OBJECTIVE: The aim of this study was to investigate the changes of biomarkers of DNA damage，total p53，phospho-p53 (p-p53) and phospho-H2AX (p-H2AX or γ-H2AX) following exposure of TK6 cells to acetaldehyde and to explore the roles and the sensitivity of p53，γ-H2AX in DNA damage detection by comparison in the indicators of DNA strand breaks (comet assay). METHODS：TK6 cells were exposed to acetaldehyde with different concentrations of 0.5-20 mmol/L for 20 min，2 and 12 h. Flow cytometry was applied to investigate the changes of relevant biomarkers of DNA damage. Alkaline comet assay was used to detect the indicators of DNA strand breaks (percentage of cells with tails，tail length，percentage of tail DNA，tail moment). RESULTS： After exposure to acetaldehyde for 12 h，a significant increase of γ-H2AX expression was observed at acetaldehyde doses of 15 and 20 mmol/L. A significant dose-dependent increase of p-p53 expression rate was found at acetaldehyde dose between 0.5-7.5 mmol/L. In parallel，a similar trend of expression was observed in total p53，but no significant increase was shown. For the comet assay，significant dose-dependent increases of percentage of cells with tails，tail length，percentage of tail DNA，tail moment were obtained after 12 h exposure acetaldehyde dose between 5.0-12.5 mmol/L. 2 h exposure showed that both total p53 and p-p53 expression rates increased significantly at concentrations of 15 and 20 mmol/L of acetaldehyde，and significant increases of percentage of cells with tails，tail DNA，tail moment accurred at the concentration of 20 mmol/L acetaldehyde，but no response was observed in γ-H2AX. For 20 min exposure，no clear trend for all the three biomarkers and no significant change were shown with the indicators of DNA strand breaks. CONCLUSION： Acetaldehyde could induce increase of p-p53 expression rate，γ-H2AX expression amount，percentage of cells with tails，tail length，percentage of tail DNA，tail moment and total p53 expression rate to a less extent. The biomarker p-p53 may be more sensitive reflecting the DNA damage caused by acetaldehyde than γ-H2AX and indicators of DNA strand breaks.

OBJECTIVE: We attempted to investigate the function and regulation mechanism of histone H3 modifications in the toxicity induced by sodium arsenite. METHODS：We constructed histone H3 lysine modified defective cell lines by expressing the site-specific H3 mutation plasmids in HBE cells. MTT assay was used to test the cytotoxicity of these cells when exposed to sodium arsenite. Cytokinesis-block micronucleus (CBMN) assay was conducted to examine the function of H3K4 methylation on the effect of the DNA damage. Meanwhile，the HBE cells were treated with sodium arsenite and the mRNA levels of H3K4 methyltransferases and demethyltransferases and the protein levels of the H3K4 methylation were measured by qRT-PCR and Western blot assay to explore the regulation of H3K4 methylation when exposed to sodium arsenite. RESULTS：We found that the cell vitality was reduced about 60% and the rate of cytokinesis-block micronucleus increased more than 2 times when H3K4 methylation defective HBE cells were exposure to low concentrations (1 μmol/L) of sodium arsenite (P<0.01). The reduction of H3K4 methylation could increase the sensitivity of HBE cells treated with sodium arsenite. We also found that H3K4me2/3 were hypermethylated when exposed to 1 μmol/L sodium arsenite and H3K4 demethylase (lysine-specific demethylase 1，LSD1) was decreased in this process (P<0.05). CONCLUSION：The increased H3K4 methylation might be regulated by reduced LSD1，which may be involved in the cytotoxicity and genotoxity induced by sodium arsenite.

OBJECTIVE: To screen the aberrant methylation genes in esophageal squamous cell carcinoma for Kazakh ethnic group in Xinjiang. The aberrant methylation pattern would provide a clue for in-depth study on esophageal squamous cell carcinoma mechanism. METHODS：We used Illumina Human Methylation 450K chip to carry out the genome-wide methylation screening on 6 cancer tissues and 6 adjacent normal tissues of esophageal squamous cell carcinoma in Kazakh people， to explore aberrant the methylation genes. Genome-wide sequencing were carried out on 2 cancer tissues and 2 adjacent normal tissues by Hiseq2000. Meanwhile，mRNA database was established. With the association study between the methylation profile and expression profile，aberrant DNA methylation genes were identifed and were uploaded to the GoMiner and the KEGG database，completing the bioinformatic analysis. RESULTS：There were 227 hypermethylated genes and 6 hypomethylated genes in cancer tissue，mRNA expression varied from 0.031 2 to 8 192 in cancer tissues and from 0.031 2 to 1 024 in adjacent normal tissues. The association study indicated that there were 10 loci，10 down-regulated genes hypermethylated in promoter region in the negative group. Additionally，there were 11 loci，10 up-regulated genes in the negative group. Using GoMiner to do GO analysis on aberrant DNA methylation genes，revealed that ALDH1L1 ralated to folinic acid catabolism and CAPN1 was associated with positive regulation of cell proliferation. During pathyway analysis，we found that ALDH1L1 was involved in one carbon metabolism and CAPN1 participated in the apoptosis process. CONCLUSION：The aberrant DNA methylation profiles were estabilsed and provided a clue for further studies on ESCC of Kazakh ethnic group.

