【Objective】 To explore the therapeutic mechanismof arsenic trioxide(AS2O3),the active component of arsenolitumininhibitingtumor.【Methods】Humanlung adenocarcinoma cells(LAC) at logarithmic growth phase were culturedin vitro for 24 hours,and then were cocultured with arsenic trioxide(AS2O3) in the dosages of 0.75,1.0,1.5 and 2.0 mg/Lrespectively.Meanwhile,model control group was set up.The morphological features of LAC in different groups were observed under reverse microscope,growth-inhibitory rate of LAC was examined by methylthiazolyltetrazolium(MTT) assay,andthe expression of p53 gene was detected by enzyme-linked immunosorbent assay(ELISA) method.【Results】AS2O3in different dosages obviously inhibited the proliferation of LACin a time-effect manner,and markedly increased the expression of p53 gene(P

Objective To investigate the effect of folic acid on the DNA methylation of tumorrelated genes promoters in healthy human peripheral blood mononuclear cells(PBMC). Methods Ten healthy volunteers were divided into two groups, and were randomized to receive either 5 mg folic acid (n=5)or placebo(n = 5) , one time per day for 3 months. The serum folic acid concentration was detected with chemiluminescence enzyme immunoassay kit before and after the intervention. The methylation statuses of five tumor-related genes promoter, including oncogenes c-myc, c-Ha-ras,tumor suppressor genes p16INK4A, E-cadherin and mismatch repair gene hMLH1 in PBMC were detected by bisufite sequencing. Results After folic acid intervention, the level of serum folic acid increased significantly in intervention group (t= -4. 739,P<0. 05) , however no significant difference in control group. After three-month folic acid intervention, the level of methylation of oncogene c-myc promoter increased from 4%, 3. 3%, 4. 1% before intervention, one week after intervention, one month after intervention respectively to 8%(t= -4. 079,P<0. 05), while no significant change in placebo taken group. Before and after the folic acid intervention, there was no significant difference of DNA methylation of other tumor-related genes promoter, including c-Ha-ras、E-cadherin、p16INK4Aand hMLH1. Conclusion Folic acid intervention can up-regulate DNA methylation of oncogene c-myc promoter, but can not affect the promoter methylation status of tumor suppressor genes E-cadherin,p16INK4Aand hMLH1.

Objective To study the effects of extracellular-signal regulated kinase mitogenactivated protein kinase (ERK-MAPK) signaling pathway inhibition on histone phosphorylation and the related gene expression in human colorectal cancer cells.Methods Two human colorectal cancer cell lines (SW1116 and HCT116) were cultured and treated with gradient(0,20,40/μmol/L) doses of ERK-MAPK signaling pathway inhibitor U0126.Cell viability was determined by cell counting kit 8 (CCK-8) assay.Cell cycle distribution was assessed by flow cytometry.The expression levels of histone H3 kinases including ribosomal S6 serine-threonine kinase (RSK-2) and mitogen-and stressactivated protein kinase 1 and 2 (MSK1 and MSK2),and the levels of histone H3 (Ser10) phosphorylation and c-Fos protein were detected using Western blotting.Results Treatment of these two human colorectal cancer cell lines with ERK-MAPK inhibitor resulted in a time and dose-dependent inhibition of cell proliferation significantly. Proliferation rate of HCT116 was reduced to 47% at 72 hours after 40/μmol/L U0126 treatment. Cell cycle analysis showed that the percentage of phase G0/G1 cells significantly increased (P<0. 01) and the percentage of phase S cells decreased (P<0.01) after treatment with ERK-MAPK inhibitor. The expression of MSK1 and RSK2 reduced obviously in both of human colorectal cancer cell lines treated with U0126, which resulted in a 28% and 40% reduction of levels of MSK1 and RSK2 as compared with control HCT116 cells respectively,while no detectable change in the expression of MSK2 was found. Consistent with this, the expression level of histone H3 (ser10) phosphorylation was markedly down-regulated by ERK-MAPK inhibitor, and the related protein c-Fos expression decreased accordantly. Conclusions Decreased ERK-MAPK signaling pathway may reduce histone H3 (Ser10) phosphorylation via suppression of the activity of histone H3 kinase including MSK1 and RSK2, but not MSK2, consequently decrease the expression of c-Fos protein, which results in the inhibition of colorectal cancer cells proliferation.

