Objective To detect the expressions of microRNA-126 (miR-126) and microRNA-7 (miR-7) in esophageal squamous cell carcinoma (ESCC) and to analyze their correlations with clinicopathologic features and prognosis of ESCC.Methods The expressions of miR-126 and miR-7 in 116 ESCC samples and matched normal tissue samples were detected by real-time PCR.Statistical analysis was used to find the relationships among the expressions of miR-126 and miR-7,pathological characteristics and prognosis.Results Low expression,normal expression and high expression of miR-126 were found in 73 (62.9％),35 (30.2％) and 8 (6.9％) carcinoma samples respectively.Low expression,normal expression and high expression of miR-7 were found in 52 (44.8％),35 (30.2％) and 29 (25.0％) carcinoma samples respectively.The disease-free survival in patients with low expression of miR-126 and miR-7 was shorter than that in patients with non-low expression (x2 =4.268,P ＜0.05 ; x2 =4.993,P ＜0.05).The low expression of miR-126 was correlated with tumor location,family history and drinking (x2 =14.564,P ＜ 0.05 ; x2 =5.691,P ＜ 0.05 ; x2 =4.971,P ＜ 0.05),but was uncorrelated with gender,age,diferentiation,infiltration,lymphatic metastasis and smoking (all P ＞ 0.05).The low expression of miR-7 was uncorrelated with pathological characteristics of ESCC (all P ＞ 0.05).Conclusion The low expressions of miR-126 and miR-7 may be related to the prognosis of patients with ESCC,and have a certain clinical detection significance.

Background and purpose:Connective tissue growth factor(CTGF) is a member of CCN family,it has been reported that CTGF involve in many biological processes and various pathological conditions.In our study,the correlation of CTGF expression and biological effects on breast cancer cell lines were investigated.Methods:The eukaryotic expression vectors containing CTGF open reading frame(ORF) pcDNA3.0/CTGF were constructed and transfected into breast cancer cell lines.The relationship between CTGF expression and breast cancer cell growth ability and proliferation,cell cycle distribution,apoptosis,cell motility and invasive capacity in vitro were observed.Results:Upregulation of CTGF expression in MCF-7 cell line could inhibit its growth ability and proliferation,increased the proportion of G0G1 phase cells,enhanced apoptosis and inhibited its invasive ability in vitro.Downregulation of CTGF expression in MDA-MB-231 cell line increased its growth ability and proliferation,decreased apoptosis and promoted its invasive ability in vitro.There were no differences of cell motility among different groups in MCF-7 and MDA-MB-231 cell lines.Conclusions:CTGF can inhibit breast cancer cell growth by increasing cell apoptosis and/or the proportion of cells in G0G1 phase.CTGF can also inhibit breast cancer cell invasive ability.

Objective: To investigate the inhibition of tanshinoneⅡA (TanⅡA) on the proliferation of lung cancer cells. Methods: Human lung cancer cell lines A549, SK-MES-1, H446 and H460 were cultured in vitro and treated with TanⅡA at concentrations of 0.3, 0.6, 1.3, 2.5, 5.0, 10.0 mg/mL, while untreated cells served as controls. Cell proliferation was measured by using CCK-8 assay, and apoptosis was measured using flow cytometry, while the expression of apoptosis-related proteins was determined using Western blotting. The apoptotic rate of lung cancer cells was compared between Tan ⅡA-treated cells and untreated cells. Results: Tan ⅡA inhibited lung cancer cell proliferation in a time-dependent and concentration-dependent manner, and the survival rates of A549, SK-MS-1, H446 and H460 cells reduced with the concentration of TanⅡA (t=4.503, 2.114, 2.103 and 3.567; all P<0.05) and the duration of TanⅡA treatment (t=5.189, 3.079, 3.023 and 3.845; all P<0.05). The 48 h half maximal inhibitory concentrations (IC50 values) of TanⅡA were (1.18±0.12), (0.78±0.08), (1.55±0.16) and (1.27±0.14) mg/mL against A549, SK-MES-1, H446 and H460 cells, respectively. Following 48 h treatment with Tan ⅡA at a concentration of 2.5 mg/mL, the apoptotic rates of A549, SK-MS-1, H446 and H460 cells were (34.97±3.78)%, (37.62±2.48)%, (18.27±2.98)% and (19.17±2.30)%, which were significantly significantly higher than those of untreated cells [(4.86±0.36) %, (3.21±0.48) %, (3.25±0.26)% and (2.66±0.19)%, all P<0.05]. Reduced Akt1 protein expression, elevated Bax/Bcl-2 expression ratio, and elevated caspase-9 and caspase-3 protein expression were detected in lung cancer cells treated with 2.5 mg/mL TanⅡA for 48 h relative to untreated cells Conclusion TanⅡA may inhibit lung cancer cell proliferation in a time- and concentration-dependent manner via the Bax/Bcl-2/caspase-9/caspase-3 pathway.

