OBJECTIVE: Quinocetone induced S phase arrest，caused DNA and chromosome damage in HepG2 cells. Our purpose was to screen and analyse the mechanism of S phase arrest induced by Quinocetone. METHODS： The Agilent whole genome gene expression microarray was used to detect gene expression profile of HepG2 cells treated and untreated with Quinocetone. Futhermore，pathway analysis by Genespring software was carried out based on the differentially expressed genes，real time qPCR was used to verify some dramatically changed genes in DNA replication process. RESULTS：1 750 differentially expressed genes，with 446 genes up-regulated and 1 304 genes down-regulated，were found in HepG2 cells treated with Quinocetone in the total of 41 000 oligonucleotide fragments. Pathway analysis revealed that the differentially expressed genes gathered in functional pathways like cell cycle checkpoint，MAPK signaling pathway，DNA replication and DNA repair，etc. Moreover，the results of real time qPCR focused on the changed DNA replication genes were consistent with cDNA microarray. CONCLUSION：The down- regulation of DNA replication genes caused incomplete DNA synthesis，and the DNA repair after its damage. These two events lead to S phase arrest after Quinocetone treatment. Moreover，the whole genome cDNA microarray analysis provided us much useful information for further research on the mechanism of S phase arrest induced by Quinocetone.

OBJECTIVE: To construct eukaryotic expression vectors with FLAG epitope that can highly express human cyclin D1 and cyclin B1 genes，then transiently transfect into human cervical cancer HeLa cell line. METHODS：cDNA of cyclin D1 and cyclin B1 genes from total RNA isolated from HeLa cells was amplified by RT-PCR. After sequencing，the open reading frame (ORF) of cyclin D1 and cyclin B1 cDNA was cloned into eukatyotic expression vector p3XFLAG-CMVTM-14 to form the recombinant plasmid named as p3XFLAG-cyclin D1 and p3XFLAG-cyclin B1. Then eukatyotic expression vectors were transfected into HeLa cells by Lipofectamine 2000. Expressions of human cyclin D1 and cyclin B1 in HeLa cells were measured by Western blot. RESULTS：The result of restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmids were correct. And we found that the human cyclin D1 and cyclin B1 fusion protein，with the right molecular weight，was expressed in transiently transfected HeLa cells. CONCLUSION：Recombinant cyclin D1 and cyclin B1 were successfully expressed，which laid foundation for further studies of these and their related proteins.

OBJECTIVE: To analyze linkage between 8 short tandem repeat (STR) markers of chromosome 12 and Kashin-Beck disease (KBD)in nuclear families. METHODS：The probands in 23 nuclear families and their 90 members diagnosed by national clinical criteria to diagnose KBD，and their blood samples were collected. The types of the 8 STR locus (D12S1613，D12S1725，D12S1663，D12S1697，D12S1675，D12S358，D12S1638 and D12S1682) polymorphisms were measured by gene scan，and gene frequencies and linkage analyses calculated between these polymorphisms and KBD. RESULTS：11，10，5，5，10，8，7 and 11 alleles on 8 STR locus (D12S1613， D12S1725，D12S1663，D12S1697，D12S1675，D12S358，D12S1638 and D12S1682) of chromosome 12 were detected，and the LOD scores of D12S1725 was 2.52. CONCLUSION：The susceptable gene linkage locus of KBD may be close to maker D12S1725.

OBJECTIVE: To compare the cytotoxicity and DNA damage caused by nano- and micro- zinc oxide particles. METHODS: Cultured A549 human lung adenocarcinoma cells were exposed to nano- (30 nm) and micro-scale ZnO particles(≤1 μm) at the concentrations of 25, 50 and 100 μg/ml for 6, 12, 24 and 48 h. In the mean time, DMEM culture medium was set up as control. Morphological changes were examined after exposure, methyl tetrazolium cytotoxicity (MTT) assay and the single cell gel electrophoresis(comet assay) were used to determine cytotoxicity and DNA damage. RESULTS: The IC50 of the nano-ZnO and micro-ZnO on the A549 cells were 53.91 and 190.15 μg/ml, respectively. It was found that nano-ZnO inhibited A549 cells significantly at 50 μg/ml, showing a dose- and time- dependent manner. But micro-ZnO inhibited A549 cells significantly only when the concentration up to 100 μg/ml. The slope of the dose-response curve and time-effect curve of the nano-and micro-ZnO was significantly different. In addition, nano-ZnO particles demonstrated a DNA-damaging potential compared to control group in the Comet assay after an exposure of 6 h. Micro-ZnO did not induce any DNA damage. CONCLUSION: Nano- and micro-ZnO particles both demonstrated cytoxicity in A549 cells, but the toxic effects of nano-ZnO occurred earlier in time and lower in dose compared with micro-ZnO. Nano-ZnO particles could induce DNA damage, but the micro- ones did not.

