Objective To construct the pGEX-4T-2-CblN/Grb2, express and purify the corresponding chimeric protein; To investigate whether the chimeric CblN/Grb2 possesses ubiquitin ligase activity. Methods Total RNA of SK-BR-3 cells were isolated and reversely transcribed into cDNA, which were used as templates to amplify Grb2 SH2 by PCR. The gene fragment encoding the N-terminal part of Cbl protein (named as CblN) was amplified by PCR using pEFHACbl plasmid encoding human Cbl as templates. BamH I and EcoR V restriction enzyme digestion sites were introduced into both flanks of SH2 by overlapping extension PCR and the modified gene were cloned into pcDNA3.1(+). The pcDNA3.1(+)-CblN/Grb2 was obtained by replacing SH2 of CblN with Grb2SH2 and then used as templates to amplify CblN/Grb2 by PCR. The pGEX-4T-2-CblN/Grb2 was constructed by subcloning CblN/Grb2 into the prokaryotic expressing vector pGEX-4T-2. The GST-CblN/Grb2 fusion protein was expressed in E. coli of DH5? under IPTG induction and further purified with Glutathione Sepharose 4B. In vitro ubiquitination assay was performed to investigate whether the GST-CblN/Grb2 fusion protein is able to mediate auto-ubiquitinating reaction, namely whether it possesses ubiquitin ligase activity. Results The fusion expressing vector of pGEX-4T-2-CblN/Grb2 was successfully constructed; The GST-CblN/Grb2 fusion protein was correctly expressed and purified; In vitro ubiquitination assay indicated that GST-CblN/Grb2 fusion protein is able to mediate auto-ubiquitinating reaction and therefore possesses ubiquitin ligase activity. Conclusion Expression, purification of GST-CblN/Grb2 and identification of its′ activity have laid the foundation for further study of chimeric ubiquitin ligase′ effects on the growth of HER2 positive tumor cells.

Objective To evaluate the clinical value of medical thoracoscopy in the diagnosis of exudative pleural effusions with unknown etiology.Methods We analyzed retrospectively the clinical data of 180 patients with undiagnosed pleural effusions who underwent medical thoracoscopy and appraised clinical value regarding validity,effectivity and applicability of thoracoscopy.Result In 180 cases of pleural effusion,there were 178 cases of patients with a clear diagnosis,including 87 cases of tuberculous pleurisy,28 cases of malignant tumor,malignant breast bildes mesothelioma in 2,lung cancer in 24,bile duct carcinoma in 1,cercival cancer in 1),pneumonia 58 case fo pyothorax,5 caseso of pyothorax,and 2 cases withou clear diagnosis.No check failure and terminate,the 95％CI was 5 cases of 97.78％-99.98％.Conclusions Medical thoracoscopy is a safe and effective method in the diagnosis of exudative pleural effusions with unknown aetiology.The early application of this method is very helpful for the management of the pleural diseases in suitable patients.

Objective To study the effects of cinobufotalin capsule combined with transcatheter arterial chemoembolization in patients with hepatocellular carcinoma.Methods 68 patients with hepatocellular carcinoma from January,2013 to March,2016 were selected and divided into the combination group and chemotherapy group according to the random number table method,each with 34 cases.The combination group was treated with transcatheter arterial chemoembolization and hepatic artery infusion chemotherapy embolization.The levels of D-dimer,tumor markers,clinical efficacy and quality of life scores were observed before and after treatment in 68 patients.Results There was no significant difference in plasma fibrinogen(FIB)in combination group before and after treatment.Before treatment,the levels of FIB)was similar to chemotherapy group.After treatment,the FIB content in the chemotherapy group was significantly higher than before treatment and that in the combination group the differences were statistically significant(P<0.05).After treatment,the levels of cytokeratin 19,carcinoembryonic antigen and neuron-specific enolase in two group were significantly higher than those before treatment(P<0.05).But there was no significant difference between the two groups after treatment.The rate of disease control in the combination group was significantly higher than that in the chemotherapy group(77.5％vs.65.4％,P<0.05).Cases of diarrhea and insomnia in chemotherapy group were similar to the combination group,but the differences of cases of pain and fatigue between two group were statistically significant(P<0.05).Conclusion Cinobufotalin capsule combined with transcatheter arterial chemoembolization can improve the hypercoagulability of the patient's blood,and is beneficial to the treatment of cancer,improve the disease control rate and improve the life of patients.

