OBJECTIVE:To investigate the effect from exposure to 1,4-benzoquinone (1,4-BQ) and of low expression of calcium/calmodulin dependent protein kinase 2 beta (Camk2b) on mitochondrial toxicity in K562 cells. METHODS:Expression of Camk2b mRNA in K562 cells was determined after 1,4-BQ exposure. Then,the appropriate concentrations of Camk2b inhibitor,KN93,was selected. The gene and protein expressions of Camk2b were verified by real-time PCR and Western blot,respectively,and the low expression of Camk2b was successfully constructed. After K562 cells were exposed to 0,10,and 20 μmol/L 1,4-BQ,the following assays were conducted:cell proliferation,reactive oxygen species,ATP production,mitochondrial membrane potential,calcium ion and apoptosis. The same assays were conducted in K562 cells and control cells which showed low expression of Camk2b. RESULTS:Expression of Camk2b mRNA in K562 cells was reduced after 1,4-BQ exposure. Compared with the control cells,significant changes of the followings were detected:reduction of the ATP production of Camk2b low expression cells,reduction of the mitochondrial membrane potentials,increase of the calcium ion concentrations and increase of the apoptotic rates (all P < 0.01). After 1,4-BQ exposure and compared with the same concentrations of 1,4-BQ-treated control cells,significant changes were observed:increase of the ROS level of Camk2b low expression cells,reduction of ATP production,decrease of mitochondrial membrane potentials,increased of the calcium concentrations and increase of the apoptotic rates (P < 0.05 or 0.01). CONCLUSION:After the K562 cells were exposed to 1,4-BQ,expression of Camk2b mRNA was reduced. Compared with the control cells,Camk2b low-expression cells showed more obvious mitochondrial damage after the 1,4-BQ exposure and Camk2b was apparently important for antioxidant damage to these cells.

OBJECTIVE:To explore the effects of 2-acetyl-4-hydroxy-butylimidazole(THI) on cellular immune functions in mice. METHODS:BALB/c female mice were exposed to THI for 7 and 30 days. The doses were 0,0.5,2.5,12.5 mg/kg and 0,0.2,0.5,2.5 mg/kg,respectively. After the end of the exposure,peripheral blood leukocyte differential count,T cell proliferation function test and NK cell activity test were performed. Flow cytometry was used to detect T cell number and lymphocyte subset changes in thymus and spleen. Transwell chamber method was used to detect T cell chemotaxis. RESULTS:The results show that after exposure for 7 days,the number of peripheral blood leukocytes was decreased,the numbers of CD4⁺ T cells and CD8⁺ T cells were increased,the NK cell activity was decreased,and the T cell chemotactic function was impaired in the 2.5 and 12.5 mg/kg dose groups. The number of peripheral blood lymphocytes was decreased,the number of thymic T cells was increased,and the T cell proliferation function was decreased in the 12.5 mg/kg dose group. After exposure for 30 days,the number of peripheral blood leukocytes was decreased,the number of thymic T cell was decreased,the T cell proliferation function was decreased,the NK cell activity was decreased and the T cell chemotactic function was impaired in the 0.5 and 2.5 mg/kg dose groups. CONCLUSION:THI inhibited cellular immune function in BALB/c female mice,and caused lymphopenia in the blood. These effects also showed dose and time response relationships.