OBJECTIVE: To observe the changes of liver function and ultrastructures induced by trichloroethylene in sensitized guinea pigs. METHODS： White female guinea pigs (250-300 g) were randomly divided into blank control group， solvent (olive oil) control group and TCE treatment group. Guinea pigs were treated with guinea pig maximization test (GPMT). Specimens were collected at 24 h and 72 h after last stimulation， liver tissue pathology and ultrastructures examined，and AST，ALT，TP，ALB，GLB and A/G in serum measured. RESULTS： Sensitization rates were 65.38% in TCE treatment groups. Compared with TCE sensitized 24 h group and TCE non-sensitized 72 h group， ALT and AST levels in TCE sensitized 72 h group increased significantly(P<0.05). Compared with solvent group and TCE non-sensitized 72 h group， ALB levels in TCE sensitized 72 h group increased significantly(P<0.05). TCE sensitized group 72 h revealed more edema in liver cells with nucleus ruptured and disintegrated. TEM observation showed： a few mitochondria vacuoles， degeneration， rough endoplasmic reticulum decreased in TCE non-sensitized group. TCE sensitized group demonstrated reduction in the number of mitochondria in liver cells， rough endoplasmic reticulum fracture， expansion， decreased glycogen granules. In the group of 72 h after the last challenge，more serious damage was visible than those in 24 h group. CONCLUSION： Liver dysfunction and ultrastructural damage occurred in sensitized guinea pigs induced by trichloroethylene. The extent of injury gradually increase with time.

OBJECTIVE: To study mRNA expression of hepatic metabolic enzyme genes and apoptosis genes in L02 cells after treatment with trichloroethylene (TCE). METHODS：Liver cells (L02 cells) were cultured with various doses of TCE (0.25，0.5，1.0 and 2.0 mmol/L) for 24 h，or with a single dose of TCE (1.0 mmol/L) for different periods of time (3 h，6 h，12 h，24 h)，the control was treated with DMSO. Real-time fluorescent PCR assay was applied for detection of mRNA expression of hepatic metabolic enzyme genes (CYP1A2，CYP3A4，CYP2E1) and apoptosis genes (BAX，BAD，Bcl-2). RESULTS：The relative levels of mRNA expression of hepatic metabolic enzyme genes and apoptosis genes markedly increased after TCE treatment at various doses for 24 h. The elevation of mRNA expression in TCE treatment groups was significant in comparison with control (P<0.05，or P <0.01). Additionally，when the cells were treated with a single dose of TCE (1.0 mmol/L) for different periods of time，the mRNA expression also increased significantly compared with control (P<0.05，or P <0.01). CONCLUSION：Trichloroethylene could induce changes in mRNA expression of hepatic metabolic enzyme genes and apoptosis genes. These genes might play important roles in cytotoxicity of trichloroethylene.

