Objective To investigate the expressions of microRNA-21 in cholangiocarcinoma tissues and the relationship between epithelial-mesenchymal transition (EMT) and the prognosis of patients.Methods Forty-one samples of cholangiocarcinoma and 10 samples of adjacent tissues from 10 patients who received radical resection of cholangiocarcinoma at the Provincial Hospital of Anhui Medical University from January 2005 to January 2010 were collected.The expressions of microRNA-21,E-cadherin and N-cadherin were detected by in situ hybridization and immunohistochemistry,and effect of their expressions on the prognosis was analyzed.Enumeration data were analyzed using chi-square test.The correlation between microRNA-21 and EMT markers was analyzed using the Spearman correlation coefficient.The survival curve was drawn by Kaplan-Meier method,and the survival rate was analyzed using the Log-rank test.Results The expression rate of microRNA-21 in the cholangiocarcinoma tissues was 63％,which was significantly higher than 30％ of that in the adjacent tissues (x2 =0.324,P ＜ 0.05).The expression of microRNA-21 was closely related with the tumor differentiation degree,lymph node metastasis,perineural invasion (x2 =6.365,0.552,11.896,P ＜ 0.05),but not with gender,age,tunor location and tumor type (x2 =0.322,0.588,0.510,0.256,P ＞ 0.05).The expressions of E-cadherin and N-cadherin were related with lymph node metastasis and perineural invasion (x2 =4.630,5.512;6.600,7.152,P ＜0.05),but not with gender,age,tumor location,tumor differentiation degree and tumor type (x2 =0.266,0.013,0.067,0.666,0.003; 1.036,0.997,1.808,2.997,0.812,P ＞0.05).A positive correlation between the expression of microRNA-21 and EMT related markers E-cadherin and N-cadherin was detected (r =0.373,0.614,P ＜0.05).The results of survival analysis showed that the overall survival rate and tumor-free survival rate of patients with low expression of microRNA-21 were significantly higher than those of high expression of microRNA-21 (x2 =3.999,4.376,P ＜ 0.05).Conclusion Over expression of microRNA-21 in cholangiocarcinoma and metastatic lymph nodes may accelerate the invasion and metastasis of cholangiocarcinoma through inducing EMT,microRNA-21 might predict the prognosis of patients.

The effects of targeted silencing of heparanase gene by small interfering RNA (siRNA) on invasiveness and metastasis of osteosarcoma cells (MG63 cells) were investigated in the present study. Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene. The expression vector containing short hairpin RNA (pGenesil-shRNA) was constructed successfully. MG63 cells were randomly allocated into 3 groups: blank group, empty vector (pGenesil) transfected group and expression vector (pGenesil-shRNA) transfected group. Under the induction of Lipofectamine 2000, the recombinants were transfected into MG63 cells. Heparanase gene expression level was detected by RT-PCR and Western blotting. Cell proliferation was measured by MTT assay. Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays. HUVECs migration assay was applied for the detection of angiogenesis. As compared with negative controls, the mRNA and protein expression levels of heparanase were down-regulated by 76.1% (P<0.01) and 75.3% (P<0.01) respectively in the pGenesil-shRNA transfected group. Meanwhile, the proliferation, adhesiveness, invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited. It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Objective To identify the variation in the Env region of SHIV-XJ02170 during passaging in Chinese origin Rhesus Macaques.Methods Fragments of the SHIV-XJ02170 gp160 and gp120 gene were amplified by PCR and RT-PCR separately from the blood samples of SHIV-XJ02170 infected animals at the peak viral load time point.Purified RT-PCR product was ligated into T easy vector and transformed into JM109 competent cells,18 clones were selected by PCR method and sequenced by ABI 3730DNA sequencers.The gene distances(divergence,diversity)were calculated using DISTANCE.Results In all,the SHTV-XJ02170 gp120 gene evolved forward along the virus passaging.It could be found that viral divergence from the founder strain serially enhanced during in vivo passaging,but in the early phase of each passage,SHIV-XJ02170 gp120 gene evolved toward ancestral state upon transmission to a new host.All of the SHIV-XJ02170 strains had V3 loop central motif(GPGQ)and were predicted to be using CCR5 on the basis of the critical amino acids within V3 loop.Conclusion There was significant increase in the genetic distance during serial passaging,and SHIV-XJ02170 gp120 gene evolved forward along passaging.This could partly explain why the virus infectivity was enhanced during in vivo passaging.

Objective To analyze the virologic and immunologic properties during SHIV-XJ02170passage in vivo and construct the clade C SHIV/Chinese origin Rhesus macaques AIDS model . Methods SHIV-XJ02170 cell-free virus tranfected in 293T was adapted by serial passage in nine Chinese-origin Rhesus macaques. CD4/CD8 ratio was detected by flow cytometry to analyze the changes in viral pathogenicity. Real-time RT-PCR and IFN-γsecreting ELISPOT methods were used to analyze changes in characteristics of virology and immunology. Results During in vivo passage, CD4/CD8 ratio did not deeply decline. However,the peak and setpoint viral load in the line 3 show a continuous upward trend. The strong humoral and cellular immune responses were induced after SHIV-XJ02170 infection. Meanwhile, there was significant positive correlation between the viral load and binding antibody titer. Conclusion There were no pathogenic viral strains, and upward trend in virulence of SHIV-XJ02170 was found during in vivo passaging. SHIVXJ02170/Chinese origin Rhesus macaques model will play an important role in effect evaluation of candidate AIDS vaccines in China.