OBJECTIVE:To construct red-green fluorescent protein dual signals which are regulated by RNR3 promoter in yeast cells for rapid screening of chemical mutagens. METHODS:DsRed-Express-2 red fluorescent protein was amplified by PCR from YIPlac204TC-NLS-DsRed-Express-2 and inserted into pGPD vector to construct a yeast red fluorescent protein reporter vector (pGPD-DsRed-Express2),which was driven by the GPD promoter. The method of lithium acetate was used to transform the vector into green fluorescent protein (yEGFP) cells (W303-1A/RNR3-yEGFP),which was regulated by the RNR3 promoter. Thus,red and green fluorescent protein dual signal fluorescing yeast cells were constructed. Red fluorescent protein (DsRed-Express-2) signal was used to normalize the effect of different cell numbers and the "states" on the fluorescence of yEGFP. Fluorescent intensity of red and green fluorescent proteins was measured in 96-well black microplate at 0,4,8,12,16 and 20 hours after the cells were treated with different mutagens. The relative fluorescence intensity (yEGFP/DsRed-Express2) was used to describe the dose-effect relationship between the mutagens and the cells. RESULTS:Actinomycin D and ethidium bromide exhibited negative results among DNA intercalating compounds;methyl mesylate (MMS) and chlorambucil were positive among DNA-alkylating compounds,while mitomycin C was negative;cisplatin,bleomycin and fleomycin were negative among DNA cleaving agents,while 4-nitro-N-oxyquinoline (4-NQO) was negative. Among the inhibitors of polymerases or topoisomerases,5-fluorouracil appeared positive,while hydroxyurea,camptothecin and aphidicolin were negative. As for non-genotoxic compounds such as colchicine,concanavaline and tetracycline,all of them exhibited negative results. CONCLUSION:The recombinant dual-signal fluorescent yeast cells (W303-1A/yEGFP/DsRed-Express2) can possibly be used as a complement to the traditional mutagenic assays,and have the characteristics of fast,convenience and high throughput.

OBJECTIVE:To investigate the effects of resveratrol (RSV) on the genome instability (GIN) and proliferation of human colonic epithelial cell line NCM460 and colon cancer cell line SW620. METHODS:Effects of different concentrations of RSV (3.125,6.25,12.5,25,50,100 μmol/L) on cell proliferation were detected using the trypan blue exclusion method. CBMN-Cyt and colony formation experiments were performed to examine the effects of different concentrations of RSV (25,100 μmol/L) on GIN and clonal formation ability. RESULTS:Compared with the control group,RSV of 6.25-100 μmol/L significantly inhibited the proliferation of SW620 cells (P < 0.05). RSV of 100 μmol/L significantly increased the apoptosis rates,increased the GIN of SW620 cells and decreased the ability of clonal formation (P < 0.05 or 0.01). On the other hand,the same concentration of RSV decreased the GIN of NCM460 cells,enhanced cell division and cell clonality (all P < 0.05). CONCLUSION:The data show that RSV was able to maintain the genomic stability of the normal NCM460 epithelial cells but significantly increased the incidence of GIN in the SW620 colon cancer cells which could be the molecular basis for its excellent anticancer effect and normal cell protection in colon cancer therapy.

OBJECTIVE:To investigate the effect of phenylethanol glycoside liposomes from Cistanche (CPhG) on proliferation,apoptosis and cell cycle of HSC-T6. METHODS:HSC cultures were divided into untreated control and treated with low,medium and high concentrations of CPhG liposomes (7.36,14.72,29.45 μg/mL,respectively). Growth inhibition was detected by using the MTT assay,apoptosis was evaluated by Annexin V-FITC/PI staining and cell cycle changes were detected by flow cytometry. Protein expressions of cleaved caspase-3,p27 were detected using Western blotting. RESULTS:Compared with the control group,all treatment doses significantly inhibited cell proliferation and promoted apoptosis (P < 0.05). In addition,cells were arrested in the G0/G1 phase and showed significant up-regulated expression of caspase-3 and p27 proteins (P < 0.05). CONCLUSION:CPhG liposomes inhibited HSC cell proliferation,induced apoptosis and caused cell cycle arrest by up-regulating the protein expression of caspase-3 and p27.