Objective To investigate the predictive value of 4 183 Da peptide of dermcidin protein in the early diagnosis and differential diagnosis of ischemic heart disease.Methods A prospective controlled study was conducted.Serum samples were drawn from 161 patients with acute coronary syndrome [ACS,including 46 patients with unstable angina (UA),23 with acute non-ST elevation myocardial infarction,and 92 with acute ST segment elevation myocardial infarction],111 subjects for routine physical examination,including 45 patients with hypertension history,42 with coronary heart disease,22 with diabetes,and 54 patients with non-ACS (including pulmonary embolism,aortic dissection aneurysm,arrhythmia,myocarditis,coronary myocardial bridge,pleurisy,pneumothorax,pneumomediastinum,rib fracture,reflux esophagitis,peptic ulcer,and pancreatitis) to serve as controls.4 183 Da peptide of dermcidin protein was assessed with matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology,and myeloperoxidase [MPO,determined by point-of-care testing (POCT) and enzyme linked i mmunosorbent assay (ELISA),respectively],high sensitive C-reactive protein (hs-CRP),heart type fatty acid binding protein (H-FABP),myoglobin (MYO),cardiac troponin Ⅰ (cTnⅠ),and MB isoenzyme of creatine kinase (CK-MB) were quantitated with biochemical analysis.The power of the biomarkers above for early diagnosis and differential diagnosis for ischemic heart disease were judged by comparison of their sensitivity and specificity.Results ① It was showed by one-way ANOVA that 4 183 Da peptide was higher in ACS group than that in control group (relative abundance:22.05 ± 16.97 vs.15.52 ± 14.09,P =0.001),but no difference was found between ACS group and non-ACS group (relative abundance:22.05 ± 16.97 vs.19.99 ± 17.63,P =0.416).② The specificity and sensitivity of the 4 183 Da polypeptide and MPO for predicting ACS and UA were compared with the receiver operating characteristic curve (ROC).It was showed that the 4 183 Da polypeptide had predictive values for ACS and UA,and the areas under the ROC curve (AUC) was 0.625 and 0.651 (both P ＜ 0.01),but MPO was not found to have predictive value (AUC was 0.440 and 0.336,respectively,both P ＞ 0.05).③ It was showed by the values of multi-markers in differential diagnosis of ACS and non-ACS disease that the specificity and sensitivity of 4 183 Da peptide in the differential diagnosis of acute myocardial infarction (AMI) and non-ACS disease were less than those of MYO,cTnⅠ,H-FABP,markers of myocardial damage,which AUCs were 0.569 vs.0.796,0.833,0.838,and equal to MPO (POCT/ELISA) and hs-CRP,AUC of which was 0.569 vs.0.505 (POCT)/0.477 (ELISA) and 0.545.But both the value of 4 183 Da peptide and MYO,cTnⅠ,H-FABP in the differential diagnosis of UA and non-ACS disease was not found,where AUC was 0.456,0.525,0.658,0.568.Conclusion 4 183 Da polypeptide,a fragment of dermcidin protein,may have association with the onset of ischemic heart disease,and may be helpful in the early diagnosis of ACS.