OBJECTIVE: To analyze the effect of exogenous ATP exposure on the expression of phenotype factors in thymic regulatory T cells(Treg). METHODS：Thymocytes were separated and exposed to 0，0.01，0.1，0.5， 1 mmol/L of exogenous ATP in vitro，then at 72 h the percentage of Treg in thymocytes and their expressions of FOXP3，CD39 were analyzed using FACS. RESULTS：Both the number of thymocytes and the percentage of its Treg subset decreased，particularly in 1 mmol/L ATP exposure groups(t=5.260，P=0.034) and the mean fluorescence intensity of both FOXP3 and CD39 increased in the higher dosage ATP exposure group(0.5 and 1 mmol/L). CONCLUSION：Treg cell numbed decreased but the specificity and function-associated phenotypes of the viable Treg were enhanced after exposure to higher concentration of ATP.

OBJECTIVE: To investigate the expression of sex determining region Y-box 2(SOX2) in gastric cancer and explore the role of SOX2 in the development of gastric cancer. METHODS：Total RNA was extracted from 52 gastric cancer tissues and normal gastric mucosa tissues and reverse transcribed into cDNA. Real-time fluorescence quantitative RT-PCR was performed for evaluation of the expression of SOX2 mRNA in these tissues. RESULTS：SOX2 expression in well-differentiated and moderately differentiated gastric cancer tissues were higher than that of poorly differentiation(t=3.09 and t=2.87，P<0.05). Gastric cancer tissues showed higher SOX2 expression of invasion(T1+T2) (t=2.16，P=0.037)，lymph node metastasis (t=2.54，P=0.017) and Ⅰ-Ⅱ clinical stages (t=2.59，P=0.014) compared to deeper invasion(T3+T4)，lymph node metastasis and Ⅲ-Ⅳ clinical stages. There was no significant difference in SOX2 expression in the cancerous tissues of patients with different sex or age (P>0.05). CONCLUSION：Low SOX2 expression might be associated with the development，invasion and metastasis of gastric cancer and may potentially be used as marker for diagnosis and predicting the prognosis of gastric cancer.

OBJECTIVE: To establish a mitoxantrone-resistant MCF-7 breast cancer cell line，and to explore the relationship between breast cancer cells developing drug resistance and their genomic methylation levels. METHODS：Using cells infected with 0，0.005，0.010 and 0.020 μmol/L mitoxantrone，different levels of drug resistance of MCF-7/Mit cell line was established. Flow cytometer was used to detect the accumulation of mitoxantrone in MCF-7 cells to confirm drug resistant cell lines. Then，we used capillary electrophoresis to evaluate genomic DNA methylation levels of wide type and cells with different degrees of resistance. RESULTS：Compared with the control group，with increasing mitoxantrone concentration，drug accumulation gradually decreased in MCF-7 cells. In the wild-type control group，0.005，0.010 and 0.020 μmol/L -treated groups the drug D(755) values were 16.1±0.04， 14.3± 0.12，13.3±0.07 and 9.7±0.08，respectively. The 0.02 μmol/L exposure group was significantly reduced compared with the control group，indicating that drug resistance was significantly increased (P<0.05). In the wild-type control group，0.005，0.010 and 0.020 μmol/L -treated groups，the DNA methylation levels were 42.25%± 0.64%，37.97%± 1.18%，34.27%±0.14% and 31.16%±0.80%， respectively. Compared with the control group， genomic DNA methylation levels in each group of treated cells gradually decreased (P<0.05)，with significant differences between each two groups (P<0.05). CONCLUSION：Mit drug-resistant MCF-7 cell line was successfully set up. The separation condition of capillary electrophoresis when measuring genomic DNA methylation levels was determined.

