Abstract:Objective:To explore the role of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) in drug resistance and metastasis of anaplastic thyroid carcinoma. Methods:Human anaplastic thyroid cancer cell line,SW1736,was sorted for side population (SP) cells which had stem cell characteristics by Fluorescent Activated Cell Sorting (FACS). Non-SP cells were treated with doxorubicin to establish drug resistant cell modes. Among SP,non-SP and non-SP drug resistant cells,the clonal formation assay was performed to evaluate the self-renewal potential;3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was adopted to examine effects of doxorubicin on the proliferation;Genes expression of stem cell markers-nestin and ATP-binding cassette superfamily G member 2(ABCG2),the gene related to cancer resisitance and relapse-multidrug resistance gene (MDR1) and some EMT associated genes-E-Cadherin,β-catenin,vimentin,Slug and N-cadherin,were compared by performing real-time PCR. Results:SW1736 line cells contend 0.8% side population cells. Clonal formation assay revealed that SP cells displayed markedly higher clonogenic potential than non-SP cells. MTT cell proliferation assay showed that the half maximal inhibitory concentration (IC50) of non-SP drug resistant cells was obviously higher than that of non-SP cells. SP cells displayed higher genes expression of nestin,ABCG2,MDR1,slug,β-catenin,vimentin and N-cadherin,compared with non-SP cells. The gene expressions of slug and MDR1 were higher in non-SP drug resistant cells than those of non-SP cells. E-cadherin was not detected in all cell types. Conclusion: Doxorubicin can’t make the transition of non-CSCs (namely non-SP) to acquired CSCs (namely SP),but can induce the aberrant activation of EMT. SP cells may have significant impact on tumor drug resistance,recurrence and metastasis.

Abstract:Objective:To determine whether and how glycogen synthase kinase-3β (GSK-3β), an important component of phosphoinositide 3-kinases (PI3K) signaling pathway, regulates the methylation of protein phosphotase 2A (PP2A). Methods:By using molecular biological and pharmological approaches to alter the activity of PI3K signaling pathyway and its key molecule, GSK-3β, we investigated the regulation of PI3K pathway and GSK-3β on PP2A methylation by using Western blot analysis in cultured HepG2 and HEK-293T cells,By using GST-pull down, we determined the interaction between LCMT-1 and GSK-3. Results:We found that inhibition of PI3K pathway by PI3K inhibitor LY294002 increased the methylation level of PP2A catalytic subunit. In addition, inhibition of GSK-3β in cells leaded to the less methylation of PP2A catalytic subunit. We found that GST-GSK-3β could pull-down LCMT-1. Conclusion:PI3K signaling pathway is involved in the regulation of PP2A catalytic subunit methylation. Inhibition of GSK-3β suppresses the methylation. GSK-3β has a mutual effect with LCMT-1 and may regulate the methylation of PP2A through LCMT-1.

