Objective:To investigate the effect of GTPBP4 silencing by RNA interference on the radiosensitivity of esphageal cancer EC9706 cells line.Methods:The expression data of GTPBP4 in esophageal cancer tissues was obtained from public Gene Expression Omnibus (GEO) database. Recombinant plasmid-mediated RNA interference (RNAi) was employed to transfect the esophageal cancer EC9706 cell to evaluate the influence of GTPBP4 silencing on the proliferation, apoptosis and radiosensitivity of esphageal cancer EC9706 cells. The expression levels of GTPBP4 mRNA and protein and apoptosis-associated proteins of Bax, cleaved caspase-9, cleaved caspase-3 and Bcl-2 were determined by qRT-PCR and Western blot. The cell proliferation was determined by MTT assay. The changes in cell apoptosis were detected AnnexinⅤ-FITC/PI double staining flow cytometry. The variations in radiosensitivity after radiation exposure were assessed by clone formation assay.Results:The expression level of GTPBP4 in the esophageal cancer tissues was significantly higher than that in the normal adjacent esophageal tissues ( P<0.001). qRT-PCR and Western blot demonstrated that the expression levels of GTPBP4 mRNA and protein in the GTPBP4-siRNA group were significantly lower than those in the blank and negative control groups (both P<0.001), suggesting that the plasmid was successfully transfected into the EC9706 cells. MTT assay indicated that the EC9706 cell proliferation rate was significantly inhibited ( P<0.001). Flow cytometry found that the apoptosis rate was significantly increased in the GTPBP4-siRNA group ( P<0.001). After GTPBP4 gene interference combined with radiotherapy, the cell sensitivity enhancement ratio was 1.716. The apoptosis rate of EC9706 cells was significantly increased in the GTPBP4-siRNA group ( P<0.001). The expression levels of apoptosis-associated proteins including cleaved caspase-9, cleaved caspase-3 and Bax were significantly up-regulated, whereas that of Bcl-2 was significantly down-regulated in the EC9706 cells in the GTPBP4-siRNA group ( P<0.001, P=0.001, P=0.001 and P=0.005). Conclusions:GTPBP4 gene is highly expressed in human esophageal cancer tissues. RNAi technology can effectively inhibit the expression of GTPBP4 gene in the EC9706 cells, thereby suppressing cell proliferation, inducing cell apoptosis and enhancing the radiosensitivity of cells.

AIM: To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. METHODS: The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6?his taq (pQE-31), the recombinant expression vect or was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containin g GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF -A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-in duced platelet aggregation. RESULTS: The rvWF-A1 was expressed successfully in E coli, comin g up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA a garose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibi tive rate was 84 7%. CONCLUSION: The above results indicated that high-level expressi on of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effectiv e antithromotic agent in preventing thrombus formation.

Objective To investigate the effect of Puerarin on expression of C-reactive protein(CRP)in iliac artery of atherosclerotic rabbits.Methods Twenty male New Zealand white rabbits were divided into the normal control group(n=6,fed on a normal diet),basic control group(n=8,fed on a cholesterol diet)and Puerarin group(n=8,fed on a cholesterol diet).The balloon injury of iliac arteries was performed in animals of the basic control group and Puerarin group,and the animals of the Puerarin group were injected with Puerarin(25 mg/kg/d per rabbit)after injury.Four weeks after balloon injury,untreated and balloon injury iliac arteries were harvested for immunocytochemical staining.The mRNA and protein expression of CRP were tested by RT-PCR and immunucytochemistry.Results The mRNA and expression of CRP in atheroseclerotic plaque of iliac artery in the Puerarin group was significantly lower than that in the basic control group(P<0.01).Conclusion Puerarin can make atheroseclerotic plaque stable by regulating of the expression of CRP in iliac artery of atherosclerotic rabbit.