Objective: To investigate the effect of atorvastatin on plasma microRNA-143/145 expression in patients with stable angina pectoris (SAP).
Methods: A total of 74 SAP patients taken atorvastatin at ifrst time were enrolled in this study, the patients were assigned into 2 groups by the dose of medication:Low dose group, the patients received atorvastatin 20 mg/day, n=36 and Moderate dose group, the patients received atorvastatin 40 mg/day, n=38. Plasma levels of LDL-C and microRNA-143/145 were examined before medication and at 1 month, 12 months after medication respectively. The patients were further divided into another 2 groups by plasma levels of LDL-C:Reach the standard group, plasma LDL-C<2.60 mmol/L and Not reach the standard group, plasma LDL-C≥2.60 mmol/L. Plasma levels of microRNA-143/145 were measured and compared between 2 groups.
Results: ① Compared with baseline condition, plasma levels of microRNA-143/145 were increased in both groups after medication, P<0.001 the levels in Moderate dose group were higher than those in Low dose group at both 1 month and 12 months of time points.②Plasma levels of LDL-C were decreased with medication in both groups, P<0.05. Micro RNA-143/145 expression was similar between Reach the standard group and Not reach the standard group, P>0.05.
Conclusion: Atorvastatin could up-regulate plasma microRNA-143/145 expression, which was not related to lipid-decreasing effect.

Aim To investigate the effects of diallyl di-sulfide( DADS) on G2/M arrest in Chk1/MGC803 and Chk2/MGC803 cells so as to establish stable human gastric cancer MGC803 cells with overexpression of Chk1/2 gene. Methods The colony formation, flow cytometry, RT-PCR and Western blot were used to de-tect the proliferation, cell cycle, and expression of Chk1/2 mRNA and protein, p-Chk1/2, CDC25C and cyclinB1, respectively. Results The colony formation showed that the colony forming efficiency in Chk1/MGC803 and Chk2/MGC803 cells treated by 30 mg· L-1 DADS was lower than in control group and vector group ( P <0. 05 ) . Flow cytometry demonstrated that 41. 3%, 57. 4%, 68. 9% and 42. 9% of G2/M cells in Chk1/MGC803 were increased than in MGC803 and Chk2/MGC803 , respectively after treated by DADS in 12,24, 36 and 48 h(P <0. 05). At the same time, RT-PCR disclosed that expression of Chk1 and Chk2 mRNA had no marked change. Western blot showed that total proteins of Chk1 and Chk2 and p-Chk2 had invisible change, but expression of p-Chk1 was up-reg-ulated, and CDC25C and cyclinB1 were down-regula-ted time-dependently in Chk1/MGC803 cells ( P <0. 05 ) . Conclusion DADS arrests MGC803 cells at G2/M by increasing p-Chk1 expression to cause down-regulation of CDC25C and cyclinB1 simultaneously.

Objective To investigate the potential mechanism that melatonin at higher concentrations inhibits the proliferation of human MG-63 osteosarcoma cells,so as to provide a certain experimental basis for the better application of melatonin in the treatment of diseases in Department of Orthopedics. Methods MG-63 cells cultured in vitro were treated with melatonin at a concentration of 4 mmol/L . Western blotting and real-time PCR method were used to analyze the effect of melatonin on the expression of cyclins and CDKs at protein and mRNA levels ,respectively. Results Western blotting and real-time PCR analyses showed that melatonin's inhibitory effect was possibly through the downregulation of cyclin D1 and CDK4 that related to the G1 phase,and downregulation of cyclin B1 and CDK1 that related to the G2/M phase. However,there was no obvious dif-ference of cyclin E,CDK2,and cyclin A,which were related to G1/S transition and S phase. Conclusion Melatonin may significantly inhibit hu-man osteosarcoma cell proliferation by inducing cell cycle arrest in a time-dependent manner,which is related to the downregulation of cyclin D1, CDK4,cyclin B1 and CDK1.