To investigate the invasive ability of the residual tumor cells after immunotherapy and explore the feasible approach suppressing the invasion, mice were inoculated with B16 cells, and then treated by gene therapy with p4-1BBL/psPD-1 or IFN-gamma. The production and activities of MMP-9 and MMP-2 in residual tumor tissues were analyzed with gelatin zymography 1 day and 7 days after the termination of the immunotherapy. The production of MMP-9 and MMP-2 by B16 cells treated with IFN-gamma was also analyzed. IFN-gamma-treated B16 cells were inoculated to mice via subcutaneous injection. The invasion of tumor to muscular tissue was analyzed. Gene therapy with CH50 was used to suppress the invasive growth of tumor. The results showed that the expression and the activities of MMP-9 and MMP-2 were significantly increased 7 days after the end of immunotherapy. The response of tumor cells to ECM molecules was intensified after the removal of IFN-gamma, resulting in significant increase of both the production and activities of MMP-9 and MMP-2, and the increased invasion of tumor. Gene therapy with CH50 effectively suppressed the invasive growth of tumor. It is concluded that the termination of immunotherapy may result in a higher metastatic potential of residual tumor cells. Suppressing tumor invasion by suitable treatment will improve the efficacy of immunotherapy.

Objective To investigate the effects of sinomenine on hind limb ischemia?reperfusion ( I∕R) injury and expression of Bcl?2 and Bax in skeletal muscle cells of rats. Methods Fifty?four healthy adult male Wistar rats, aged 6-8 weeks, weighing 180-220 g, were divided into 3 groups ( n=18 each) using a random number table: sham operation group ( group S) , group I∕R and sinomenine group ( group SIN) . The rats were subjected to 4 h of ischemia on the proximal part of the right hind limb using elastic rubber bands followed by reperfusion in I∕R and SIN groups. Sinomenine 60 mg∕kg was injected intraperito?neally at 30 min before reperfusion in group SIN, and the equal volume of normal saline was given instead of sinomenine at 30 min before reperfusion in S and I∕R groups. Immediately after onset of reperfusion and at 4 and 24 h of reperfusion, blood samples were collected from the cardiac apex to measure the concentra?tions of serum lactate dehydrogenase ( LDH) and creatine kinase ( CK) . The animals were sacrificed imme?diately after blood sampling, and the gastrocnemius specimens of the hind limb were immediately removed for determination of the wet to dry weight ratio ( W∕D ratio) and expression of Bcl?2 and Bax in gastrocnemi?us cells ( by immunohistochemistry) and for examination of the pathological changes after haematoxylin and eosin staining. The Bcl?2∕Bax ratio was calculated. Results Compared with group S, the gastrocnemius W∕D ratio and concentrations of serum LDH and CK were significantly increased, the expression of Bcl?2 was significantly down?regulated, the expression of Bax was significantly up?regulated, and the Bcl?2∕Bax
ratio was significantly decreased in I∕R and SIN groups ( P<0?05) . Compared with group I∕R, the gastroc?nemius W∕D ratio and concentrations of serum LDH and CK were significantly decreased, the expression of Bcl?2 was significantly up?regulated, the expression of Bax was significantly down?regulated, and the Bcl?2∕Bax ratio was significantly increased in group SIN ( P<0?05) . The pathological changes of the gastrocne?mius were significantly attenuated in group SIN as compared with group I∕R. Conclusion Sinomenine can attenuate hind limb I∕R injury, and the mechanism may be related to maintenance of the balance between Bcl?2 and Bax and to inhibition of apoptosis in skeletal muscle cells of rats.