OBJECTIVE: To explore the differences of cytokine-induced killer (CIK) cells from healthy persons and patients with primary cervical cancer，in their phenotype，proliferation activity，cytotoxic activity and antitumor effects in vitro and in vivo. METHODS：CIK cells were generated by IFN-γ，CD3McAb，IL-2，IL-1 induction of cultured peripheral blood mononuclear cells (PBMC) of both 8 healthy blood donors and 8 cervical cancer patients. These treated cells were followed at different intervals by MTT assays and flow cytometry analysis. The antitumor activity of the CIK，LAK and PBMC cells were evaluated in BALB/c nude mice bearing HeLa cervical cancer. RESULTS：There was no significant difference in proliferation performance of CIK cells between healthy persons and cervical cancer patients (P>0.05). The flow cytometry analysis showed that the percentage of CD3+CD56+ cells increased from 0.13% on day 0 to 25.8% on day 28. CIK cells generated from patients with cervical cancer patients possessed a higher antitumor cytotoxic activity in vitro than PBMC. In addition，CIK cells had a stronger suppressive effect on tumor growth in BALB/c nude mice bearing cervical cancer than LAK cells (median inhibitory rates 80.6% vs 59.1%， respectively，P <0.01) or PBMC (median inhibitory rates 80.6% vs 38.3%，respectively，P <0.01). The tumor size in the experiment group was smaller than that in the control group after CIK treatment (P<0.05). CONCLUSION：CIK cells had a significantly stronger suppression on growth of cervical cancer cells，providing an experimental basis for CIK clinical use as an adoptive immunotherapy.

OBJECTIVE: To explore the changes of superoxide dismutase (SOD) activity，DNA damage and apoptosis of cultured rat Sertoli cells after exposure to 2，2’，4，4’，5-hexachlorobiphenyl (PCB153) ，and the effect of N-acetyl-L-cysteine (NAC) pre-treatment. METHODS： Sertoli cells were isolated from 18- to 20-day-old male Sprague-Dawley rats. Contaminated groups were exposed to PCB153 at the doses of 10，20，30 μmol/L. NAC group was pre-treated with 300 μmol/L NAC for 1h，then exposed to 30 μmol/L PCB153. Control group was exposed to DMSO with the volume equal to the highest concentration of PCB153. Then all groups were cultured for 24 h. SOD activity，DNA damage，apoptosis and morphological examination were determined by SOD assay kit，comet assay，flow cytometry and fluorescence photomicrographs，respectively. RESULTS：After 24 h exposure，SOD activity decreased as the PCB153 dose increased，significant difference was observed between 20，30 μmol/L groups and control group. NAC pre-treatment was able to increase the activity of SOD. No significant difference was observed between treated groups and control group for comet assay. There was a significant dose -response relationship between the level of PCB153 exposure and the apoptotic rate，NAC pre-treatment was able to decrease the apoptotic rate. CONCLUSION： PCB153 in the concentration of 10-30 μmol/L was able to decrease the activity of SOD and induce apoptosis in cultured rat Sertoli cells，while DNA damage was not evident. NAC pre-treatment has protective effect on SOD activity and apoptosis.

OBJECTIVE: To explore the association between COX-2 expression in human esophageal cancer and its clinicopathologic characteristics of esophageal cancer. METHODS：Published studies were searched in the CBMdisc(1978-2011)，CNKI(1979-2010)，VIP(1989-2010) database，CMA Digital Periodicals(1998-2010)，other relevant journals were manually searched to identify all the related case-control studies in mainland China. The quality of the included studies was assessed. Cochrane Collaboration’s Software Revman 5.0.2 was utilized to test the heterogeneity， the overall effect and the publication bias of the combined studies as well. RESULTS：Seven studies were included. For the positive rate of COX-2 expression，significant difference was tested between esophageal cancer and normal esophageal tissues[OR=31.44，95%CI(10.47，94.38)]. In addition，cell differentiation G1+G2 vs. cell differentiation G3 revealed significant difference[OR=3.60，95%CI(1.63，7.99)]. However，there was not any significant difference shown in terms of gender，age，depth of invasion，lymph node metastasis，of which the overall effect were 1.56 (0.61，3.98)，0.59(0.27，1.30)、1.21(0.49，2.98)，1.47 (0.38，5.64)，respectively. CONCLUSION：According to the currently available national evidence，higher COX-2 expression may be associated with esophageal cancer and cell differentiation.