Objective To explore the correlation between CT characters and epidermal growth factor receptor (EGFR) gene mutations in lung adenocarcinoma. Methods Two hundred and three lung adenocarcinoma patients (from September,2014 to March,2015) confirmed by pathology were divided into effective mutation group (97 cases) and non?effective mutation group (106 cases) on the basis of the site and the response to tyrosine kinase inhibitors. Among all the CT characters, rank?sum test was adopted to analyze the difference of diameter between the two groups; Fisher's exact test was applied to explain the difference of density type and Chi?square test was applied to analyze the difference of lobulation, spiculation, necrosis, pleural indentation, cavitation and air?brochogram signs. Logistic regression was used to analyze the significant signs and evaluate the odds ratio (OR). Results There were 65, 67, 45, 74 cases of lobulation, spiculation, necrosis, pleural indentation in the EGFR gene effective mutation group and 56, 51, 26, 61 cases in non?effective mutation group with statistically significant difference between the two groups (χ2=4.230, 9.141, 10.646, 7.986, P<0.05). However, there were no significant difference between the two groups in the diameter, density, cavitation and air?brochogram (P>0.05). Logistics regression analysis showed that, spiculation (OR=2.120), necrosis (OR=2.853) and pleural indentation (OR=2.094) were in correlation with EGFR effective mutations, and lobulation was not in correlation with EGFR effective mutations. Conclusions Among all the CT sings, spiculation, necrosis and pleural indentation were in correlation with EGFR gene effective mutation, they were more likely to appear in lung adenocarcinoma patients with EGFR gene effective mutation.

Objective To analyze the profile of dermcidin (DCD) changes in different stages of acute coronary syndrome (ACS) by quantifying the serum 4 183Da DCD peptide fragment deriving from different ACS patients treated with early antithrombotic therapy.Methods A total of 118 patients with confirmed diagnosis of ACS were enrolled. Immediately after visiting a doctor, the venous blood was collected and afterwards instantly the patient was given orally 300 mg of aspirin and 300 mg clopidogrel, and according to the patient's condition and the consent of his/her or acknowledgement of family members achieved, emergency percutaneous coronary interference (PCI) or thrombolysis or conservative treatment was adopted separately. After anti-thrombotic treatment, at 2, 4, 6, 8, 10, 12, 16, 20, 24, 32, 40, 48, 60 and 72 hours, venous blood was collected and serum isolated respectively. The concentration of 4 183Da DCD fragment in serum was determined by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Simultaneously, the myoglobin (Myo), cardiac troponin I (cTnI) and MB isoenzyme of creatine kinase (CK-MB) were also detected.Results The mean relative strength of nature logarithmic transformations of 4 183Da DCD fragment of 118 patients with ACS was 2.75±1.02 before treatment on admission, and after intervention therapy (mainly antithrombotic therapy) it was decreased to 1.84±1.19 (P = 0.005) and 1.74±1.12 (P = 0.000) at 2 hours and 4 hours, respectively, and then after 4 hours it was slightly elevated. 4 183Da polypeptide increased earlier than myocardial injury markers.Conclusion Aspirin and clopidogrel can significantly decrease the concentration of 4 183Da DCD peptide fragment in serum in patients with ACS, which indicates that the DCD fragment could be used as one of the indexes for observation on early efficacy of antithrombotic therapy.

Objective To explore the correlation of oral chemotherapy adherence and illness perception among colorectal cancer patients at different stages of chemotherapy in order to provide evidence for improving oral chemotherapy adherence among colorectal cancer patients in clinical practice.Methods A prospective longitudinal study was employed.Convenience sampling was used to recruit 132 cases of colorectal cancer patients.Data of general information,oral chemotherapy adherence and illness perception were collected by questionnaire.Results At different stages of chemotherapy,patient's score of oral chemotherapy adherence were 7.24±1.15,6.97±1.43,6.95± 1.17;patients' illness perception scores were 40.12±9.47,39.91±8.65,37.01±6.83.Oral chemotherapy adherence was negatively correlated with illness perception (r=-0.225,P＜0.05;r=-0.279,P＜0.01;r=-0.277,P＜0.01).Conclusion Higher level of patient's illness perception was related to worse oral chemotherapy adherence.Nurses should instruct patients to understand the disease and the treatment correctly,to reduce the adverse impact on patients' life and emotion,and to improve patients' compliance with oral chemotherapy.