Abstract:Objective:To explore the pathogenesis of ovarian cancer by investigating the expression of Toll-like receptor 1 (TLR1),TLR2 and TLR6 in patients with ovarian cancer. Methods:We collected peripheral blood mononuclear cells (PBMC) from 13 ovarian cancer patients,13 benign diseases controls,and 13 healthy normal controls. The expression levels of TLR1,TLR2 and TLR6 were measured by flow cytometry in monocytes,CD4+T lymphocytes,CD8+T lymphocytes and B lymphocytes. Quantitative real-time PCR was used to measure IL-1β,IL-6,IL-8,and TNF-α in the collected from cells treated with TLR1,TLR2 or TLR6 ligands. Results:TLR1,TLR2 and TLR6 were all expressed in PBMC and they were elevated in monocytes from all three groups. The expression of TLR1,TLR2 and TLR6 from monocytes were higher (P < 0.05) in ovarian cancer patients(69.13%,59.43%,52.99%,respectively) as compared with benign disease controls (34.34%,25.32%,15.21%,respectively) and healthy normal controls(36.31%,26.63%,16.43%,respectively),there was no difference between benign disease controls and the healthy normal controls. While between CD4+T cells,CD8+T cells,as well as among B cells,no significant difference was found. In concordance with the above results,there was an observable increased expression of inflammatory cytokine interleukin interleukin-1β (IL-1β) upon stimulated by HKLM (TLR2 ligand) in ovarian cancer patients compared to benign disease controls and healthy normal controls (F = 2.05,P < 0.05;F = 2.19,P < 0.05),while no significant difference was noticed between benign disease controls and healthy normal controls;IL-6 was also up-regulated in ovarian cancer patients(F = 1.40,P < 0.05;F = 1.99,P < 0.05) when compared with benign disease controls and healthy normal controls,while no significant difference was obvioused between benign disease controls and healthy normal controls;Furthermore,there were differences in IL-8,TNF-α among three groups. Conditioned medium with FSL-1 (TLR6 ligand) induced higher amounts of IL-6 expression in ovarian cancer patients than benign disease controls and healthy normal controls (F = 1.30,P < 0.05;F = 1.69,P < 0.05),while no difference was found between benign disease controls and healthy normal controls. In particular,all three groups did not show significant up-regulation of IL-1β,IL-8 or TNF-α production in response to FSL-1 stimulation. Moreover,stimulated with Pam3CSK4 (TLR1/2 ligand),IL-1β,IL-6,IL-8 or TNF-α didn’t show significant up-regulation among all three groups. Conclusion:TLR1,TLR2 and TLR6 were highly expressed in monocytes from ovarian cancer patients,and inflammatory reaction mediated by IL-1β,IL-6 may play an important role in driving ovarian tumor progression.

Abstract:Objective:To investigate the effects of melatonin on adhesion and migration abilities of human SGC-7901 gastric cancer cells and its role in reorganization of actin cytoskeleton. Methods:SGC-7901 cells were treated with different concentrations of melatonin for 24h. The contents of G-actin(in cytosolic fraction)and F-actin(in cytoskeletal fraction)in cells were measured by Western blot. Cellular F-actin stained with FITC-phalloidin was examined by fluorescence microscopy. Adhesive assay was used to detect the adhesive ability of SGC-7901 cells to HUVECs. Migration rate was measured by wound healing assay. Results:After treated with melatonin for 24h,the celluar F-actin was depolymerized and actin cytoskeleton was rearranged. Consistently,the abilities of adhesion and migration in SGC-7901 cells were significantly reduced compared to the control cells. Conclusion:Melatonin inhibits the abilities of adhesion and migration in SGC-7901 cells,which may be via reorganization of cellular actin cytoskeleton.

Abstract:Objective:To investigate the effects of advanced glycation end products (AGEs) on apoptosis and approach the mechanism. Methods:The primary cultured mouse chondrocytes were incubated with different concentration of AGEs for 24 h or 48 h. Apoptosis of chondrocytes was detected by FCW;expression of ColⅠ,MMP-3,MMP-9 and p53 was measured by Western blot. Results:After the chondrocytes were treatment with AGEs(50,100,200 mg/L) for 24 h,the apoptosis was increased compared with the control groups;the maximum stimulation of chondrocytes was 200 mg/L AGEs. After the chondrocytes were treatment with AGEs(50,100,200 mg/L) for 48 h,expression of Col was significamtly decreased(P<0.05,n=3) while the levels of MMP-3,MMP-9 and p53 were increased,compared to control groups and BSA group. Conclusion:AGEs can increase the apoptosis of mouse chondrocytes in a dose-dependent manner, thus damage in chondrocytes via decrease of the expression of extracellular matrix.