OBJECTIVE: To study the effects of baohuoside-I in combination with 5-FU on the proliferation and apoptosis of human esophageal carcinoma cell Eca-109. METHODS：The cells were divided into 4 groups： combination group (12.5 mg/L baohuoside-I and 12.5 mg/L 5-FU)，negative control group (0.1% DMSO)，25 mg/L baohuoside-I group and 12.5 mg/L 5-FU group，with 3 holes in each group and 100 μl each hole (1×104 cells ). After treatment for 24，48，72 h，the inhibitory effect on proliferation of Eca-109 cells was analyzed by MTT. After treatment for 48 h，apoptosis of Eca-109 cells was measured with flow cytometry；the expression of Bcl-2 protein and Bax protein of Eca-109 cells was measured with Western blot. RESULTS：Baohuoside-I，5-FU alone and baohuoside-I in combination with 5-FU inhibited proliferation of Eca-109 cells (P all <0.05)，the inhibitory effect of the combination group was superior to either alone (P <0.05). After treatment for 48 h，apoptosis of Eca-109 cells was induced significantly，the expression of Bcl-2 protein was decreased and Bax protein was increased significantly than control group (P all <0.05). T he combination group showed more significant effect than either alone (P <0.05). CONCLUSION： Baohuoside-I in combination with 5-FU exerted a synergistic effect on inhibiting proliferation and promoting apoptosis. This effect associated with decreased expression of Bcl-2 and increased expression of Bax.

OBJECTIVE: To investigate the expressions of IL-28 and mac-3 in liver of chronic alcoholism mice and withdraw from alcohol. METHODS：The dose of 40% alcohol [8 mg/ (g·d)] was administered via gastrolavage once daily for 12 weeks in Kunming mice. The control mice were given the same volume of saline. The experimental mice were sacrified at the end of 4th，8th，12th week and after withdrawal from alcohol 24 hours. Pathologic changes of liver were examined under light microscope after HE staining，expressions of IL-28 and mac-3 were examined by immunohistochemitry. RESULTS：The tissues of treated mice showed various changes of chronic alcoholic hepatitis at the end of 4，8，12 week，such as fatty degerneration，inflammatory and fibrosis，more severe with longer alcohol administration. The expressions of IL-28 and mac-3 were decreased in alcoholic mice but increased in mice withdrawal from alcohol. CONCLUSION：Alcohol could injure Kupffer cells or inhibit their functions. Withdrawal of alcohol may introduce the rebound of IL-28 and mac-3 expressions or temporary inflammation of liver.

OBJECTIVE: To observe the anticancer effect of the serum thymic factor(FTS) 9 peptide on human colon cancer HT-29 transplantation model in nude mice. METHODS： Nude mice were inoculated subcutaneously with human colon cancer HT-29 cells，grouping was performed when tumor reach a volume of approximately 100 mm3. Tumor-bearing nude mice were divided into 5 groups：FTS 9 peptide high，medium and low dose group (1.25， 0.625，0.312 mg/kg，respectively)，positive control (cyclophosphamide，30 mg/kg) and blank control (saline)，10 mice in each group. Mice were treated subcutaneously once a day for 28 days. Body weight，length and width of tumor were measured twice a week. Mice were sacrificed 24 h after the last treatment，body weight，length，width，volume and weight of tumor were measured，the relative tumor proliferation and inhibition rates were calculated. Experiment was repeated 3 times. RESULTS：Relative tumor volumes of FTS 9 peptide groups were significantly reduced compared with blank control (P<0.01 or P<0.05)，relative tumor proliferation rates of FTS 9 peptide high，medium and low dose groups were 50%± 20%，62%±20% and 77% ± 35%， respectively. Tumor weights of FTS 9 peptide groups were significantly reduced compared with blank control (P<0.01)， inhibition rate of FTS 9 peptide high，medium and low doses group were 45% ± 3%，35% ± 1% and 27 %±3%， respectively. Body weights of nude mice in each group were not significantly different (compared with blank control，P>0.05). CONCLUSION：The results showed that the FTS 9 peptide had an obvious inhibitory effect on transplanted tumor of human colon cancer HT-29.