Objective To analyze clinical features and prognosis of primary intestinal T cell lymphoma (ITCL)which was misdiagnosed as Crohn′s disease (CD),and summarized the key points of differentiation between ITCL and CD.Methods From January 2003 to January 2014,clinical data of patients with ITCL once misdiagnosed as CD were retrospectively analyzed,which included demographic,clinical,pathological and prognostic data.The data of 177 patients diagnosed as CD from January 2012 to January 2014 were collected. The demographic,clinical,pathological and prognostic data of these two groups were analyzed and compared. The continuous variables were compared with t test or Mann-WhitneyU test,and the differences of classification variables between two groups were analyzed by Chi-square test or Fisher exact probability method.Results A
total of 18 patients (17 males and one female)with ITCL misdiagnosed as CD were enrolled in the study,and the median age at diagnosis was 38.5 (28.8 to 42.5)years and the median duration of diagnosis was 6.00 (3.75 to 13.25)months.The common primary symptoms were abdominal pain (12/18),diarrhea (13/18)and anemia (13/18).Intestinal perforation was primary symptom in two cases (2/18).However,B symptoms of lymphoma was observed in 16 patients,which included fever in 13 patients,weight loss in 16 patients and night sweat in one patient.One or more serious complications appeared in 12 patients,which included intestinal perforation in nine patients,severe gastrointestinal bleeding in seven patients and intestinal obstruction in two patients.In 177 patients with CD,104 patients were male (58.8%),and the median age at diagnosis was 22.0 (18.0 to 29.0) years.The primary symptoms were abdominal pain (88.7%,157/177),diarrhea (55.9%,99/177),anemia (63.8%,113/177),fever (33.3%,59/177)and weight loss (59.9%,106/177).During the disease course,30 patients (16.9%)had intestinal perforation (mainly chronic),12 patients (6.8%)had intestinal obstruction and seven patients (4.0%)had severe gastrointestinal bleeding.Compared to CD patients,male patients were more common in ITCL (χ2 =8.837,P <0.01),age at diagnosis was older (χ2 =314.5,P <0.01),the disease course was shorter (U=385.0,P <0.01),weight loss (χ2 =5.867,P <0.05)and fever (χ2 =10.609,P <0.01)were more common in clinical symptoms and intestinal perforation and severe gastrointestinal bleeding were more common in complications (χ2 =9.185,24.908,both P <0.01).The lesions of ITCL were multiple lesions, small bowel involved in eight cases,colon involved in 14 cases and one case with esophagus involved.Under endoscopy examination,most lesions appeared as ulcerations and were segmentally distributed.Compared to CD, lymphocyte proliferation was more common in the intestinal histopathological findings of ITCL (17/18 vs 19.7%(35/177);χ2 =42.844,P <0.01)and granuloma was rare (0 vs 42.8%(76/177),χ2 =12.665,P <0.01). Among 18 patients with ITCL,nine received chemotherapy and the median survival time was two months. Conclusions Primary ITCL had non-specific symptoms and was easily misdiagnosed as CD.More attention should be paid to the differential diagnosis of the two disease.

Objective To detect anti-M_2 autoantibody using recombinant BCOADC-E_2.Methods We purified recombinant BCOADC-E_2 by Ni~(2+)affinity chromatography column and then detect anti-M_2 autoan- tibody in the sera of patients with primary biliary cirrhosis(PBC)by Western blot test(WBT)and enzyme linked immunosorbent assays(ELISA).Results Among 60 sera from PBC patients,33 were positive,all of controls were negative.Conclusion The recombinant BCOADC-E_2 can be used to detect anti-M_2 autoanti- body specifically and sensitively.It is helpful for the diagnosis of PBC.