ObjectiveTo investigate the long-term therapeutic effect of transjugular intrahepatic portosystemic shunt (TIPS) in patients with intractable cirrhotic ascites and prognostic factors. MethodsA retrospective analysis was performed for the clinical data of 57 patients with intractable cirrhotic ascites who were received TIPS in our hospital from January 2009 to June 2014. Regular telephone follow-up was performed in all patients. Laboratory testing results and abdominal ultrasound and CT findings were examined. The improvement in ascites and survival were evaluated. The χ2 test was applied for comparison of categorical data between groups. The Kaplan-Meier method was used to calculate the cumulative probability of survival and other cumulative probabilities, the log-rank test was used for survival difference analysis, the Cox regression model was used to analyze prognostic factors, and the receiver operating characteristic curve(ROC) and the area under the curve(AUC) were used to determine the optimal cut-off values of prognostic factors. ResultsThe 1-year ascites remission rate after TIPS was 93%, and the 1- and 2-year survival rates were 60% and 43%, respectively. The multivariate Cox regression analysis showed that Child-Pugh score (HR=268, 95%CI: 1.009-1.594, P=0.042) and urea nitrogen (HR=1.143, 95%CI: 1034-1.264, P=0.009,) were predictive factors for 1-year survival rate after TIPS in patients with intractable cirrhotic ascites. The area under the ROC curve of Child-Pugh score was 0.699 (P=0011, 95%CI: 0.558-0.840), and the optimal cut-off value of Child-Pugh score was 8, with a sensitivity of 75% and a specificity of 67%. The Kaplan-Meier survival analysis demonstrated that the 1-year survival rates of patients with Child-Pugh scores of ≤8 and ＞8 were 82% and 38%, respectively (χ2=10.888, P=0.001). ConclusionTIPS is safe and effective in the treatment of intractable ascites, and Child-Pugh score ≤8 is a predictive factor for 1-year survival rate in such patients.

Although liver cirrhosis is the most common cause of portal hypertension (PH), about 20% of PH cases are caused by non-cirrhotic reasons, which are referred to as non-cirrhotic portal hypertension (NCPH), with a high incidence rate in developing countries. NCPH is a group of heterogeneous hepatic vascular diseases, including idiopathic portal hypertension (IPH) and extrahepatic portal vein obstruction (EHPVO), as well as the rare diseases in clinical practice such as Budd-Chiari syndrome, congenital hepatic fibrosis, and nodular regenerative hyperplasia. The patients with NCPH usually have the symptoms of portal hypertension, such as recurrent variceal bleeding and splenomegaly, but liver function is well preserved in these patients. At present, the diagnosis of NCPH lacks a universally accepted standard and remains a challenge. In clinical practice, the method of exclusion is usually applied for the diagnosis of HCPH, and liver biopsy is performed when necessary to make a confirmed diagnosis. This paper introduces the pathogenesis and pathological manifestations of IPH and EHPVO, as well as the selection of diagnostic methods and therapeutic strategies. If upper gastrointestinal bleeding can be effectively controlled, NCPH is considered to have a relatively good prognosis.

Objective: To investigate the effects of c-Met inhibitor AMG-102 on the proliferation and apoptosis of laryngeal squamous carcinoma Hep-2 cells and the underlying mechanism. Methods: Laryngeal squamous carcinoma cell line Hep-2 cells were treated with 2.5, 5 and 10 μmol/L AMG-102, respectively. The proliferation activities of Hep-2 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). The apoptotic rate of Hep-2 cells was detected by flow cytometry analysis and Hoechst staining. The mRNA expression levels of apoptosis-related genes were detected by real-time quantitative polymerase Chain reaction (RT-qPCR), and the protein expressions of c-Met/PI3K/AKT pathway were detected by western blot. Results: Compared with the control group, the proliferation rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 24 hours were (89.8±1.1)%, (79.8±1.0)% and (69.1±1.2)%, respectively; for 48 hours were (76.8±2.0)%, (60.2±1.1)% and (49.8±1.2)%, respectively; for 72 hours were (50.1±2.0)%, (41.5±1.1)% and (33.6±1.0), respectively, with significant differences (all P<0.05). The apoptotic rates of Hep-2 cells treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours were (16.09±1.53)%, (27.51±2.02)% and (36.57±1.42)%, respectively, which were significantly higher than (3.62±0.10) % in the control group (all P<0.05). After treated with 2.5, 5 and 10 μmol/L AMG-102 for 48 hours, the relative expression levels of Bcl-2 mRNA in Hep-2 cells were 0.58±0.13, 0.38±0.12 and 0.20±0.13, respectively; the relative protein expression of p-Met were 80.0±3.8, 50.6±4.2 and 28.5±1.3, respectively; the relative protein expression of p-PI3K were 87.1±0.9, 54.2±1.2 and 21.0±1.2, respectively; the relative protein expression of p-AKT were 98.7±5.6, 56.9±3.2 and 32.2±4.3, respectively; which were significantly lower than those in the control group (all P<0.05). The relative expression levels of Bax mRNA were 1.78±0.13, 2.37±0.14 and 3.05±0.13, respectively, and the relative expression levels of caspase-3 mRNA were 1.98±0.14, 2.47±0.14 and 3.15±0.13, respectively, which were significantly higher than those in the control group (all P<0.05). Conclusion c-Met inhibitor AMG-102 could inhibit the proliferation and induce apoptosis of laryngeal squamous carcinoma Hep-2 cells by regulating the c-Met/PI3K/Akt pathway.