OBJECTIVE:The aim was to investigate tumorigenic effects of interleukin-17 (IL-17) overexpressing gastric cancer cells in mice. METHODS:MFC,MFC/pcDNA3.1 and MFC/IL-17 cells were inoculated subcutaneously into 615 mice,with 10 mice in each group. Three weeks after the inoculation when tumors became obvious,mRNA and protein expressions of IL-17,vascular endothelial growth factor (VEGF) and Podoplanin in tumor tissues were detected using RT-PCR and Western-blots. Micro-vessel density was detected by immunohistochemistry. RESULTS:Tumor nodules developed on 5-7 d after inoculation and grew rapidly in mice inoculated with MFC/IL-17 (MFC/IL-17 mice). The average tumor weights in MFC/IL-17 mice[(5.384±0.391) g] were significantly more than that in MFC mice[(1.726±0.445) g] and MFC/pcDNA3.1 mice[(2.064±0.397) g] (P < 0.05). In addition,the mRNA and protein expressions of IL-17,VEGF and Podoplanin in MFC/IL-17 mice were significantly higher than that in MFC and MFC/pcDNA3.1 mice (P < 0.05). The micro-vessel densities were significantly increased in MFC/IL-17 mice than that in MFC and MFC/pcDNA3.1 mice (P < 0.05). CONCLUSION:Our data show that expression of IL-17 accelerated tumor growth in vivo by up-regulating the expression of VEGF,Podoplanin and CD31,thus promoting angiogenesis and lymphatic vessel formation.

OBJECTIVE:To screen the key genes and signal pathways in the development of gastric cancer and to provide the basis for finding valuable molecular markers of gastric cancer. METHODS:Five gastric cancer gene chip datasets were downloaded from the GEO database:GSE35809,GSE54129,GSE79973,GSE66229,and GSE51105. The samples in the five data sets were combined,the batch effect between the data sets was removed,the combined gene expression data was normalized,and the data standardization was monitored by principal component analysis. The limma package in the R language was used to screen for differentially expressed genes in gastric cancer tissues and normal tissues. The DAVID database was used to analyze the differential genes in the development of gastric cancer,and the interaction network between the differentially encoded proteins was analyzed and visualized by STRING database and Cytoscape. RESULTS:A total of 1205 differential genes were screened,including 480 up-regulated genes and 725 down-regulated genes. The biological functions of differential genes are mainly enriched in cell-cell signaling,regulation of inflammatory responses,cell adhesion,apoptosis,and transmembrane transport of ions. Analysis of KEGG signaling pathway revealed that differential genes are mainly enriched in the p53,PI3K-Akt and NF-κB signaling pathways. The 29 Hub genes such as CENPE,KIF15,MELK,KIF2C,CENPF,KIF11,NUSAP1,UBE2C,TTK,AURKB,DLGAP5,TOP2A were screened by constructing a protein interaction network. CONCLUSION:By combining different data sets,key genes and signal pathways for the development of gastric cancer were screened by bioinformatics method,providing new research targets for the diagnosis and treatment of gastric cancer.

OBJECTIVE:Apply bioinformatics analyses to explore relationships between hub genes and diagnosis/prognosis of gastric cancer and to possibly enhance molecular diagnosis,targeted therapy and prognosis of gastric cancer. METHODS:Three gastric cancer-associated mRNA expression profiles were down-loaded from the GEO database. The Limma package of R software was used to screen out differentially expressed genes (DEGs) in microarrays. Then,Funrich software was employed to take the intersection of the three expression profiling chips,and further obtain DEGs in all three expression profiles. DAVID (Database for Annotation,Visualization and Integrated Discovery) database was utilized to do the GO function annotation and KEGG enrichment analysis for DEGs,STRING online website and Cytoscape software network analysis,plug-in CytoHubba were applied to construct protein interaction network (PPI) and visual analysis,and then screen out the core genes (Hub Gene). The relationship between core genes and prognosis of patients with gastric cancer was analyzed in KM database. The diagnostic value of core genes with prognostic significance was quantified and visualized by GraphPad software. Finally,Pearson method was used to examine correlations among core genes. RESULTS:In the three datasets,there were 1 839 DEGs,851 up-regulated genes,and 988 down-regulated genes. After screening out overlapping genes,there were 66 genes with significant differential expression in the three expression profiles,consisting of 24 up-regulated genes and 42 down-regulated genes. GO enrichment analyses show that the functions of DEGs were mainly concentrated in extracellular space,extracellular exosomes,digestion,extracellular matrix tissue,and collagen fibrous tissue. KEGG enrichment analyses show that involved pathways were mainly in protein digestion and absorption,gastric acid secretion,nitrogen metabolism,ECM-receptor interaction,mineral absorption. In the PPI network,Cytoscape visual analyses show that differential expression of the 10 core genes was closely related to the occurrence of gastric cancer. KM database searches identify that patients with low expression of FN1 and COL1A1 had better prognosis than those with higher expression. The AUC of FN1 and COL1A1 were 0.93 and 0.90,respectively,indicating that both had high diagnostic values. The correlation coefficient,R=0.59,was obtained through the Pearson Correlation analyses which suggest that the expression of COL1A1 and FN1 in gastric cancer was positively correlated. CONCLUSION:The data show that both FN1 and COL1A1 can potentially be important targets for prognosis,early diagnosis and development of targeted drugs for gastric cancer.

