OBJECTIVE: To explore the effects of titanium dioxide (TiO2) nanoparticles on Natural Killer (NK) cell activity and related cytokines in rats. METHODS：Sprague Dawley rats were randomly divided into 0.5，4 and 32 mg/kg TiO2 nanoparticles exposure groups，control group (0.9% sodium chloride solution)，and TiO2 micro-sized particles exposure group (32 mg/kg). Rats were treated by intratracheal instillation of these substances in suspension twice a week，a total of 28 consecutive days. NK cell population in peripheral blood was measured by flow cytometry. NK cell killing activity in the spleen was analyzed by lactate dehydrogenase (LDH) release assay. Cytokines IL-2 and INF-γ were measured by liquid phase chips method. RESULTS：The NK cell activity increased with the rise of TiO2 exposure dose. Significant difference in NK cell activity in the spleen was found in the group exposed to 32 mg/kg TiO2 nanoparticles compared with the control group (P＜0.05). No significant difference was found between control group and micro-sized TiO2 group (P＞0.05). Compared with micro-sized TiO2 group，NK cell activity was found to be significantly increased in 32 mg/kg TiO2 nanoparticles exposure group (P＜0.05). No significant change of NK cell populations，IL-2 or INF-γ was observed compared with control group and micro-sized group (P＞0.05). CONCLUSION：TiO2 nanoparticles could trigger systemic immune responses，and enhance NK cell killing activity in the spleen in the studied dosage range. However，further research is needed to confirm the long-term changes in NK cell populations，NK cell killing activity and related cytokines，as well as the potential mechanisms.

OBJECTIVE: To study the effects of hydroquinone(HQ) on monocytic differentiation of HL-60 cells and explore the regulatory role of miR-146a in the mechanism of toxicity. METHODS: Using the model of monocytic differentiation induction with phorbol 12-myristate 13-acetate (PMA)，HL-60 cells were transfected with miR-146a-5p inhibitor or negative control and then treated with various doses of hydroquinone(0，0.5，1.0，2.5 and 5.0 μmol/L) for 3 h，and cells collected after induction with PMA for 72 h. The expressions of miR-146a and its target gene TRAF6，were measured by real-time quantitative PCR. CD11b and CD14 levels were also determined by flow cytometry. Western blot was used to detect protein expression levels of TRAF6，and its downstream regulatory factor IκBα. RESULTS： Hydroquinone could inhibit the expressions of CD11b and CD14 in HL-60 cells induced by PMA. Compared with control group(0 μmol/L)，5.0 μmol/L of hydroquinone decreased the expressions of CD11b and CD14 by 44% and 38%，respectively (both P <0.01)；the expression of miR-146a increased nearly 2-fold (P<0.01). The expressions of TRAF6 mRNA and protein were decreased by 40% and 74%(P<0.01)，respectively. The expression of phosphorylated IκBα protein was reduced by approximately 45%，and the total IκBα protein increased nearly 73% (P<0.01). After inhibiting the expression of miR-146a，there were no significant effects of hydroquinone on TRAF6，phosphorylated IκBα and total IκBα protein expression. CONCLUSION: miRNA-146a may be one of the regulatory factors in which hydroquinone influence HL-60 cells differentiation.

OBJECTIVE: To explore the relationship of Toll-like receptors 2 (TLR2) and TLR4 Thr399Ile gene polymorphism and the susceptibility to EBV-associated gastric carcinoma (EBVaGC) and EBV-negative gastric carcinoma (EBVnGC). METHODS：TLR2 gene promoter region - 196 to - 174 del polymorphism was detected by using the allele-specific polymerase chain reaction in 52 EBVaGCs，157 EBVnGCs and 94 normal controls. TLR4 gene Thr399Ile genotype and allele distribution were evaluated by PCR and restriction fragment length polymorphism (PCR-RFLP) in 50 EBVaGCs，67 EBVnGCs and 71 normal controls. The relationship between gene polymorphism in TLR2 and TLR4 coding genes and the susceptibility to EBVaGC and EBVnGC was analysed. RESULTS：There was no significant difference (χ2=3.180，P=0.075) between gastric cancer group and control group in genotype distribution of TLR2(-196 to -174 del)，but the del allele frequency of gastric cancer group was obviously higher than that of control group (χ2=4.875，P=0.027)，The risk of developing gastric carcinoma in del allele carriers was significantly higher than that in controls(OR=1.491，95%CI= 1.045-2.126). TLR2 (-196 to -174 del) genotype and the frequency of del allele showed no significant difference in EBVaGC and EBVnGC group (χ2=0.05，P=0.867). TLR4 gene Thr399Ile locus mutation was not found in EBVaGC，EBVnGC or normal control groups. The genotype distribution and allele frequency also revealed no statistical differences . CONCLUSION：TLR2 -196 to - 174 del alleles could be risk factors for gastric carcinoma，but its effects were not different in EBVaGC and EBVnGC. There was no obvious correlation in the genotype distribution of TLR2(-196 to -174 del) and TLR4 gene Thr399Ile and EBVaGC genetic susceptibility.