Objective: To investigate the effect of c-Met inhibitor AMG-102 on proliferation and radiosensitivity in laryngeal squamous carcinoma cells. Methods: The effects of AMG-102 on proliferation and radiosensitivity of laryngeal squamous carcinoma cell lines Hep-2 and KBV200 were detected by 3-(4, 5-dimethy-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and colony formation assay, respectively. The apoptosis of Hep-2 and KBV200 cells was detected by flow cytometry. The expression levels of c-Met, phospho-Met (p-Met), cleaved caspase-3 and Akt/p-Akt, Erk/p-Erk were detected by Western blot. Specific small interfering RNA targeting c-Met or plasmid of c-Met were transfected into Hep-2 and KBV200 cells to investigate the cell sensitivity to AMG-102. Results: Compared with KBV200 cells, Hep-2 cells were more sensitive to AMG-102 with IC₅₀ of 14 and 9 μmol/L, respectively. The relative expression levels of c-Met and p-Met proteins in Hep-2 cells were 194.48±0.57 and 177.76±1.53, respectively, which were significantly higher than those in KBV200 cells (171.24±1.00 and 115.37±0.56, respectively, P<0.001 for both). Exogenous hepatocyte growth factor (HGF) was added to increase the expression level of p-Met protein in KBV200 cells. The results showed that AMG-102 significantly reduced the expression of p-Met in KBV200 cells treated with HGF (P<0.001). Compared with the dimethyl sulfoxide (DMSO) group, AMG-102 treatment combined with radiotherapy significantly increased the radiosensitivity of Hep-2 cells (SER=1.28, P<0.001). However, AMG-102 had little effect on the radiosensitivity of KBV200 cells (SER=1.18, P=0.002). Compared with the 4 Gy radiotherapy alone group and the 5 μmol/L of AMG-102 alone treatment group, the apoptosis rate of Hep-2 cells in the combined treatment group was significantly increased. Meanwhile, the expression level of cleaved caspase-3 protein was also markedly increased. However, there were no significant changes in the apoptotic rate and cleaved caspase-3 expression in each treatment group of KBV200 cells. Compared with DMSO treatment group, the expression levels of p-Met, p-Akt and p-Erk were significantly decreased in the 4 Gy radiotherapy group, 5 μmol/L of AMG-102 treatment group and combined treatment group of Hep-2 cells. And the levels of p-Met, p-Akt and p-Erk in the combined treatment group were significantly lower than those in the 4 Gy radiotherapy alone group and 5 μmol/L of AMG-102 treatment alone group. By contrast, in KBV200 cells, the expression of p-Met, p-Akt and p-Erk in each group was not changed. The relative expression of p-Met in Hep-2 cells before and after radiotherapy at 30 min, 1 h, 4 h, 8 h, 24 h were 99.89±0.61, 138.62±1.00, 163.07±5.00, 87.80±1.85, 90.67±0.65 and 94.09±1.41, respectively. The level of p-Met was slightly increased after radiotherapy at 30 min and 1 h (P<0.001 for all), whereas it was significantly decreased from 4 h to 24 h after radiotherapy (P<0.05 for all). By contrast, the expression of p-Met in KBV200 cells did not change with time after radiotherapy (P>0.05). The sensitivity of Hep-2 cells to AMG-102 was decreased after silencing of c-Met, while the sensitivity of KBV200 cells to AMG-102 was not significantly changed (P>0.05). Moreover, the radiosensitivity of Hep-2 cells in c-Met knockdown group had a slightly increasing trend (SER=1.07, P=0.068). After the treatment with 10 μmol/L of AMG-102, the proliferation rate of c-Met ectopically expressed KBV200 cells was 60.05%±3.23%, It was significantly lower than that of the blank control 90.08%±1.04% and siRNA negative control (90.12%±1.01%, P<0.001). The results suggested that the overexpression of c-Met in KBV200 cells increased the radiosensitivity to AMG-102, whereas depletion of c-Met resulted in resistance to AMG-102 in Hep-2 cells. Furthermore, the radiosensitivity of KBV200 cells that overexpressed c-Met showed a decreased trend (SER=0.7, P=0.005). Conclusions c-Met inhibitor AMG-102 has a significant inhibitory effect on the proliferation of c-Met overexpressing laryngeal squamous carcinoma cells, leading to increased radiosensitivity. It suggests that molecular targeted therapy against c-Met receptor is more effective in c-Met overexpressed subtype of laryngeal squamous cell carcinoma.