Objective To investigate the cytotoxicity of vascular endothelial growth factor (VEGF) siRNA combined with fusion suicide gene yCDglyTK on human gastric cancer cell line in vitro.MethodsThe gastric cancer cell line SGC7901 was transfeeted with blank plasmid pcDNA3.1 (-) null [pcDNA3.1 (-) group], or VEGF-siRNA expression plasmid pGenesil-shVEGF (SGC7901/shVEGF group),or fusion suicide gene plasmid pcDNA3.1 (-)CV-yCDglyTK (SGC7901/CDTK group),or combined gene plasmid pcDNA3.1 (-)shVEGF-yCDglyTK (SGC7901//shVEGF-CDTK group) with calcium phosphate nanoparticles (CPNPs).Un-transfected gastric cells were set as control group.The stable transfected cells were selected by G418.The target gene expression was verified by RT-PCR and Western-blot.After given prodrug 5-fluorocytosine (5-FC), the biologic characters variation, apoptotic morphology and apoptotic rate of cells in each group were observed through cell growth curve by MTT assays, by-stander effect, Hoechst 33258 staining and flow cytometry.The data was analyzed with SPSS 13.0 software and multiple groups’ comparison was analyzed with LSD test.ResultsFour gastric cancer cells lines transfected with different plasmids were successfully established.The expression of gene yCDglyTK was detected both in SGC7901/CDTK cells and SGC7901/shVEGF-CDTK cells.By MTT assays, the cell growth curve indicated that the A570 value of SGC7901/shVEGF cells, SGC7901/CDTK cells and SGC7901/shVEGF-CDTK cells decreased significantly compared with that of SGC7901 and SGC7901/null cells after a 24-hour 5-FC treatment (P＜0.01).When the percentage of stable gene trasfected SGC7901 cells was 60％, 80％and 100％, the cell relative viability was 13.09％±2.40％, 9.74％±2.83％ and 5.68％±1.03％,respectively. A large number of cells in SGC7901/CDTK and SGC7901/shVEGF-CDTK group appeared typical apoptotic morphology under fluorescence microscope.The result of flow cytometry showed that the apoptosis rates in SGC7901/shVEG group、 SGC7901/CDTK group and SGC7901/shVEGF-CDTK group were 16.40％ ±4.68％, 57.63％ ± 4.96％ and 69.07％ ± 4.69％,respectively, and there were significant differences compared with control (P＜0.01).Conclusion VEGF siRNA combined with suicide gene can effectively kill gastric cancer cell line SGC7901.Apoptosis induction may be one of the important mechanisms of killing tumor cells.

The response of glueagon to the change of glucose and its relation to blood pressure in 71 eidely patients with type 2 diabetes was investigated. The results showed that the postprandial increment of glucagon in the group of patients with postprandial 2h plasma glucose increment<2.5 mmol/L was significantly higher than that in the group with plasma glucose inerement≥ 2.5 mmoi/L (P<0. 05). The postprandial increment of glucagon in patients with normal blood pressure was significantly higher than that in patients with hypertension (P<0.05). The results suggest that the decreased response of glucagon to the change in plasma glucose in elderly patients with type 2 diabetes is related to increased blood glucose and high blood pressure.

Objective To investigate the changes and the clinical significance of serum concentration of soluble Fas (sFas) and Fas ligand (FasL) in the elderly. Methods Fifty elderly subjects and forty-seven adults were recruited. Serum concentration of sFas and EasL were detected by enzyme-linked immunosorbent assay (ELISA) . Psychosocial stress and coping styles were also evaluated. Results The sFas level of the elderly was significantly higher than that in control group,whereas the EasL level was decreased with ageing (P<0.01). A positive correlation between sFas level and psychosocial stress, and a negative correlation between sFas level and positive coping were also found (all P<0. 05). Conclusions Age-related changes occur in serum sEas and EasL levels.The interaction of sFas/FasL system with psychosocial stress and coping styles seems to play important roles in immunosenescence. Our results also suggest that during aging a subtle balance between sFas and FasL could exist.