OBJECTIVE:To investigate the expression of interleukin-17 (IL-17) and eqithelial-mesenchymal transition (EMT)-related markers in colon cancers which came from the left-and right-sides of the colon. METHODS:Expressions of IL-17 and EMT-related markers,E-cad and Vim,were determined in colon cancers and adjacent healthy tissues using qPCR. Their relationships with clinical pathological indicators of patients were also determined. RESULTS:The mRNA expression levels of IL-17 and Vim in 43 colon cancers were higher than those in normal tissues but the expression of E-cad was decreased,and the difference was statistically significant (P < 0.05). There was no significant difference in the correlation between IL-17 and EMT in colon cancers (P > 0.05). The expressions of IL-17 and EMT-related markers in colon cancer were not statistically different in gender,age,TNM stage and lymph node metastasis (P > 0.05). The mRNA expression of IL-17 was higher in left colon cancer than right colon cancer (P < 0.05),while the expression of EMT-related markers was not statistically different. CONCLUSION:IL-17 and EMT may promote the development of colon cancers but there was no correlation between them and no correlation with clinicopathological parameters. The mRNA expression of IL-17 in cancers from the left colon was higher than that in the right colon. Therefore,IL-17 may serve as one of the targets for monitoring and treatment of colon cancer (especially left colon cancer).

OBJECTIVE:To investigate the effects of di-(2-ethylhexyl)phthalate (DEHP) on spermatogenic function and expression of p-CREB in testes of mice. METHODS:40 newly weaned ICR male mice were treated with DEHP by gavage in doses of 0,250,500,1 000 mg/kg,6 times a week for 8 consecutive weeks. The weights of testes,epididymis and sperm counts were determined. Histomorphology and expressions of p-CREB of testicle tissues were also analyzed. RESULTS:Results:Compared with the control group,the epididymis coefficient and sperm counting of DEHP treated groups were all reduced significantly (P < 0.05). The testicular coefficients were also decreased in the 1000 mg/kg group (P < 0.05). Pathological alterations of testes were also observed. Reduced seminiferous epithelium,loose and anomalistic organization were observed in all treatment groups. Sperm counts were reduced in the lumina of seminiferous tubules. p-CREB protein expressions were down-regulated in the 500 and 1 000 mg/kg exposure groups (P < 0.05). CONCLUSION:Our data show that exposure in mice to DEHP damaged epithelial cells of seminiferous tubule,decreased the number of spermatozoa and the expression of p-CREB protein.

OBJECTIVE:To establish a method for the biotoxicity detection of paralytic shellfish toxins. METHODS:Cultured mouse neural stem cells (NSC) were exposed to ubunin,resveratrol and different concentrations of standard solution of clam toxins. The in vitro detection method was established based on evaluation of the dose-response relationship between the content of standard liquid and D(450) value of the clams. RESULTS:In culture,the normal NSC started as single cell suspensions which formed small neurospheres in 2-3 days and large neurospheres in the fourth day. After adding ouabain and cucurbitine,the cells showed swelling and rupture. After adding STX,the cells became swollen or ruptured but the cell morphology was mostly normal. Consequently,the in vitro biotoxicity detection of paralytic shellfish toxins was established within the range of 0.2-1.0 ng and a standard curve was established. The regression equation was:y=1.252+0.495x (r=0.998 0,P < 0.01). CONCLUSION:An in vitro method for the biotoxicity detection of paralytic shellfish toxin was successfully established. The method can be further developed for in vitro detection of neurotoxins.