Objective To explore the growing development and community succession of main sarcos-aphagous insects on pig carcasses in summer indoor and outdoor environment in Shenzhen area and to estimate the postmortem interval (PMI). Methods From early May to August in 2013, in Forensic Med-ical Examination Center of Shenzhen Public Security Bureau, the main insect species and the decomposi-tion process were observed in two adult pig carcasses of simulative indoor and outdoor environment. The different decomposition stages and the community succession of insects were recorded. Results The indoor and outdoor pig carcasses showed skeleton 412.5 and 325 hours after death, respectively. The main species of flies on pig carcasses were Chrysomya megacephala, Chrysomya rufifacies and Chrysomya chani. The main species of beetles were Crecphilus maxillosus, Necrobia ruficollis, Saprinus splendens and Dermestes maculatu. The dominant species of flies in the outdoor pig carcasses obviously produced the second generations due to the effect of mass rainfall, nor in the indoor pig carcasses. Conclusion There are regular patterns on the community succession of insects on pig carcasses in summer indoor and out-door environment in Shenzhen area. The activity patterns of seven typical insects and their larva show important value for estimating PMI.

Objective To investigate the expression of transcription factor Oct 4 in gastric carcinoma and its effects on gastric cancer cell proliferation and apoptosis after Oct 4 gene interfered by lentivirus vector.Methods Real time PCR and Western blot were used to observe the expression of Oct 4 in different differentiated gastric cancer cell lines.Gastric cancer cell lines with high expression of Oct 4 was cultured and infected by siRNA-Oct 4-lentivirus vector.Cell proliferation and apoptosis were observed after Oct 4 gene was interfered.Results Oct 4 was highly expressed in poorly and moderately differentiated gastric cancer cells.Gene interfered with siRNA inhibits the expression of Oct 4 in gastric cancer cells and show significant effects on cell proliferation and mobility as well as apoptosis after down-regulation of Oct 4.Condusions Oct 4 expression is in close relationship with gastric cancer cell proliferation and invasive ability.