OBJECTIVE: To study the effect of retinoic acid on expression of nuclear matrix protein in human esophageal cancer cell EC9706， in order to further understanding of the relationship between esophageal carcinogenesis and nuclear matrix proteins. METHODS：Through cell culture and induction of differentiation treatment for 48 h， nuclear matrix protein selective extraction， two dimensional gel electrophoresis analysis to detect the expression of nuclear matrix protein in human esophageal carcinoma EC9706 before and after retinoic acid treatment. RESULTS：The expression of nuclear matrix protein showed great changes after treatment with retinoic acid in EC9706 cells. There were 18 marked changes during apoptosis induced by retinoic acid， with 11 up-regulated proteins，the 4 down-regulated proteins， and 3 new spots. CONCLUSION：This study suggested that the types and expression levels of nuclear matrix protein could change significantly after treatment by retinoic acid in human esophageal cancer EC9706 cells.

OBJECTIVE: To explore the toxicity of sodium hyaluronate on SD rats in sensitive period of teratogenesis. METHODS：Pregnant rats were divided randomly into five groups，three sodium hyaluronate groups (1 333， 667 and 333 mg/kg)，one negative control given deionized water，and one positive control given Aspirin (250 mg/kg). There were at least 12 pregnant rats in each group. During the sensitive period of teratogenesis (the 7th to 16th day of gestation)，the pregnant rats of each group were given test sample orally once a day for ten days. On the 20th day of gestation，the pregnant rats were sacrificed. Embryonic and fetal development were evaluated，appearance，viscesal and skeletal abnormalities were observed. RESULTS：In sodium hyaluronate groups，the weight of pregnant rats and uterus including fetuses，living fetus rate，dead fetus rate，body weight，length of body and tail of fetuses were not significantly different from negative control. No abnormality of skeleton，organ and body was identified in fetuses of groups treated with sodium hyaluronate. CONCLUSION：At dosages used in this study，sodium hyaluronate had neither maternal toxicity nor teratogenecity in pregnant SD rats.

OBJECTIVE: To evaluate genetic toxicity of recombinant human lactoferrin (rhLf) expressed in milk from genetically modified cow. METHODS：In Ames test，TA97，TA98，TA100，and TA102 strains were treated with 62，185，556，1 667 and 5 000 μg recombinant human lactoferrin (rhLf) per plate. In mice bone marrow cell micronucleus test and mice sperm abnormality test，1.88，3.75 and 7.50 g/kg rhLf groups and control groups were set. RESULTS：Back mutation colonies of rhLf groups did not exceed more than twice that of the control in Ames test and there was no dose-response relationship. In mice bone marrow cell micronucleus test，no significant difference was found in PCE/RBC and micronucleus rate between rhLf-treated groups and negative control group. In mice sperm abnormality test，sperm abnormality rate of rhLf-treated groups was lower than that of negative control. CONCLUSION：No genetic toxicity was observed in recombinant human lactoferrin in this study.

OBJECTIVE: To study the teratogenicity of copper-bearing intrauterine device(Cu-IUD) using Sprague-Dawley rats. METHODS： Timed-pregnant rats were divided into 4 groups (24 for each group)，including three treatment groups (0.1，0.3，and 0.9 g/ml，IV) and vehicle control group (NS，IV.). Rats received extract solution of Cu-IUD or vehicle on gestational days (GD) 6 through 15. Body weight and clinical signs of maternal rats were monitored at regular intervals throughout gestation. At termination (GD 20)，pregnant females were evaluated for clinical status and gestational outcome；live fetuses were examined for external appearance，visceral，and skeletal malformation and variations. RESULTS：There were no maternal deaths and no dose-related clinical signs. Maternal weight gain，liver weight，number of corpora lutea，implantation and live fetuses，body length and tail length of fetuses were not affected. IUD did not affect prenatal viability or incidences of fetal malformations or variations. Percent of resorbed embryos in high dose group was significantly higher than that in control group. CONCLUSION：No maternal toxicity and teratogenicity，but toxic effects in early embryo development were found when rats were treated with extract solution of Cu- IUD at a dose of 0.9 g/ml.

OBJECTIVE: To study the genetic toxicity of NiKeAn. METHODS：the mutagenicity of NiKeAn was examined using Ames test， micronucleus test and CHL cell chromosome aberration test. RESULTS： No increase in the number of revertant colonies was found in the Ames test with and without S9 mixture. The micronucleus rates and chromosome aberration rates at all doses were not significantly different from the control group(P>0.05). CONCLUSION：Mutagenicity of NiKeAn was not observed in this study.