OBJECTIVE: To explore the effects and mechanisms of oxymatrine on anti-influenza A virus (IAV) activity. METHODS: MDCK and A549 cells were cultured and divided into 4 groups: blank control (DMSO， 0.5%)， virus-treated group，positive group (ribavirin，200 μmol/L) and oxymatrine-treated group (12.5，25，50 and 100 μmol/L) ，with 6 repeats in each group. The anti-IAV activity of oxymatrine was determined by plaque inhibition assay. The influence of oxymatrine on the IAV-induced transcription of TLR4，MyD88 and TRAF6，the activation of TLR4-Myd88-TRAF6-NF-κB signal pathway，and the release of inflammatory cytokins were determined by luciferase reporter plasmid，Western blot and ELISA assays，respectively. RESULTS: Plaque inhibition assay showed that oxymatrine， at a range of 12.5－100 μmol/L，could significantly inhibit the replication of IAV in vitro，and its EC50 was 19.95 μmol/L. Luciferase reporter assay showed that oxymatrine，at the concentration of 12.5 μmol/L，could significantly inhibit the transcription of TLR4，MyD88 and TRAF6 induced by IAV infection (compared with virus-treated group，P < 0.05). Western blot assay showed that oxymatrine (12.5 μmol/L) could significantly inhibit the IAV-induced activation of TLR4-Myd88-TRAF6-NF-κB pathway (compared with virus-treated group，P<0.05). ELISA assay showed that oxymatrine (12.5 μmol/L) could significantly inhibit the IAV-induced release of inflammatory cytokins (compared with virus-treated group，P< 0.05). CONCLUSION: Oxymatrine possessed anti-IAV activity，and its mechanism may be related to its ability to inhibit the IAV-induced activation of TLR4-MyD88-TRAF6-NF-κB pathway.

OBJECTIVE: To evaluate the effects of nano-silica(nm-SiO2) on apoptosis of neuroblastoma cells，and compare the advantages and disadvantages of Hoechst33342/PI staining and TUNEL assay in apoptosis measurement. METHODS：Cultured human neuroblastoma SK-N-SH cells were treated with nm-SiO2 (15 and 30 nm) and micro-sized SiO2 at different doses (2.5，5 and 10 μg/mL) for 24 h. Apoptotic cells were detected by Hoechst33342/PI and TUNEL staining. RESULTS：Compared with normal control，the percentages of total apoptosis in nm-SiO2-treated cells were significantly increased by both methods. CONCLUSION：nm-SiO2 increased apoptosis of SK-N-SH cells in a dose- and size-dependent manner. Hoechst33342/PI is more specific，cheaper and simpler，while TUNEL assay is more sensitive. It is recommended to combine these two methods to assess apoptosis in neuronal cells.

OBJECTIVE: To explore the relationships of the dynamic changes of plasma apoAⅠcontent and mTOR activity with cervical carcinogenesis development in Uyghur women. METHODS：We collected 82 plasma samples from Uyghur women with cervicitis (CV) (22 cases)，cervical intraepithelial neoplasia (CIN) Ⅱ-Ⅲ (20 cases)，early stage (Ⅰ-Ⅱ) (20 cases) and advanced stage (ⅡB-Ⅳ) (20 cases) cervical squamous cell carcinoma， quantitatively determined the content of plasma proteins apoAⅠand the activity of mTOR by enzyme linked immunosorbent assay (ELISA) and statistical analysis，in order to analyze the correlation with the progression of cervical lesion and evaluate the accuracy，specificity and sensitivity of candidate plasma protein markers for early warning of cervical cancer. RESULTS：With the development of cervicitis to CINⅡ-Ⅲ，early stage or advanced stage cervical cancer，the changes of two plasma proteins altered gradually，with obvious decrease in the content of apoAⅠ，and increase in the activity of mTOR，with statistical differences for all (P<0.05). Correlation analysis of cervical cancer-specific protein indicators found that mTOR and apoAⅠshowed a low negative correlation (r＜0.4，P＜0.05). The sensitivity，specificity，and accuracy of detection，for early warning of cervical cancer of the mTOR protein were 94.0%， 60.0% and 80.0%，respectively；and for apoAⅠwere 70.1%， 60.0% and 29.0%，respectively. apoAⅠ/mTOR combined detection of sensitivity，specificity， and accuracy were 82.1%，60.0% and 54.5%，respectively. CONCLUSION：The dynamic changes of apoAⅠcontent and mTOR activity were important indicators of the procession of cervical lesions，and expected to assist with the early diagnosis of cervical cancer.