Objective To investigate the clinical efficacy of laparoscopic radical gastrectomy in elderly patients with advanced gastric cancer.Methods The clinical data of 85 elderly patients with advanced gastric cancer who were admitted to the Ningbo First Hospital from January 2012 to June 2014 were retrospectively analyzed.Laparoscopic radical gastrectomy was performed on 46 patients (LRG group) and open radical gastrectomy on 39 patients (ORG group).All the patients underwent primary tumor resection for gastric cancer + D2 lymph node dissection,and the postoperative recovery plans were done according to enhanced recovery program.The volume of blood loss,number of lymph node dissected,operation time,intraoperative arterial partial pressure of carbon dioxid (PaCO2),time to anal exsufflation,indwelling time of gastric tube,time for out-off-bed activity,time for fluid diet intake,postoperative hemoglobin,duration of hospital stay and occurrence of complications in the 2 groups were analyzed.The follow-up by outpatient examination and telephone interview was carried out on patients up to August 2014.The count data were analyzed by the chi-square test and Fisher exact probability.The measurement data with normal distribution were presented as x ± s and analyzed using the t test.The t' test was used if the data were deficient.Results Surgical procedures in the 2 groups were successfully carried out and no perioperative death occurred.There was no conversion to open surgery in the LRG group.The resection margins in all the patients were negative.The operation time and number of lymph node dissection in the LRG group were (239 ±68)minutes and 27 ± 10,compared with (227 ±50)minutes and 26 ± 10 in the ORG group,with significant differences (t =0.919,0.179,P ＞ O.05).PaCO2 in the LRG group was (41 ± 5) mmHg (1 mmHg =0.133 kPa),which was significantly higher than(36 ± 5) mmHg in the ORG group (t =4.745,P ＜ 0.05).The volume of blood loss was (102 ± 44)mL in the LRG group,which was significantly less than (200 ± 120) mL in the OPG group (t' =-4.807,P ＜ 0.05).The postoperative level of hemoglobin in the LRG was (110 ± 15) g/L,which was significantly higher than (98 ± 27)g/L in the ORG group (t' =2.471,P ＜ 0.05).The time to anal exsufflation,indwelling time of gastric tube,time for out-off-bed activity,time for fluid diet intake,duration of hospital stay in the LRG group were (2.6 ± 0.7) days,(2.1 ± 0.7) days,(1.1 ± 0.3) days,(4.1 ± 0.7) days and (11 ± 4) days,which were significantly different from (4.8 ± 1.5) days,(4.0 ± 1.8) days,(4.5 ± 0.6) days,(5.9 ± 1.8) days and (18 ± 3) days in the OR G group (t' =-8.415,-6.206,-33.831,-5.879,t =9.632,P＜0.05).Eight patients in the LRG group and 15 patients in the ORG group had complications,with the incidence of complications of 17.4％ (8/46) and 38.5％ (15/39),respectively,showing a significant difference (x2 =4.748,P ＜ 0.05).Forty-four patients in the LRG group and 36 patients in the ORG gorup were followed up for 2-25 months,1 patient in the LRG group and 2 patients in the ORG group died and others had full recovery.Conclusions Laparoscopic radical gastrectomy could provide a safe and complete tumor resection for elderly patients with advanced gastric cancer compared with open radical gastrectomy,meanwhile,it can improve postoperative recovery and reduce postoperative complications in elderly patients with advanced gastric cancer.

Objective To construct short hairpin RNA (shRNA) expression plasmids against the inhibitor of differentiation 2 (Id2) gene and establish a suitable cell model for the role of Id2 in proliferation and differentiation of human Ewing sarcoma cell.Methods Three shRNA sequences targeting Id2 gene were designed and inserted into the pGPU6/GFP/Neo (-shRNA-Id2) expression vectors.The recombinant pGPU6/GFP/Neo-shRNA-Id2 plasmids were introduced into Ewing sarcoma RD-ES cells by liposome-mediated transfection.The knock-down efficiency of Id2 in infected RD-ES cells was verified by Western blot assay.The cell growth and cell cycle changes were evaluated by cell counting and flow cytometry between transfected cells and control cells.Results The Id2 expression decreased 54 ％ and 57 ％,respectively,in RD-ES cell line which were transfected with the shRNA-Id2-543 and shRNA-Id2-593 plasmids compared with the control group cells by Western blot analysis.The cell growth assay demonstrated that the cell number in transfected cells was significantly decreased during 6-7 d compared with the control group (P ＜ 0.05).The cells at the S-phase of cell cycle were increased [(36.60±1.53) ％ and (44.89E2.46) ％ vs (29.73±2.03) ％,P ＜ 0.05],and no significant changes at the G2 phase or even reduction in the transfected cells.Conclusions Id2 stable knock-down cell lines are successfully established.The reduced expression of Id2 is related with decreased cell growth and cell cycle arrest in the S-phase.Id2 maybe plays an important role in proliferation of Ewing sarcoma cell.

AIM:To establish a fast , accurate and economical technique for culturing mouse pulmonary arte-riolar smooth muscle cells ( PASMCs ) , and to explore the effects of hypoxia on the proliferation and apoptosis of the PASMCs.METHODS:In sterile condition, the pulmonary artery was isolated from the male BALB/c mice by digesting with collagenase I, and the cells were cultured in fetal bovine serum-coated flask.Centrifugal procedure was not used dur-ing the cell passage .The cell morphology was observed under an inverted phase-contrast microscope .α-Smooth muscle ac-tin was identified by immunocytochemistry and immunofluorescence .The effects of hypoxia on the proliferation and apopto-sis of the PASMCs were detected by CCK-8 assay and TUNEL assay .RESULTS:PASMCs were identified by the methods of immunocytochemistry , immunofluorescence staining and observation of morphology .Unlike the rat PASMCs with typical subcultured peak-vally pattern, the mouse PASMCs showed a lot different without a peak-vally pattern.The cells could be subcultured after 5 d to 7 d and there was 3 to 5 generations depending on the activity of the cells .CCK-8 assay demonstra-ted that the A values of PASMCs exposed to hypoxia increased after 24 h ( P<0.05) as compared with normoxia .TUNEL result showed that the apoptotic index of the PASMCs in hypoxia decreased after 24 h (P<0.05).CONCLUSION:This technique for obtaining cultured mouse PASMCs is simple , fast, accurate and economical .The digestion time is easy to control.Hypoxia promotes the proliferation and inhibits the apoptosis of PASMCs .

