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 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 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 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.

Objective To clinically observe and verificate the advantages of treating infantile anorexia of spleen-dysfunction in transportation with infantile massage and Chinese medicine based on the thought of “invigo-rating spleen and nourishing qi”. Methods Multi-central large-sample randomized controlled trial was carried out in Guangzhou, Shenzhen, and Foshan. Five hundreds cases of infantile anorexia of spleen-dysfunction in transportation were randomized into infantile massage group, Chinese medicine group, infantile massage combined with Chinese medicine group and Western medicine group and two sessions of treatment were required in each group. Separately, during the enrollment, after 2 sessions of treatment, infantile anorexia signs and symptoms scale , the related therapeutic effect criterion from clinical guideline of new drugs for traditional Chinese medicine, the expression level of hemoglobin and blood zinc were adopted for efficacy assessment. Results A mong 4 groups , the recovery rate and the total effective rate of infantile massage combined with Chinese medicine group were better than those of other groups (P < 0.05). The expression level of hemoglobin and blood zinc of 4 groups both increased obviously (all P < 0.01), but more improvement was found in infantile massage combined with Chinese medicine group (both P < 0.05). Conclusion The treatment of infantile anorexia with infantile massage and Chinese medicine based on the thought of invigorating spleen and nourishing qi has a significant effect.

Objective:To explore the therapeutic effect and safety of thrombus aspiration catheter in young patients with acute ST-segment elevation myocardial infarction (STEMI) during primary percutaneous coronary intervention (PCI) .Methods :According to using thrombus aspiration catheter during emergency PCI or not ,a total of 79 young patients with acute STEMI were divided into aspiration group (n= 37 ,received thrombus aspiration ) and routine treatment group (n=42 ,didn't receive aspiration catheter ) .Coronary TIMI flow ,angina pectoris symptoms ,cardi-ac function and major adverse cardiovascular events (MACE) etc .after PCI were observed and compared between two groups .Results:Compared with routine treatment group after treatment ,there were significant rise in TIMI flow [ (2.33 ± 0.48) grade vs .(3.00 ± 0.00) grade] ,2h ST segment regression >50% rate (45.24% vs .70.27% ) and left ventricular ejection fraction on the first week [ (47.21 ± 9.28)% vs .(52.16 ± 7.87)% ];significant reduc-tion in angina pectoris symptom (50.00% vs .27.03% ) ,and NYHA cardiac function during follow-up [ (1.52 ± 0.71) class vs .(1.22 ± 0.42) class] in aspiration group , P<0.05 or <0.01. There was no significant difference in incidence of MACE between two groups , P>0.05 all .Conclusion:Application of thrombus aspiration catheter could improve coronary blood flow ,reduce symptoms of angina pectoris and improve cardiac function during primary PCI in young patients with acute STEMI ,and it's safe .

Objective:To determine the expression of BMP4 in hepatocellular carcinoma (HCC) and to study the role of BMP4 in inducing epithelial-mesenchymal transition (EMT) to analyze the effect of BMP4 on the migration and invasion of HCC cells. Methods: The expression of BMP4 in HCC specimens was examined by immunohistochemistry staining, and the correlations were analyzed between the expression of BMP4 and clinicopathological data. The BMP4 expression plasmid was transfected into HepG2 cells to induce exogenous overexpression of BMP4 protein. The changes of HepG2 cell morphology were detected after BMP4 transfection by using a microscope; the changes of the expression of BMP4, EMT-related protein (E-cadherin, Vimentin) in HepG2 cells were detected by Western blot after transfection of BMP4;the wound healing assay in vitro was used to detect the effects of BMP4 gene transfection on the ability of migration of HepG2 cells;the invasion assay was used to determine the role of transfection of BMP4 on the invasive potential of HepG2 cells. Results: Immunohistochemistry staining method displayed that BMP4 expression was positively associated with age, histological differentiation, stage, and poor prognosis. After BMP4 overexpression, the morphology of HepG2 cells showed significant changes from a paving stone structure with cell-cell adhesion to a fibroblastic shape, which showed typical EMT change; Western blot exhibited that the expression of E-cadherin was downregulated and the Vimentin expression was upregulated in HepG2 cells;the wound healing and invasion assay showed that the migration and invasion potentials of HepG2 cells were significantly enhanced. Conclusion: BMP4, which displayed a high expression in HCC specimens, was closely associated with clinicopathologic data, and BMP4 may promote migration and invasion of HCC cells by inducing epithelial-mesenchymal transition.