OBJECTIVE: To study the significance of arginase-1 (Arg-1) in identifying primary hepatic carcinoma (PHC) and hepatic metastasis (HM). METHODS：Fine-needle aspiration biopsies (FNABs) were performed in 123 patients，cell block were made and immunohistochemical staining were applied. The expression of arginase-1 was measured. All patients received surgical resection and histopathologic diagnosis were obtained. Cytology，serum tumor markers，clinical and radiographic follow-up results were used for a final decision. The sensitivity and specificity of Arg-1 in identifying PHC and HM were calculated. RESULTS：123 patients diagnosed to have PHC (78 cases) or HM (45 cases). High expression was present in PHC group (83.3%) and stained strongly (60.3%). Positive expression rate of highly and moderately differentiated patients (92.9%) were higher than lowly differentiated patients (76.9%)，with no expression in cholangiocarcinoma patients. In the hybrid liver cancer patients the positive expression rate was 75%. In patients with HM，Arg-1 positive expression rate was low (8.9%) with weak staining，and were found in HM from colorectal cancer，pancreatic cancer. HM from other primary sites showed no Arg-1 positive expression. The sensitivity and specificity of Arg-1 in identifying PHC and HM were 83.3% and 91.1%，respectively. CONCLUSION： Arginase-1 may be a more sensitive marker of hepatocellular carcinoma (HCC) and hepatic metastasis. FNABs with Arg-1 immunohistochemical staining was sensitive and specific in identifying PHC and HM and warrant further promotion.

OBJECTIVE: To identify whether the mRNA and protein expression of Zwint-1 v7 existed in human cancer cell line. METHODS：To identify the mRNA with the deleted fragment in HeLa cells，we designed primers on the each side of the deleted fragment for PCR and sequencing. Therefore，we constructed prokaryotic expression vector pGEX-KG-GST-Z7，the inserted fragment Z7 was chosen basen on the different sequences between Zwint-1 and Zwint-1 v7. This vector was transformed into BL21 strain for the expression of fusion protein GST-Z7. After ultrasonication and purified from the SDS-PAGE gels，GST-Z7 was used as antigens and emulsified with adjuvants to immunize C57BL/6 female mice for the preparation of polyclonal antibody Anti-Z7. The serum titer was validated by Dol blot test. The Zwint-1 v7 protein in the total proteins of HeLa cells was evaluated by Western blot with Anti-Z7. RESULTS：The PCR and sequencing results showed mRNA with 37 bp deletions in HeLa cells. SDS-PAGE confirmed that GST-Z7 was correctly expressed in BL21 in the inclusion body. 12 ng GST-Z7 could be detected by the 5 000 times diluted Anti-Z7. Zwint-1 v7 from HeLa cells could be detected by the 200 times diluted Anti-Z7. CONCLUSION：We confirmed that Zwint-1 v7 protein existed in HeLa cells at the levels of translations of nucleic acid and protein structure.

OBJECTIVE: To establish a rapid fluorescence in situ hybridization (FISH) method and to analyze the chromosome aberrations induced by irradiation. METHODS：Human centromeric alpha satellite DNA was amplified and directly labeled with Cy3-dUTP or Fluorescein-12-dUTP by degenerate oligonucleotide priming-PCR (DOP-PCR) to prepare two pan-centromeric probes. Chromosome aberrations in lymphocytes of healthy irradiated peripheral blood samples with 60Coγ-rays were analyzed with FISH. RESULTS：Two directly labeled pan-centromeric probes hybridized to the centromeres of all 46 chromosomes. The established rapid FISH method could easily detect chromosome aberrations after hybridizing 5 h to 12 h in 37 ℃ and simple washing. CONCLUSION： FISH method established in this study could be use to rapidly detect the dicentrics，centric rings，and translocations induced by irradiation.

OBJECTIVE: To construct the lentivirus vector with CYP2E1 gene overexpression，lentivirus was packaged，then transduced into normal human liver cells (L02 cells)，finally the CYP2E1 overexpressing cells (CYP2E1 OE cells) were constructed and identified. METHODS：Primers were designed according to cDNA sequence of CYP2E1 gene from GenBank. The gene was amplified using PCR and ligated into the lentiviral vector pLVX-acGFP-C1. 293FT cells were transfected with the recombinant vector，viral supernatant was collected，L02 liver cells were then transfected. After puromycin screening，L02 cells with CYP2E1 gene transfection were constructed. Finally，gene sequencing，real-time fluorescent quantitative PCR and western blot assays were performed for identification. RESULTS：The sequence contained in the recombinant vector was exactly the same as CYP2E1 gene from GenBank. CYP2E1 gene expression level in L02 cells with CYP2E1 gene transfection was 273 times higher than that of normal L02 cells. CYP2E1 protein level in L02 cells with CYP2E1 gene transfection was 3.2 times higher than that of normal L02 cells. CONCLUSION：CYP2E1-overexpressing cell strain was successfully constructed by using lentiviral vector，this cell strain expressed CYP2E1 with high efficiency，and could become a useful tool for metabolism research of environmental or occupational pollutants.