Objective: To study the axonal effect and the expression of integrin α6β4 during Schwann cell(SC) differentiation and myelination. Methods: Schwann cells were dissociated from the sciatic nerve of neonatal Waster rats and neurons dissociated from spinal cord. Singal cultures and purified populations of SC were cocultured with NC. Four methods (contrast microscope, scanning electron microscopy(SEM), immunocytochemistry method and in situ hybridization ) were used. Results: The separately cultured Schwann cells showed MBP negetive by immunocytochemistry method. But cocultured SC were shown positive. SEM showed that Schwann cells' membrane loop progressively circumnavigated around the axon during myelination, which suggested that the non-myelinating SC(nMSC) transformed to myelinating SC (MSC). In situ hybridization showed integrin α6β4 positive signals only on the outer surface of the Schwann cell-axon unit in SC coculture with NC. Conclusion: The differentiation and maturation of SC depend on axon, and the activity of integrins is expressed by axon. Axonal contact induces the expression of α6β4 during SC myelination, which suggests that integrin α6β4 is an important mediator of interactions of myelinating SC with the basal limina.

Objective To compare the performance of four commercial anti-dsDNA antibody assays,i.e,BioPlex 2200 (BioPlex),Farr radioimmunoassay (Farr),MESACUP DNA-Ⅱ TEST ds [MBL-enzyme linked immunosorbent assay (ELISA)] and Anti-dsDNA-NcX ELISA (IgG) (EURO-ELISA),Antoantibodies Profile Assay Kit (HOB-Chemiluminescent Immunoassay) in disease activity assessment of systemic lupus erythematosus (SLE).Methods SLE patients (n=119) as well as healthy controls (n=200) and disease controls (n=100) were recruited and their serum anti-dsDNA antibodies were detected by BioPlex,Farr,MBL-ELISA,EURO-ELISA,and a standard Crithidia luciliae indirect immunofluorescence test (CLIFT).The consistency between above four methods to CLIFT was analyzed.The correlation of anti-dsDNA antibody level of these four methods to SLE disease activity was assessed.All data analyses were performed with Statistical product and service solutions (SPSS) 16.0 (SPSS.Inc) and GraphPad Prism 4.0.3 (GraphPad).Unless otherwise specified,all data in this study were expressed as mean±standard deviation.Cut-off values of the anti-dsDNA quantification methods were set by the manufacturers.Chi square and kappa coefficients were adopted to assess the agreement determination and correlation analysis between anti-dsDNA level and SLE disease activity (SLEDAI).Receiver-operator characteristic (ROC) curve analysis was used to compare the specificity and sensitivity of the anti-dsDNA assays.Student's t test was adopted for the comparison of anti-dsDNA levels by different methods between SLE and SLE+LN groups.A p value small than 0.05 was considered statistically significant.Results Using cut-off values set by the manufacturers,BioPlex demonstrated the highest overall agreement with CLIFT,while MBL-ELISA and EURO-ELISA showed the highest positive agreement with CLIFT.Disease activity correlation analysis showed that SLEDAI score correlated poorly with anti-dsDNA level in Farr assay,but strongly with the other three assays.Bioplex had a better performance in terms of SLE activity index corelation (r=0.297 6,P=0.001 2).Moreover,anti-dsDNA level differed in SLE patients with renal lupus nephritis in BioPlex assay (P=0.026 8),but not in the other assays.In ROC curve analysis,BioPlex showed the largest area under the curve (AUC) over other assays.Conclusion Bio Plex assay has better sensitivity and specificity than Farr,MBL-ELISA and EURO-ELISA and correlates well with SLE disease activity.