Aim To investigate the specific binding sites that histone deacetylases 1(HDAC1) and estrogen receptor α(ERα)can be recruited to regulate the transcriptional activity of p21WAF1/CIP1 promoter in the breast cancer MCF-7 cells, and to clarify the molecular mechanism of suberoylanilide hydroxamic acid(SAHA) and leptin regulating p21WAF1/CIP1 promoter function.Methods The breast cancer MCF-7 cells in logarithmic growth phase were starved with FBS free medium for 24 hours, and treated with 20 μmol·L-1 SAHA(SAHA group) or 0.625 nmol·L-1 leptin(Leptin group) for 24 hours.The cells that were cultured in complete RPMI 1640 medium without any treatment were assigned as control group(Basal group).The cell lysis was prepared and incubated respectively with anti-HDAC1 and anti-ERα antibody by chromatin-immunoprecipitation(ChIP) method overnight at 4℃.The DNA-ChIP was followed the manufacturer′s protocol for the assay.DNA fragments binding anti-HDAC1 and anti-ERα antibody were gathered and purified.The relative expression level of DNA fragments from TSS to the upstream of the p21WAF1/CIP1 promoter region(+2~-4 000 bp) binding with antibody was detected by real-time PCR and analyzed by 2-△△CT method.Results In basal group, HDAC1 and ERα had high affinity with the f1 and f8 fragments of p21WAF1/CIP1 promoter compared to the f4 fragment.In SAHA group, the binding ability of HDAC1 and ERα to the f1 and f8 fragments of p21WAF1/CIP1 promoter was significantly lower than that of the control, while reversing to reach the peak after leptin treatment.Conclusions HDAC1 and ERα can be recruited to p21WAF1/CIP1 promoter by the cell proliferation signal during the proliferation of breast cancer MCF-7 cells.The DNA f1(from 0 to-400 bp) and f8(from-2 800 to-3 200 bp) fragment in the upstream of p21WAF1/CIP1 promoter are the target functional region for the binding with HDAC1and ERα.

To clarify the molecular mechanism of SAHA in the cell proliferation of ER-positive breast cancer cell line MCF-7 induced by leptin. Methods Human breast cancer cell MCF-7 was incubated with SAHA and/or leptin, and cell viability, apoptosis and cell cy-cle of MCF-7 cells were detected by Muse Cell Analy-zer. The expression of proteins related with apoptosis was determined by apotosis antibody array. Results Real-time cell proliferation assays indicated that the in-duction effect of leptin for MCF-7 cells reached the peak at a concentration of 0. 625 nmol · L-1 . SAHA reduced the viability of MCF-7 cells, induced G0/G1 phase arrest in the cell cycle, and triggered the apopto-sis. Meanwhile, SAHA significantly induced the pro-tein expressions of some apoptotic factors, including Bax, Caspase-3, TRAIL DR5, p21CIP1, and inhibited the expressions of Claspin, Clusterin, x-linked inhibi-tor of apoptosis protein(XIAP) and survivin. Howev-er, leptin had reverse effects on the related expression of the proteins. Conclusion The effects of cell prolif-eration by leptin and SAHA treatment in breast cancer ER positive cell line MCF-7 may involve in the activa-tion of apoptosis pathway, in particular with releasing of Caspase-3 trigged by endogenous mitochondrial ap-optosis pathway.

Objective To explore the relationship of serum level of Cystatin C and suPAR with tumor infiltration, metastasis, and treatment of patients with malignant tumor. Methods: The serum levels of Cystatin C was detected by particle enhanced nephelometic immunoassay (PENIA) by 7600-010 full-automatic biochemical analyzer made in Japan. The level of suPAR was detected by ELISA. The serum levels of Cystatin C and suPARof 82 normal adults and 172 patients with malignant tumor were measured and compared. Results: The serum level of Cystatin C and suPAR in patients with malignant tumor was significantly increased compared with that of normal adults (P＜0.01 and P＜0.01). The level of Cystatin C and suPAR in terminally ill patients or patients with metastasis was significantly higher than that in the control group. The levels of the two indices in postoperative patients were lower than those in preoperative patients. No significant difference was found in the levels of the two indicies before chemotherapy or radiotherapy and after therapy. Conclusion: The serum levels of Cystatin C and suPARin patients with malignant tumor are correlated with tumor invasion, metastasis and surgical intervention. Detection of Cystatin C and suPAR levels in patients with malignant tumor is valuable for disease monitoring and treatment evaluation.