Abstract:Objective:The purpose of this study was to study the distance of iliotibial band(iliotibial band,ITB)and lateral femoral condyle and the angle of ITB long axis with long femoral shaft in the condition of different knee flexion in normal young subjects. Method:One hundred asymptomatic volunteers (50 men and 50 women)participated in the study. The thickness and width of ITB were measured by sonographic and physical examination. The distance of ITB and lateral femoral condyle and the angle of ITB long axis with long femoral shaft were also measured in different the knee joint positions,which were extension,30°,90° and 135° flexion. Result:ANOVA shown that the average distance of ITB and the lateral femoral epicondyle varied significantly according to different positions of knee joint (P < 0.01)in either group. At the level of the lateral femoral condyle,ITB center gradually shifted from anterior to posterior of the lateral femoral condyle with the increasing of knee joint flexion. There was a significant difference in the average angle of ITB long axis with long femoral shaft according to different positions of knee joint using ANOVA(P < 0.01)in both groups. ITB axis gradually shifted from posterior to anterior of femoral shaft axis with the increasing of knee joint flexion. ITB average width and average thickness were statistically increased in male group using t-test(P < 0.01). ITB gradually shifts from anterior to posterior of femoral shaft with the incensement of knee joint flexion in healthy subjects. Conclusion:When knee joint occurs near 30° of flexion,the iliotibial band and the lateral femoral condyle friction is most prominent,which may one of causes of iliotibial band syndrome.

Abstract:Objective:The aim of this study was to evaluate 5 creatine (Scr)-based glomerular filtration rate (GFR)estimating equations on renal function evaluation in Chinese living related donor transplantation recipients. Methods:We compared 5 Scr-based GFR estimating equations using GFR measurement of 99m Tc-DTPA as the reference test in 39 stable renal transplantation populations from living related donor in our hospital. Bias,precision,accuracy within 30% and 50% ranges from the reference method and agreements of each test were compared. Results:The Scr-based Japanese Society of Nephrology-Chronic Kidney Disease Initiatives (JSN-CKDI) equation had the highest accuracy (71.8% within 30% of the reference)with a bias of only -0.7 ml/(min-1.73m2)and a precision of 12.0 ml/(min-1.73m2). Conclusion:Scr-based JSN-CKDI equation may be the best choice for assessing and monitoring GFR of long-term postoperative Chinese renal transplant recipients.

Abstract:Objective:To compare the impacts of different sample collection methods and RNA extraction methods from thyroid fine needle aspiration samples on the results of target gene detection. Methods:Trizol and RNeasy Plus Micro Kit (Qiagen) were used to extract RNA from thyroid FNA samples obtained from a separate puncture or leftover material in the needle; the thyrocyte-relevant genes were detected by reverse transcription-polymerase chain reaction; and the influence of different sample types and RNA extraction methods on RNA quality,quantity and RT-PCR outcome was analyzed. Results:RNA was isolated from 102 FNA samples,the RT-PCR results showed that the positive detection rate of GAPDH and TG were 97.06% and 93.14% respectively,followed by TPO,TSHr,and NIS. The concentration and quantity of RNA extracted by Trizol were higher than that by RNeasy Plus Micro Kit in samples from a separate puncture,but the differences were not statistically significant(P > 0.05). The OD ratio of RNA extracted by the two methods had no significant difference,so as the success rate of RT-PCR(P > 0.05). In samples obtained from leftover material in the needle,no significant differences were found in RNA concentration,quantity,OD ratio and success rate of RT-PCR between the two RNA extraction methods (P > 0.05). In the group that RNA was extracted by Trizol,there were no significant correlation between sample collection methods and RNA concentration,quantity,OD ratio or success rate of RT-PCR (P > 0.05). The same results were observed in the group that RNA was extracted by RNeasy Plus Micro Kit,different sample collection methods had no significant impact on RNA concentration,quantity,OD ratio and success rate of RT-PCR (P > 0.05). Conclusion:RNA can be successfully isolated from FNA samples to detect genes. Samples from a separate puncture or leftover material and RNA extraction methods of Trizol or Qiagen can all be used in RNA extraction from thyroid FNA samples.

