might be one of the causes of increased SF.

Abstract:Objective:To examine the association between nutritional status of pregnant mothers and the risk of astigmatism in twins. Methods:Twenty-eight twin pairs were recruited from Jiangsu Province Hospital. Noncycloplegic refraction was measured by vision screening instrument 3 and 6 months after birth. Detailed perinatal history and postnatal data were collected. The relationship between astigmatism and related factors of pregnant mothers and infants was analyzed. Meanwhile 56 cases of singleton were randomly selected as control group. Results:Detection rate of astigmatism in observed twins’ group was 53.57% (cylinder power of 2.0 D or worse),which was significantly higher than that of their parents (14.28%,cylinder power of 1.0 D or worse),and that of singletons. The values of astigmatism were correlated with family history. Morning sickness(OR=1.40,95%CI=1.10~5.13),BMD(OR =1.44,95%CI=1.30~6.19) and haemoglobin(OR=1.40,95%CI=1.03~2.78) of mother during mid-pregnancy,maternal serum magnesium (OR=1.39,95%CI=1.03~1.89) and vitamine A during delivery (OR=1.03,95%CI=1.01~1.05),Umbilical vein vitamine D(OR=1.02,95%CI=1.00~1.04),birth weight(OR=1.02,95%CI=1.00~1.03) were correlated(P < 0.05)with the formation of twins astigmatism. Conclusion:Detection rate of twins astigmatism was significantly higher than that of singletons. The formation of astigmatism in twins was related with the level of maternal and fetal nutrition and other factors,noticeably morthers’ morning sickness,haemoglobin and twins’ birth weight. Further studies are warranted. Corresponding nutrition instruction should be given to pregnant women to decrease the risk of astigmatism.

Abstract:Objective:To evaluate the short-term therapeutic efficacy of parathyroid hormone and calcitonin in postmenopausal osteoporotic women. Methods:A total of 26 postmenopausal women with osteoporosis were randomly classified into two groups to receive open-label use of 20 μg biosynthetic human parathyroid hormone (PTH) subcutaneously or 200 U salmon calcitonin(CT) in a nasal spray per day,and all patients were supplemented daily with 500 mg elemental calcium and 200 U vitamin D throμghout the 24 weeks. The bone mineral densities (BMD) of all patients were measured before and after 24-week treatment. At the same time the biochemical marker of bone formation serum osteocalcin (OC) and alkaline phosphatase (AKP) were determined. Results:The BMD increase at the lumber spine(L2-4) in the PTH and the CT group were 5.11%(P < 0.01)and 2.13%(P > 0.05)respectively at 24 weeks,but the BMD changes at the femoral neck,trochanter and the total hip in each group were not significant,and no significant difference of final BMD was observed at any matching site between the two groups. There were obvious increases in serum osteocalcin (OC) and alkaline phosphatase (AKP) level in the PTH group(173.04%,47.48% respectively,all P < 0.001),but the levels of both markers were not changed in the CT group. Conclusion:PTH application was associated with larger increases in lumbar spine BMD,although PTH can increase bone formation markers while CT can not,PTH seems to be equally effective with CT in the short-term treatment.

Abstract:Objective:To investigate the influence of metformin on urinary albumin excretion,serum cystatin C and plasma homocysteine in patients with diabetic nephropathy. Methods:According to the levels of urinary albumin excretion,104 patients of type 2 diabetes mellitus were divided into non-albuminuria group(40 cases),microalbuminuria group(36 cases)and macroalbuminuria group(28 cases). Patients in each group were treated with insulin(INS subgroup)or insulin and metformin(INS+MET subgroup) so as to control FPG≤7 mmol/L and HbA1c≤7%. UAE,BMI,TG,TC,HDL-C,Cr,CysC and Hcy were measured before and after medication. 28 normal subjects were involved as control. Results:BMI,Hcy and TC were reduced and HDL-C was increased(P < 0.05) of whom had been medicated with metformin and insulin for 3 monthes. Cr,Ccr and CysC had no differences (P > 0.05)before and after the pharmacotherapy. Hcy was correlated independently with Cr and CysC(P < 0.05). Conclusion:Metformin had not showed an increase of nephropatic UAE and injuring effects to renal function. The decrease of Hcy by metformin showed beneficial effects on the renal function.

Abstract:Objective:To evaluate the effect of aesthetic crown lengthening surgery and restoration with precious metal porcelain crown in the treatment of upper anterior tooth,and to explore the best postoperative restoration time. Methods:40 patients with 46 upper anterior teeth were treated by crown lengthening surgery,and restored with precious metal porcelain crown affer 6 months. All evaluation indexes were recorded before surgery,6W and 6M(before restoration)afeter surgery,6M and 1Y after restoraion. Results:one year after the restoration,all of the cervical margin was good,and all of the teeth did not have peridentioclasia or other complication such as gingival stain. The esthetic effects of restorations were satisfactory. Conclusion:Aesthetic crown lengthening surgery combined with restoration with precious metal porcelain crown can achieve a good cosmetic restoration purpose.

Abstract:Objective:To study the arthroscopic technique of modified suture fixation through single bone tunnel for tibial eminence avulsion fracture and its clinical results. Methods:From May 2007 to October 2008,15 cases of tibial eminence avulsion fracture were treated arthroscopically with modified suture fixation technique through single bone tunnel. Active rehabilitation began after operation. Results:The patients were followed up 8~24 months. X-ray showed that all fractures healed without displacement in an average of five months after operation. Range of motion of the affected knee was recovered in all patients. Six month postoperatively,the Lysholm knee score was 95.4±4.5. Conclusion:Arthroscopic treatment of tibial eminence fracture with modified suture fixation technique through single bone tunnel is reliable for fracture reduction,fixation and early mobilization.