Aim To investigate the effects of caffeic acid phenethyl ester (CAPE) on activation and proliferation of murine CD3+ T lymphocytes,and to study the mechanisms of its immunomodulative effects. Methods Lymphocytes suspension was isolated and prepared sterilely from murine lymph nodes, and was incubated with different concentrations of CAPE for 2 h, then polyclonal activator Con A was added to activate the lymphocytes; double-fluorescence staining and flow cytometry were used to detect CD69 and CD25 expressions to evaluate activation level of CD3+ T lymphocyte, and CFDA-SE staining plus flow cytometry were used to analyze CD3+ T lymphocyte proliferation. Results CAPE (0.5, 1, 5, 10 mg?L-1) could inhibit the expression of CD69 and CD25 of CD3+ T lymphocytes in a dose-dependent manner, and it also could inhibit T lymphocyte proliferation stimulated by Con A in a dose-dependent manner. Conclusion In vitro, CAPE could exert notably inhibition on activation and proliferation of murine CD3+ T lymphocytes,and its immunomodulative effects might result from affecting PLC-? signaling and MAPK-signaling pathways. So CAPE was a potentially effective immunoinhibitory agent.

The hardship and differentiation degrees are important judgments about test questions.The key purpose about analyzing hardship and differentiation degrees is not only to keep the qualified test questions,eliminate the unqualified and refine the test questions,but also to accumulate valuable data to make exams more scientific,standardized and specified.

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.

AIM: To study the effects of [8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate] (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88?1.88)%. 10, 20 and 40 ?mol/L of TMB-8 inhibited the expression of CD69 (P

AIM: To investigate the expression of B lymphocyte stimulator(BLyS) and CD38 molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus(SLE). METHODS: Twenty-two patients with SLE and fourteen healthy subjects entered the study. Isolated peripheral blood lymphocyte were stained for the lymphocyte surface markers BLyS, CD19, and CD38, and then was measured by flow cytometry(FACS). RESULTS: BLyS + lymphocytes, CD19 + lymphocytes, and CD19 +CD38 + lymphocytes were increased significantly in patients with SLE( P

Objective To explore the association of serum bilirubin level at the time of admission with the compos?ite outcome（disability or death）in discharged patients with acute ischemic stroke. Methods In a retrospective cohortstudy from June 1st 2009 to May 31st 2012, we continuously included 3151 patients with acute ischemic stroke and col?lected demography,lifestyle,clinical manifestations and laboratory test data. Functional outcome was measured with themodified Rankin scale (mRS) when subjects were discharged. Disability was defined as mRS≥3 and composite outcomewas defined as mRS≥3 or death. Serum bilirubin was divided into four groups according to the quartile. Multiple Coxregression analysis was used to assess the independent relation between serum bilirubin and disability death and the com?posite outcome. Results There were 407 disabled patients，the disability rate was 12.9%；and 104 patients were dead，the fatality rate was 3.3%.After adjusting for multiple factors, we found the risks of composite outcome with total bilirubin in the four quartile were higher than that in the first quartile, aHR and 95%CI were 1.335(1.047～1.702) respectively；The risks of composite outcome with indirect bilirubin in the four quartile were higher than that in the first quartile, aHR and 95%CI were 1.355(1.062～1.728) respectively; The risks of composite outcome with bilirubin direct in the third and the forth quartile were higher than that in the first quartile, aHR and 95% CI were11.403(1.089～1.807)and 1.431 (1.118～1.833) respectively.With the increase of total bilirubin，indirect bilirubin and direct bilirubin level，the compos?ite outcome of discharged patient was on the increase. Conclusions The study indicated that higher serum bilirubincould increase the risk of composite outcome in ischemic stroke patients, there was dose-response relationship ,and bili?rubin was a independent risk factor.

Objective To explore the efficacy of triple therapy and sequential therapy in the eradication of Helicobacter pylori (Hp) in patients receiving long-term non-steroidal antiinflammatory drugs (NSAID) treatment.Methods Patients receiving long-term NSAID treatment were enrolled in this study.Patients diagnosed as Hp infection were divided into triple therapy and sequential therapy groups.The patients in triple therapy group received omeprazole,clarithromycin and amoxicillin theray for 10 days.The patients in sequential group received esomeprazole with amoxicillin for five days,and then esomeprazole with clarithromycin and metronidazole for another five days.All patients were given mucosal protective therapy as maintenance treatment after eradication therapy and followed up for 12 weeks.Patients underwent endoscopy examination and Hp testing before and after follow-up.Hp eradication rates were compared with the intention-to-treat (ITT) and per protocol (PP) analysis.Results According to ITT analysis,the eradication rates of Hp in triple therapy group and sequential therapy group were 78.4 ％ (40/51) and 80.0 ％ (40/50) respectively,there was no significant difference between these two groups (x2 =0.038,P=0.846).According to PP analysis,the eradication rates of Hp in triple therapy group and sequential therapy group were 84.4％ (38/45) and 87.0％ (40/46) respectively,there was no significant difference between these two groups either (x2=0.117,P=0.732).Conclusion There was no significant difference in Hp eradication between triple therapy and sequential therapy in patients receiving long-term NSAID treatment.