Abstract:Objective:Genetic assessment of biomarkers in appropriate tumor samples remains to be hallmarks of the real-time individualized therapy in malignant diseases. Malignant effusion is one of the most common presentations in advanced solid cancer. The aim of this study was to characterize cell-free mRNA in malignant effusions and evaluate its association with the sensitivity of primary tumor cells to docetaxel and 5-fluorouracil (5-FU). Methods:Forty-three Malignant effusions samples were prospectively collected from 42 patients. After cell-free RNA extraction and separation of tumor cells,TS and β-tubulin Ⅲ mRNA transcripts in both supernatants and tumor cells were characterized by real-time quantitative PCR. ATP-TCA assay was used to analyze the sensitivity of primary tumor cells to docetaxel and 5-FU. Results:Cell-free TS and β-tubulin IIItranscripts were observed in 42 of 43 specimens. Compared with gene expression patterns in paired-tumor cells,TS mRNA was significantly higher in supernatant whereas β-tubulin Ⅲ mRNA remained the same(P = 0.02). Cell-free TS mRNA was inversely associated with in vitro sensitivity of cancer cells to 5-FU in the gastrointestinal cohort(P = 0.002). Although high β-tubulin III mRNA expression in supernatantsappeared is positively related with cancer cells’ sensitivity to docetaxel,the result didn’t reach the statistical significantce. Conclusion:The cell-free mRNA transcripts in malignant effusions were highly detectable and cell-free TS mRNA levels in gastrointestinal patients were correlated with 5-FU sensitivity.

Abstract:Objective:To investigate the effects of bisphenol A on proliferation and miR-19 expression in PC-3 prostate cancer cells. Methods:Cell viability of PC-3 cells was measured by MTT and Hoechest 33258 staining 6 days after treatment with different concentrations of Bisphenol A; miR-19 expression was measured by real-time PCR; expression levels of PTEN,p-AKT,AKT,Cylcin D1 and PCNA were determined by Western blot. Results:Bisphenol A 5 × 10-6 to 5 × 10-5 mol/L with concentration from increased cell proliferationof PC-3 cells. Expression levels of miR-19a and miR-19b were unregulated by Bisphenol A. Meanwhile,Bisphenol A decreased PTEN expression and increased the expression of p-AKT,PCNA and CyclinD1 in PC-3 cells. Conclusion:Bisphenol A promoted PC-3 cell proliferation,and up-regulation of miR-19 may be involved in the regulation of proliferative process induced by Bisphenol A.

Abstract:Objective:To explore the role of Oatp3 on the PFOS-induced Sertoli cell injury. Methods:The primary Sertoli cell was isolated from 20 day old male SD rat. Cytotoxicity assay,RNAi experiments and immunoblotting analysis were conduced to explore the role of Oatp3 on the PFOS-induced Sertoli cell injury. Results:There is no significant toxic effects observed after PFOS treatment with the dose range from 0~30 μM for 24 hrs(P > 0.05). In addition,compared with the control group,40 μmol/L of PFOS significantly decreased the survival rate of Sertoli cells(P < 0.01).The IC50 of PFOS on Sertoli cell is 45.6 μmol/L. Further more,40 μmol/L of PFOS significantly increased the expressions of Oatp3 on cells,which was inhibited by Oatp3 siRNA(P < 0.01). Compared with Mock group,40 μmol/L of PFOS significantly decreased the survival rate of Sertoli cells(P < 0.01),which was inhibited by Oatp3 siRNA(P < 0.01). Conclusion:Oatp3 is involved in PFOS-induced Sertoli cell injury.

Abstract:Objective:To survey and evaluate the effects of expanded programmed immunization of hepatitis B vaccine in newborns. Methods:A sero-epidemiology surveillance was conducted in infants aged 7-12 months in Huaian district of Huaian. Immunization history of hepatitis B vaccine (Hep B)was investigated and blood samples were collected at the same time. HBsAg,anti-HBs and anti-HBc was detected using chemiluminescence microparticle imunoassay (CMIA). HBsAg seropositive rate,anti-HBc seropositive rate,seroconversion rate and titer of anti-HBs were calculated and the influenced factors were analyzed using χ2 test or analysis of variance(ANOVA). Results:Twenty-three hundred and twenty infants were involved in the investigation. The timely first dose injection rate was 99.6%,and all the infants received the whole course vaccination. The HBsAg and anti-HBc seropositive rate was 0.13% and 2.1%,respectively the calculated protective rate of the Hep B was 93.4% and succession rate of blocking the maternal-infant transmission was 91.6%. The cause of the chronic HBV infection in the immunized infants was the failure of blocking maternal-infant transmission,the failure all happened in the infants with HBeAg positive mothers. The non-hyporesponsive rate was 26.3% in our surveillance population who immunized with 5 g Hep B,which was higher than other places using 10 g Hep B as the newborn immunization vaccine. Meanwhile the low birth weight infants had higher non-hyporesponsive rate than the normal birth weight infants. Conclusion:The standard vaccination rate of Hep B was very high and chronic HBV infection was very low in infants now. The chronic HBV infection in infants was mainly caused by maternal-infant transmission. The vaccine dose used in the infants should be added.