Objective To investigate the relationship of omentin-1 with adiponectin and inflammatory cytokines in type 2 diabetes (T2DM) patients with nonalcoholic fatty liver disease (NAFLD). Methods The serum levels of omentin-1 and adiponectin were assayed by enzyme-linked immunosorbent assay (ELISA) in patients of T2DM with NAFLD (group A, n=63), T2DM without NAFLD (group B, n=63)and normal control group (group C, n=70). At the same time the biochemical markers and inflammatory marker, such as tumor necrosis factor (TNF)-α, high-sensitivity C-reactive protein (hs-CRP) and interleukin 6(IL-6) were detected in three groups. The correlation analysis and multiple regression analysis were used to de-tect the association of omentin-1 with adiponectin and inflammatory markers. The logistic regression was used to analyze fac-tors influencing NAFLD in patients with T2DM. Results The serum levels of omentin-1 and adiponectin were significant-ly lower in group A [ (27.02±2.82)μg/L and (11.98±3.63) mg/L] than those of group B [(31.52±2.81)μg/L and (15.85±3.28) mg/L] and group C [(35.92±2.80)μg/L and (19.88±3.44) mg/L], and there were significantly lower levels of them in group B than those of group C (P<0.01). The plasma omentin-1 level was positively correlated with adiponectin and high density li-poprotein (HDL-C) in group A. Also the plasma omentin-1 level was negatively correlated with TNF-α, IL-6, fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), visceral adipose tissue, waist, waist-to-hip ratio (WHR) and free fatty acid in group A (P<0.05 or P<0.01). Multiple stepwise regression analysis showed that adipo-nectin, TNF-αand IL-6 were independent factors influencing the level of plasma omentin-1. Logistic regression analysis showed that omentin-1 was one of independent factors influencing T2DM combined with NAFLD (P<0.01). Conclusion The incident of NAFLD in T2DM patients is related to the lower level of omentin-1, which may be influenced by adiponectin and inflammatory factors.

ObjectiveTo observe the effect of acetylcholine (ACh) on lipopolysaccharide (LPS) induced inflammatory model of rat alveolar macrophages, and to observe the effect of the acetylcholinesterase inhibitor physostigmine (Phy) on the anti-inflammatory effect of ACh.Methods The rat alveolar macrophages NR8383 were cultured in vitro, which were divided into five groups: blank control group, LPS group (stimulated with 1 mg/L LPS for 12 hours), LPS+ ACh group (0.01, 0.1, 1, 10, 100μmol/L of ACh were added for 5 minutes before LPS stimulation), LPS+ Phy group (1 mmol/L Phy was added for 5 minutes before LPS stimulation), and LPS+ ACh+ Phy group (1 mmol/L Phy and 10μmol/L ACh were added for 5 minutes before LPS stimulation). The supernatants were collected in each group, the enzyme-linked immunosorbent assay (ELISA) was used to assay the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.Results① The contents of TNF-α (ng/L: 605.09±57.13 vs. 34.07±8.62), IL-1β (ng/L: 377.09±28.55 vs. 32.33±10.62) and IL-6 (ng/L: 558.04±77.45 vs. 42.62±11.21) in the LPS group were significantly higher than those in the blank control group (allP< 0.05). These results indicated that the inflammatory model of rat alveolar macrophages was constructed successfully.② ACh with the final concentrations of 0.01, 0.1, and 1μmol/L had less influence on the production of TNF-α, IL-1β and IL-6 in the culture supernatants of alveolar macrophages stimulated with LPS compared with LPS group (allP> 0.05). Nevertheless, 10μmol/L and 100μmol/L ACh notably reduced the production of TNF-α (ng/L: 451.19±30.67, 332.19±32.19 vs. 604.96±22.56), IL-1β(ng/L: 261.08±24.78, 143.98±28.39 vs. 367.06±10.44) and IL-6 (ng/L: 342.75±54.60, 235.48±29.75 vs. 562.69±63.34) in the culture supernatants compared with the LPS group (allP< 0.05).③ The activity of AChE in the LPS group was significantly higher than that in the blank control group (kU/L: 5.21±0.63 vs. 3.09±0.10,P< 0.05). The activity of AChE was successfully inhibited by 1 mmol/L acetylcholinesterase inhibitor Phy pretreatment compared with that in the LPS group (1.51±0.12 vs. 5.21±0.63,P< 0.05).④ The level of TNF-α (ng/L: 183.17±35.44 vs. 451.19±30.67), IL-1β (ng/L: 91.49±12.27 vs. 261.08±24.78) and IL-6 (ng/L: 108.17±22.82 vs. 342.75±54.60) in the culture supernatants of LPS+ ACh+ Phy group was significantly decreased as compared with LPS+ ACh group (allP< 0.05).Conclusions ACh with the final concentrations of 10μmol/L and 100μmol/L can inhibit the LPS induced inflammatory reaction in alveolar macrophages. The acetylcholinesterase inhibitor Phy can reinforce the ACh-mediated anti-inflammatory effect on alveolar macrophages inflammatory model.