Objective:To investigate the expression of TIP30 and its relationship with clinico-pathological characteristics in patients with extrahepatic cholangiocarcinoma (ECC).Methods:The expression of TIP30 in 78 cases of ECC tissues and 78 cases of para-cancerous tissues were detected by immunohistochemistry.Results:The positive expression rate of TIP30 was 43.59％ and 75.64％ in ECC tissues and para-cancerous tissues,respectively.Differences were statistically significant (P＜ 0.05).The expression levels of TIP30 were not correlated with age,gender,degree of differentiation and tumor size(P＞0.05),but correlated with lymph node metastasis,distant metastasis and TNM staging(P＜ 0.05).The median overall survival of 78 ECC cases was 14.8 months,and it of TIP30 positive expression cases was 20.3 months,statistically higher than 11.5 months in TIP30 negative expression cases(P＜ 0.01).Conclusion:The downregulation of TIP30 is closely correlated with the development,metastasis and prognosis of ECC.TIP30 may be used as a molecular marker to identify and predict the progression,metastasis and prognosis of ECC.

Seventy-four men aged 20-50 years with normal glucose tolerance were divided into abdominal obese group(n=36),simple obese group(n=16),and normal body weight group(n=22).One-hour postload plasma glucose(1hPG)and carotid intima-media thickness(IMT)were higher in abdominal obese group than those in simple obese group and control group(all P<0.01).IMT was positively correlated with 1hPG(P<0.05).In multiple regression analysis,waist circumference and triglycerides were independent predictors for IMT.

Objective To establish a method for rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin,and to study the possibility of using it in single radial immunodiffusion (SRID) assay for quantitative detection of antigen in H7N9 influenza vaccine.Methods Hemagglutinin proteins expressed in eukaryotic cells were used to immunize sheep.Serum samples were collected to detect antibody titers by ELISA and double immunodiffusion assay.Different concentrations of antiserum were used in SRID assay to get the optimized concentration.Results After 4 times of immunization,the antiserum titers achieved 1 ∶ 1 000 000 and 1 ∶ 32 as indicated by ELISA and double immunodiffusion assay,respectively.The antiserum could form a clear precipitation line in SRID assay.The detection of antigen in the range of 10 to 40 μg/ml showed good linearity in the standard curve.The antigen titers in six batches of H7N9 vaccine detected by this SRID assay were identical with those by SDS-PAGE assay.Conclusion The antiserum against H7N9 hemagglutinin for SRID assay was developed successfully,and could be used as a reagent for the quantitative detection of antigen in H7N9 influenza vaccine.

Forty-eight men with normal glucose tolerance were divided into fatty liver disease ( NAFLD, n =23 ) and non-NAFLD (n =25 ) groups. The blood glucose excursion was evaluated by continuous glucose monitoring system. The results showed that the mean amplitude of glucose excursion[MAGE, (2. 17± 1.13 vs 1.45±0. 42 )mmol/L]and standard deviation of blood glucose[SDBG, (0. 88 ±0. 45 vs 0. 61 ±0. 21 ) mmol/L]were significantly higher in NAFLD group than in non-NAFLD group( both P＜0. 05 ). MAGE and SDBG were positively correlated with body mass index, waist circumference, and the increased value of plasma glucose 0. 5 h after glucose loading( △G30,all P＜0. 05 ). In multiple regression analysis, △G30, waist circumference, and age were significant independent predictors for MAGE( P＜0. 05 or P＜0. 01 ). △G30 and waist circumference were significant independent predictors for SDBG( P＜0. 05 or P＜0. 01 ).

Objective To study the in vivo expression and biodistribution of Ad5-Fluc (Adenovirus carrying firefly luciferase genes) in mice.Methods The recombinant Ad5-Fluc virus was constructed and infected to BALB/c or nude mice through three different routes.The protein expression level,tissue distribution and the characteristics of infection were analyzed by in vivo bioluminescence imaging technology.Results Compared to other two routes,the BALB/c mice infected through muscular route had the longest expression cycle (over 60 days) and the highest expression level,while the virus was transferred into the liver and spleen after infection.The nude mice had a significantly extended expression cycle than BALB/c mice.Moreover,the characteristic of liver tropism was eliminated after Ad5 F35 infection in mice,while maintained similar expression efficiency.Conclusion Due to the highest expression efficiency,the muscular route would be the optimal route for Ad5 vector based vaccination.In addition,Ad5F35 virus could become an ideal alternative vaccine vector for eliminating the liver tropism.

Objective To explore the mechanism and effect of miR-146a on alveolar macrophages and to observe the changes of miR-146a expression in the LPS-induced alveolar macrophages. Method NR8383 alveolar macrophages were divided into LPS-stimulated group and control group, and the cells of former group were treated with LPS ( 1 μg/mL) and then incubated for 3 h, 6 h and 12 h, respectively. The level of TNF-α in the supernatant of cells was assayed by using enzyme-linked immunosorbent assay (ELISA), and the expression of miR-146a of cells was detected by using Real-Time PCR (TaqMan probe).Statistical analysis carried out by using SPSS 13.0 software package in which One-way ANOVA and Student's t-test were used. Results Compared with control group, the levels of TNF-α in the supernatant of cells were significantly increased 3 h, 6 h and 12 h after LPS challenge (P ＜ 0.01 ). The expression of miR-146a increased 6 h and 12 h after LPS stimulation in NR8383 cells( P ＜0.01 ), and it had an upward tendency.Conclusions The expression of miR-146a in alveolar macrophages increases after LPS-stimulation. It hints miR-146a may be involved in the regulation of the inflammatory responses produced by alveolar macrophages.