OBJECTIVE: To test expressions of Survivin，NF-κB and STAT3 in 40 cervical carcinoma tissue and 30 normal cervical tissue samples. The correlation between Survivin，NF-κB and STAT3 expression level and the clinical factors and pathology classification were analyzed，and the relationship and mechanism of Survivin，NF-κB and STAT3 in cervical carcinoma development were studied. METHODS：Trizol one step method was used to extract total RNA from both cervical carcinoma and normal tissues，mRNA reverse transcriptase cDNA，the polymerase chain reaction (RT-PCR half quantitative) method was used to measure Survivin，NF-κB and STAT3 mRNA expressions in cervical carcinoma and normal tissues. Further analysis of correlation between Survivin，NF-κB and STAT3 mRNA expression and the clinical pathological factors were performed. RESULTS：Survivin，NF-κB and STAT3 mRNA expressions in carcinoma tissue were significantly higher than the normal tissue (P<0.05). Survivin，NF-κB and STAT3 mRNA expression inⅡb-Ⅲa stage was significantly higher than Ⅰ-Ⅱa stage (P<0.01). The expression of Survivin mRNA in patients with lymph node metastasis was higher than those with no lymph node metastasis (P<0.01) . Survivin expression was related to STAT3 expression (r=0.483，P<0.05) and to NF-κB expression (r=0.377，P<0.05). CONCLUSION： Survivin，NF-κB and STAT3 expressions were upregulated in cervical carcinoma. All of these were closely related to the development and progression of cervical carcinoma.

OBJECTIVE: Esophageal squamous cell carcinoma (ESCC) is still the leading cancer-caused mortality in China，and 5-year survival remains poor. The aim of this study is to survey the ability that coexpression of Fascin and EGFR predicts ESCC prognosis. METHODS：We collected 189 formalin-fixed paraffin-embedded esophageal curative resection specimens，and then constructed tissue microarray. Immunohistochemistry (IHC) was used for detecting the co-expression of Fascin and EGFR. Statistical analyses were performed using SPSS 13.0 for windows. RESULTS：Our IHC results showed that positive rates of high-expression of Fascin and EGFR were 35.4% and 56.1%，respectively. Positive rate of co-expression of Fascin and EGFR was 29.5%. The Kendall’s tau-b correlation analysis indicated that EGFR expression was associated with regional lymph node metastasis. Positive rate of EGFR without regional lymph node metastasis was 49.6%，while it was 68.7% with regional lymph node metastasis，P=0.014. Kaplan-Meier survival analysis showed that high-expression of Fascin and EGFR were associated with poor survival，while co-expression of Fascin and EGFR presented poorer survival. Pearson’s rank correlation analysis illustrated that there was a significant correlation between Fascin and EGFR. The multivariate Cox stepwise regression analysis demonstrated that Fascin/EGFR coexpression was a passive independent poor prognostic factor for overall survival. The RR for patients with high-coexpression of Fascin/EGFR was 1.987 ，just a little higher than RR for high-expression of Fascin. The ROC curve analysis indicated that the specificity of the combination of Fascin and EGFR was more robust than that of a single biomarker. CONCLUSION：This study has successfully showed that co-expression of Fascin and EGFR could confer a poor prognosis.

