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.

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.

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.

[Abstract ] Objective At present, the majority of injectable tissue engineering bones or carrier stents are gel , whose surface area , intensity, and hardness cannot satisfy the requirements of the repair of complex and varied bone and cartilage defects .This paper evaluated the new injectable microspherical porous chitosan/biological properties of the hydroxyapatite ceramic scaffold . Methods Injectable porous chitosan /hydroxyapatite composite microspheres with mass fractions of 30%, 50%, and 70% were prepared respectively . The hydroxyapatite ceramic ball was obtained by sintering with liquid nitrogen freezing ( liquid nitrogen group ) or without liquid nitrogen pro-cessing ( non-liquid nitrogen group ) as a new carrier of bone tissue engineering scaffold material .The microstructure of the scaffold was observed and the porosity measured under the scanning electron microscope .The mechanical properties were determined through biome-chanical experiments.Human umbilical vein endothelial cells (HUVECs) were grown in the porous chitosan/hydroxyapatite ceramic scaf-fold followed by observation of the growth of the cells and validation of the biological fusion of the scaffold . Results No difference was observed with the naked eye in the ceramic scaffold of different mass fractions in the liquid nitrogen and non -liquid nitrogen groups . Scanning electron microscopy exhibited spherical shape , uniform size, and regular morphology of the ceramic scaffolds in both groups .A large number of irregular pores were seen in the surface of the microspherical ceramic scaffolds treated with liquid nitrogen but not in the surface of those not treated .With increased mass percentage of chitosan/hydroxyapatite , the internal pores were reduced and the interior structure compacted.In the liquid nitrogen group, the scaffold of 50%mass fraction had a significantly larger diameter ([0.48 ±0.11] mm), higher compression intensity ([1.75 ±0.14] MPa), and lower porosity ([79 ±2]%) than that of 30%mass fraction ([0.40 ± 0.08] mm, [1.21 ±0.12] MPa, and [87 ±1]%) (all P<0.05).Electron microscope scanning revealed well -grown HUVECs with multiple synapses in the porous tricalcium phosphate scaffold. C onclusion The porous chitosan /hydroxyapatite ceramic scaffold of 50%mass fraction treated with liquid nitrogen , with its strong mechanical intensity and high biological fusibility , can be used as a new carrier of bone tissue engineering scaffolds .

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.

ObjectiveTo investigate the protective effect of transfected microRNA-146a (miR-146a) on mice with sepsis-induced acute lung injury (ALI) in vivo.Methods Twenty-four healthy male BALB/C mice were randomly divided into sham group, sepsis group, transfection group and transfection control group, eachn = 6. Mice in transfection group were given miR-146a agomir loaded by in vivo-jetPEITM via airway before reproduction of model, and mice in transfection control group were given negative control loaded by in vivo-jetPEITM only via airway. The septic model was reproduced by cecal ligation and puncture (CLP) 12 hours after transfection , and the mice in the sham group underwent laparotomy and closure only without ligation and puncture of the cecum. The mice of each group were sacrificed at 24 hours post-operation. The expression of miR-146a in lung tissue was determined by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the quantity of tumor necrosis factor-α (TNF-α) in the bronchial alveolar lavage fluid (BALF) was determined with enzyme-linked immunosorbent assay (ELISA). The wet/dry ratio of lung (W/D) was determined. The pathohistological changes in the lung were observed and scored. Results The expression of miR-146a showed a significant increase in sepsis group, transfection group and transfection control group, which were (3.56±0.43), (27.64±3.46) and (3.72±0.54) folds of that in sham group, respectively (P< 0.05 orP< 0.01). The miR-146a expression in transfection group was significantly increased compared with sepsis group and transfection control group (bothP< 0.01), but no statistical difference in the expression was found between sepsis group and transfection control group (P> 0.05). Compared with the sham group, higher level of TNF-αin the BALF was found in the sepsis group, transfection group and transfection control group (ng/L: 511.65±43.47, 305.74±34.76, 492.27±42.21 vs. 50.72±7.23, allP< 0.01). The level of TNF-α in transfection group was significantly lower than that in sepsis group and transfection control group (bothP< 0.01). Compared with the sham group, the W/D ratio of lung in sepsis group, transfection group and transfection control group showed a significant increase (6.11±0.32, 5.02±0.29, 6.05±0.43 vs. 4.18±0.10, allP< 0.01). The W/D ratio of lung in transfection group was significantly lower than that of sepsis group and transfection control group (bothP< 0.01). The lung injury score of transfection group was significantly lower than that of sepsis group and transfection control group (6.12±0.75 vs. 10.53±1.52, 9.73±1.08, bothP< 0.01).Conclusions miR-146a agomir loaded by in vivo-jetPEITM instillation into airway was able to increase the expression of miR-146a in the lung tissue of septic mice. Up-regulation of miR-146a inhibit the release of the inflammatory cytokine TNF-α stimulated by sepsis, and alleviate inflammatory reaction and lung tissue injury in mice with sepsis-induced ALI.