OBJECTIVE: To explore the association between nonsyndromic cleft lip with or without cleft palate (NSCL/P) and genetic polymorphism of 8q24 rs1530300 in Chinese Han population located in Guangdong province. METHODS：Blood samples from 168 NSCL/P patients and 127 unrelated healthy individuals of the Chinese Guangdong population were collected. DNA was extracted and high resolution melting (HRM) was used to identify single nucleotide polymorphism of rs 1530300 in all samples. Chi square test was used to analyze the genotype and allele distribution between case group，father group，mother group and control group. Transmission-disequilibrium test was also carried out. RESULTS：The method for genotyping 8q24 rs1530300 was set up. The genotypic distribution of rs1530300 in case and control group did not deviate from the Hardy-Weinberg equilibrium. There were no significant differences in the frequency distributions of both genotypes and alleles when case group，or father group ，or mother group was compared with control group at the rs1530300 (P>0.05). We found no evidence of allele transmission-disequilibrium at rs1530300 in cleft case-parent trios(P>0.05). CONCLUSION：In our study，the genetic polymorphism of 8q24 rs1530300 was not associated with the development of NSCL/P in Chinese Han population located in Guangdong province.

OBJECTIVE: To study the genetic toxicity of 5-polymethine cyanine(Cy5)，and to provide scientific evidence for its safe use. METHODS：Mice were randomly divided into five groups，including negative group (saline)，positive group (cyclophosphamide，CP) and test groups with different doses of Cy5 (2，20，200 mg/kg). Micronucleus test in mice bone marrow polychromatic erythrocytes，mice sperm shape abnormality test，mice marrow chromosome aberration test and Ames test were used to investigate the genetic toxicity of Cy5. RESULTS：Bone marrow micronucleus test，sperm shape abnormality test and marrow chromosome aberration test，in each Cy5 test group compared with the negative control group and the differences between the test groups were statistically insignificant(P＞0.05)，while each positive group compared with the negative control group was statistically significant (P＜0.05). Ames test showed negative reaction. CONCLUSION：Under the conditions of this experiment，Cy5 has genetic toxic effect was not found in mice.

OBJECTIVE: To study the toxicity of spermiogenesis caused by MC-LR exposure.? METHODS：L4 him-5 strain males were exposed to MC-LR at 0.1，1，10 and 100 μg/L for 48 h. The ability of spermatid activation was measured by testing its rates in vitro. Spermatid size was measured to assess its competitiveness. Expression levels of the related genes were evaluated by qRT-PCR. Male fertility was measured by brood sizes of him-5 males. RESULTS：After 48-hour exposure，the 100 μg/L group showed reduced spermatid size (P<0.01). Exposures to 10 and 100 μg/L reduced the rates of spermatid activation (P<0.01). Expression levels of spe-10 and spe-15 were significantly decreased after exposure. No significant decreases in brood sizes were observed. CONCLUSION：MC-LR may impair the process of spermiogenesis.

OBJECTIVE: To evaluate the teratogenic and mutagenic effects of barley green by traditional teratogenicity test and micronucleus test. METHODS：Acute toxicity test：the method of maximum tolerated dose (15 g/kg) was used；micronucleus test：five groups (1.25，2.5，5.0 g/kg barley green dose groups，positive and negative groups) were divided. Marrow smear of the femur bone was stained after two treatments 24 h apart，and the rate of micronucleus was calculated. Teratogenicity test：five groups (1.25，2.5，5.0 g/kg barley green dose groups，positive and negative groups) were also divided. Rats were treated on the 7th day of pregnancy for 10 days. On the 20th day of pregnancy，the appearance，growth and development of fetal rats were checked. In addition，the skeletal and visceral teratogenic changes were also assessed. RESULTS：Acute toxicity test：the level of oral barley green MTD was greater than 15 g/kg. Micronucleus test：bone marrow cell micronucleus rate in each dose group was not different compared with the negative control group (P> 0.05)，but significantly different compared with the positive control group (P <0.01). Teratogenicity test：the appearance，growth and development，the skeletal and visceral teratogenic changes were not different compared with the negative control group (P> 0.05)，but significantly different compared with the positive control group (P<0.01). CONCLUSION：The teratogenic and mutagenic effects of barley green were not observed.