OBJECTIVE: We developed a standard Ames fluctuation test in our laboratory and lay the foundation for its application in detecting the genetic toxicity of compounds. METHODS：We used four positive compounds in the test，Dexon，SA，CP and 2-AF. We compared two ways of enrichment culture and two indicators to eatablish the sandand percedure. We adopted 0.1，1，10 μg/mL Dexon and 0.01，0.1，1 μg/mL SA in the non-activated condition with their menstruum as negative control；10，100，1 000 μg/mL CP and 0.1，1，10 μg/mL 2-AF in the activated condition with their menstruum as negative control. We recorded the number of positive wells，adjusting the dose of the positive compounds accordingly，until there was a significant difference (P<0.01) between the positive group and the solvent compared group. After determining the dose of the positive control compounds，we repeated the test at the same dose 3 times，to ensure that the test system was stable and reliable. RESULTS：We determined the doses of the four positive compounds for the Ames fluctuation test in our laboratory，Dexon 3 μg/mL，SA 0.05 μg/mL，CP 100 μg/mL and 2-AF 30 μg/mL. In the non-activated condition 3 μg/mL Dexon and 0.05 μg/mL SA of the 3 96-well plates were significantly different from the solvent control group，exhibiting strong mutagenic effect to the TA100(P<0.01). Similarly，100 μg/mL CP and 30 μg/mL 2-AF in the activated condition of the 3 96-well plates were significantly different from the solvent control group，also showing strong mutagenic effect to the TA100(P<0.01). We also improved the test method during the experiment，including sandardizing the doses of test substance，improving enrichment culture method and determining the indicator. CONCLUSION：We established the Ames fluctuation test in our laboratory，confirmed that the known positive compounds were good and stable，and we optimized the experimental conditions in the study.

OBJECTIVE: To construct a 16HBE cell line with low expression of hsa-miR-148a-3p. METHODS：A pair of TuD RNA sequences of hsa-miR-148a-3p was designed according to information provided in miRBase， which was ligated to lentiviral vector pLKO.1-puro afterwards. The recombinant vector was transfected into 293FT cells for lentivirus packaging. The viruses were collected and used to infect normal 16HBE cells. The target cells were selected with Puromycin and was identified by quantitative PCR. The mRNA expression and the protein expression level of DNMT1 were then tested. RESULTS：Sequencing results indicated the successful construction of recombinant lentiviral vector with hsa-miR-148a-3p TuD RNA， quantitative PCR showed that the expression level of hsa-miR-148a-3p in the target cells was 44% lower than that of normal 16HBE cells(P<0.01)， whereas a 3.4-fold increase of the mRNA expression level and a two-fold increase of protein expression level of DNMT1 were observed in the cell line(P<0.01). CONCLUSION：Low expression of hsa-miR-148a-3p of 16HBE cell line was successfully constructed， and inhibition of hsa-miR-148a-3p could lead to higher expression and higher protein levels of DNMT1.

OBJECTIVE: To establish a HepG2 cell line with stable PXR silencing effect by a lentiviral vector carrying a short hairpin RNA (sh-RNA). METHODS：Three double-stranded sh-RNA targeting the PXR gene were designed，synthesized and cloned into the psi-sH1 sh-RNA vector. The effective recombinant sh-RNA expression plasmid against PXR，which was selected of HEK293T cells with three double-stranded sh-RNA plasmids，was packaged into HEK293T cells and used to infect HepG2 cells after 200 mmol/L puromycin screening for 2-3 weeks. The expression level of PXR was detected by western blot，the CYP3A4 mRNA levels both in sh-RNA PXR HepG2 stable cells and scramble control HepG2 cells were detected by real-time PCR. RESULTS：A recombinant sh-RNA expression plasmid against PXR gene with effective interference was successfully selected and a HepG2 cell line stably transfected with the sh-RNA PXR was established. CONCLUSION：A HepG2 cell line stably transfected with the sh-RNA PXR was established.

OBJECTIVE: To establish recombinant Lac Z yeast cells regulated by RNR3 in order to screen chemical mutagens. METHODS：RNR3 promoter was amplified using touch down polymerase chain reaction (PCR) from yeast genome and inserted into the yeast report vector of pMP206/ERE to construct yeast Lac Z gene report vector regulated by RNR3. This vector was transformed into the W303-1A yeast cells to construct the recombinant Lac Z gene yeast cells. These yeast cells were treated by several kinds of chemical mutagens to assess the expression of RNR3. RESULTS： Actinomycin D，mitomycin C，aphidicolin and ethidium bromide could not induce RNR3 expression. Methyl methanesulfonate，chlorambucil，cisplatin，4-nitro-N-oxidation of quinoline，bleomycin，phleomycin，5-fluorouracil，camptothecin and hydroxyurea mutagen could induce RNR3 expression in a dose-dependent manner. CONCLUSION：Recombinant yeast can be used for rapid screening of many chemical mutagens.