Abstract:Objective:To stady the genetic variations and evolution characteristics of neuraminidase (NA) gene of the novel influenza A(H1N1) virus isolated from Nanjing in 2013. Methods:Viral RNA was extracted from 18 strains of the novel influenza A (H1N1) virus. NA genes were amplified by RT-PCR and then sequenced. The genetic variations and important functional sites of NA genes were analyzed. Results:Compared with recommended vaccine strain in and abroad, the homology of NA amino acid sequences between 18 Nanjing isolates and vaccine strains was above 98.2%. The isolats were grouped into two clusters, with some genetic distance by phylogenetic mapping with the WHO vaccine stain,while closer to the domestic vaccine stain. Compared to the vaccine strains,the variations of amino acids occurred in different sites;1 isolate had a E119K replacement in enzymatically active site;Glycosylation sites appeared to be increased or deleted in the 2013 isolats. Conclusion:High homology was observed in all the NA genes between the strains isolated from Nanjing in 2013 and vaccine strains. The vaccine can continue provide a protection. However,more mutations were accumulated in NA genes in the strains isolated in the late of 2013. Therefore,the monitoring should be strengthened.

Abstract:Objective:To analyze the epidemiological characteristics of syphilis in Qinhuai district of Nanjing,Jiangsu province from 2004 to 2012. Methods:The epidemic data of syphilis from China disease prevention and control information system in Qinhuai district was analyzed by excel 2003 and SPSS 16.0. Results:There were reported a total of 1310 cases of syphilis in Qinhuai distict from 2004 to 2012. In the reported cases of syphilis,early syphilis including primary and secondary syphilis were 71.53%; there were significant differences between the stages of syphilis cases and the different years (P< 0.05); the gender ratio was 0.91∶1.00; there were 676 cases of syphilis were aged from 20 to 39,accounting for 51.60% in total cases of syphilis. Conclusion:The incidence of syphilis in Qinhuai distict was increased from 2004,declined from 2010,and most of cases of syphilis were primary and secondary syphilis,we should strengthen the comprehensive measures of prevention diagnosis and treatment of syphilis,in order to control the spread of syphilis and AIDS.

Abstract:Objective:To evaluate the efficacy and safety of moxifloxacin as well as levofloxacin in the treatment of multidrug resistance tuberculosis (MDR-TB) in China. Methods:Databases included the CB1Mdisc,CNKI,wangfang data,and VIP from Jan,2000 to Jun,2014 were retrospectively searched to collect randomized controlled trials (RCTs) of moxifloxacin and levofloxacin for MDR-TB. The methodological quality of included studies was evaluated,and data analyses were performed with The Cochrane Collaboration’s software RevMan 5.3.0. Results:A total of 36 RCTs were included. The results showed that,compared with the levofloxacin group,moxifloxacin increased the sputum negative conversion rate after 3-month taking(OR=2.25,95%CI:1.87~2.40)and at the end of the treatment period(OR=3.70,95%CI:2.97~4.62). Moxifloxacin was more effective in focus absorption(OR=2.45,95%CI:1.90~3.17),cavity closure(OR=1.82,95%CI:1.45~2.29)and clinical curative effect(OR=5.08,95%CI:3.58~7.22). However,there was no statistical difference in incidence of adverse reaction (P=0.14). Conclusion:According to the domestic evidence,moxifloxacin is more effective for MDR-TB than levofloxacin. Its adverse reaction rate is equivalent to levofloxacin.