OBJECTIVE: To prepare anti-liver-type fatty acid binding protein (LFABP) monoclonal antibody (mAb) and identified subtype and specificity. METHODS：The LFABP recombinant protein was used to immunize BALB/c mice，spleen cells from immunized mice were fused with myeloma cell Sp2/0 . After HAT selective culture and indirect ELISA screening，we obtained hybridoma cell line secreting mouse mAb against human LFABP. The specificity was verified by ELISA and Western blotting. RESULTS：After using purified recombinant protein immunized mice and screening，we obtained two hybridoma cell lines secreting the mAb against human LFABP，named 3E6 and 5B7，both were of the IgG1 subtype. The antibody was purified，reaching a concentration of 2 mg/mL，and titer of 1∶10 000 above. Western blotting showed that mAb had a specific reaction with the LFABP endogenous expression in the cells. CONCLUSION：We successfully prepared mice mAb against human LFABP ，which could not only provide an important foundation to further studies of the biological characteristics of LFABP，mechanism involved in regulation of fatty acid metabolism and drug interaction，but also provide experimental evidence for the treatment of related diseases.

OBJECTIVE: To study the mutagenic and anti-mutagenic activities of total terpene ketones from Swertia mussotii (TTKS) at different doses. METHODS：Mutagenic experiments：conventional Ames test and micronucleus test were selected. Ames experiment was used with the incorporation method. Micronucleus test was conducted in mice which were treated orally once a day for 10 days consecutively. Anti-mutagenic experiments：methods in vitro，pre-incubated the TA100 and TA102 strains with CP (200 μg/plate) or MMC ( 2 μg/plate) for 30 minutes，then mixed the above materials with TTKS ( 156，312，625，1 250 and 2 500 μg/plate )，so as to detect the protective effects of TTKS on mutational colonies induced by positive agent. Mice were exposed to CP (40 mg/kg) or MMC (2 mg/kg) after continuous irrigation of TTKS (25，50 and 100 mg/kg ) for 10 days，the micronucleus rate was calculated in order to detect the protective effects of TTKS on non-mutation bone marrow cells. RESULTS：In mutagenic test，TTKS at a dose lower than 2 000 μg/plate induced no obvious increase in mutagenicity of the four strains. TTKS at a dose lower than 40 mg/kg did not increase the micronucleus frequencies of polychromatic erythrocytes. In anti-mutagenic test，comparing with the positive groups of CP and MMC，TTKS at 625－2 500 μg/plate could decrease the colony numbers in T100 and T102 strains. TTKS at 50-100 mg/kg could significantly reduce the rate of micronucleus of polychromatic erythrocytes in mice bone marrow induced by CP and MMC as compared with the positive control. CONCLUSION：Under these experimental conditions，TTKS showed no effect of mutagenesis and could confer significant anti-mutagenic protection.

OBJECTIVE: To determine the teratogenic effects of peristrophe roxburghiana (Schult.) brem in rats. METHODS：Pregnant rats were divided into 5 groups，each with 12 rats. Three groups were treated orally with exract from peristrophe roxburghiana for 10 days from 6th to 15th day of pregnancy with doses of 10.0，5.0，2.5 g/kg. The other two groups were fed orally with deionized water as negative control and aspirin with 0.3 g/kg as positive control. At 20th day，the rats were sacrificed to allow physical examination of the fetuses. RESULTS：In all treated groups the body weight of parental rats， litter weight， live rate， fetus body weight and fetus length were not significantly different from negative control group (P>0.05)，and no abnormality of organ or external was found in the fetuses. CONCLUSION：In these experimental conditions，peristrophe roxburghiana had no appearance maternal toxicity，embryo toxicity nor teratogenicity in rats.

OBJECTIVE: Study on effects of the ethylene oxide-sterilized Petri plate on Ames test. METHODS：In the plate incorporation test of Salmonella typhimurium，the revertant colonies of TA97， TA98，TA100，TA102 and TA1535 on the ethylene oxide sterilized Petri plates stored for different periods of time or by ventilation was counted. RESULTS：Ethylene oxide residuals in the Petri plates one month after sterilized increased the revertant colonies of TA100 and TA1535(TA100，about 1.5 fold of the spontaneous revertant colonies； TA1535，about 10 fold of the spontaneous revertant colonies)，and extending storage or ventilation could reduce the plate ethylene oxide residuals to a level that caused no mutagenetic effects. CONCLUSION：Under certain conditions，ethylene oxide-sterilized Petri plate could induce TA100 and TA1535 mutagenicity on Ames test.