BACKGROUND AND AIM: To explore the effects of transbronchial TiO2 nanoparticles(size 50 nm) poisoning on liver and kidney in rats. MATERIALS AND METHODS: 24 SD rats were randomly divided into 0.5, 5.0 and 50.0 mg/kg nano-TiO2 groups and control group(0.15％ sodium chloride solution). Rats were treated with intratracheal TiO2 nanoparticle suspension and were sacrificed after 7 days. Complete blood count and biochemical indexes and tissue coefficients were assessed. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) and the contents of maleic dialdehyde (MDA) in plasma, liver and kidney were measured. Pathological changes of liver and kidney were also examined. RESULTS: Indexes of blood count, blood biochemistry and tissue coefficients did not show statistical significance between nano_TiO2 exposure groups and control group. Kidney SOD activities of 5.0 and 50.0 mg/kg nano_TiO2 groups were obviously decreased compared with that in control group(P<0.05). GSH-PX activities in plasma and kidney of 50.0 mg/kg nano-TiO2 group were lower than that in control group(P<0.05). In liver, MDA contents of 0.5 and 5.0 mg/kg nano-TiO2 groups were significantly increased compared to the control group (P<0.05). No obvious pathological changes of liver and kidney was observed. CONCLUSION: Acute transbronchial exposure to TiO2 nanopaticles could induce oxidative stress in the liver and kidney, but did not influence hepatic or renal functions nor cause pathological changes . Its biological and toxicological significance needs further studies.

BACKGROUND AND AIM: To investigate the feasibility of gastric carcinoma gene therapy by utilizing RNA interference(RNAi) to inhibit expression of Livin in vitro. MATERIALS AND METHODS: Small interference RNA(siRNA) homologous to Livin gene were designed, pTZU6＋1-siRNA-Livin vector was constructed and transfected into SGC-7901 cells to induce RNAi in vitro.The changes of tumor cell cycles were examined with flow cytometry, Livin gene expression were measured with RT-PCR and immunochemistry, tumor cell apoptosis was teseted by TUNEL in vitro. RESULTS: After transfected with pTZU6＋1-siRNA-Livin,the percentage of S phase was reduced from 42.78％ to 19.15％(P<0.05),G1/G0 phase increased from 52.68％ to 72.98％(P<0.05). The down-regulation of Livin mRNA in group transfected with pL1-siRNA was obvious compared with the control group,with marked apoptosis. CONCLUSION: RNA interference down-regulated Livin expression, inhibited gastric carcinoma SGC-7901 cell proliferation and induced apoptosis, and may become a potential method for gene therapy of gastric cancer.

BACKGROUND AND AIM: To construct the antisense expression vector of human COX-2 gene and explore the relationship between COX-2 gene and tumor proliferation. MATERIALS AND METHODS: COX-2 gene fragment cleaved from plasmid pBOSNeo COX-2 with EcoRⅠ, restriction enzyme cutting sites EcoRⅠand Bgl II was added to its two ends in two reverse directions artificially. The target fragment was inserted into the polyclone site of plasmid eukaryotic expression vector pIRES2-EGFP. The constructed recombinant was identified by agarose gel electrophoresis. The transfection of antisense COX-2 recombinant was made to gastric cancer cell line named SGC-7901 which with high expression of COX-2 gene mediated by liposome. The transfectants were screened by G418 and idendified by detection of exogenous NEO resistant gene with PCR technique. PCR and Western Blot were used to determine if the expression of COX-2 was inhibited by antisense gene in the transfectant. MTT essay and colony formation test were used to observe the effect to the proliferation of the transfectant. RESULTS: Agarose gel electrophoresis confirmed that the sense and antisense target fragment were successfully bound to pIRES2-EGFP. The test of exogenous gene showed we obtained stable transfectants. The expression of COX-2 was downregulated in transfectant with antisence gene. MTT essay and clone formation test showed the proliferation of transfectant was inhibited. CONCLUSION: The antisense eukaryotic expression vector of COX-2 was constructed successfully by means of reversely inserting the target fragment into pIRES2-EGFP. COX-2 gene could control proliferation of SGC-7901.

BACKGROUND AND AIM: To investigate the effects of helicobacter pylori L-form (Hp-L) on apoptosis of gastric cancer cells BGC-823 and the probable mechanisms involved. MATERIALS AND METHODS: The BGC-823 cells were co-incubated with Hp-L in different ratios (1∶20, 1∶100, 1∶500) and the followings were carried out at different time points: morphology changes of BGC-823 cells were examined by inversion microscope; the ultrastructure of cells and the apoptotic morphology by transmission electronic microscopy; the apoptotic rate by flow cytometry and immunohistochemical staining (SP method) to detect the expression of Livin, bcl-2 and p53 in BGC-823 cells. RESULTS: When BGC-823 cells were infected by Hp-L, the cells proliferated well, many tumor giant cells appeared indicating obvious inhibition of apoptosis. The number of apoptotic cells and the apoptotic rate were both reduced. The expressions of Livin, bcl-2 and p53 were all increased. All the effects were in a concentration- and time-dependent manner. CONCLUSION: Hp-L could inhibit the apoptosis of gastric cancer BGC-823 cells, its mechanisms might be related to up-regulation of Livin, bcl-2 and p53 expressions.

BACKGROUND AND AIM: To explore the correlation of staphylococcus aureus L-form infection and mutation of k-ras in ovarian carcinomas. MATERIALS AND METHODS: Gram staining and PCR-FLRP were used for detection of bacterial L-forms in paraffin-embeded tissues of 97 ovarian papillary carcinomas and 23 benign tumors. RESULTS: The positive rate of L-forms in ovarian carcinomas was 25.8％(25/97)，and in this group, the mutation rate of k-ras at codon 12 was 45.5％.However the positive rate of L-forms in ovarian benign tumors was only 13.0％(3/23)，and in this group, the mutation rate of k-ras at codon 12 was only 8.7％.With Spearman analysis, the malignant group was r=0.504 7(P<0.05);and the benign group was r=-0.119 5(P>0.05). CONCLUSION: Infection of L-forms had a positive correlation with point mutation of k-ras in malignant tumors but not in benign tumors.

BACKGROUND AND AIM: To study the inhibitory effects of c9,t11-conjugated linoleic acid (c9,t11-CLA) on matrix metalloproteinase of human gastric carcinoma cell line(SGC-7901) via cyclooxygenase-2 pathway. MATERIALS AND METHODS: Inhibiting COX-2 activity by 100 μmol/L COX-2 inhibitor NS-398 in SGC-7901 cells, which were then treated with c9,t11-CLA by 200,100,50,25 μmol/L for 24 h. The expressions of MMP-2,MMP-9 mRNA were measured by RT-PCR. RESULTS: At the concentrations of 200,100,50,25 μmol/L, c9,t11-CLA decreased the expressions of MMP-2,MMP-9 mRNA. But when the SGC-7901 cells were treated with NS-398 in advance, MMP-2 and MMP-9 mRNA level were not decreased in any concentration of c9, t11-CLA groups. CONCLUSION: c9,t11-CLA could inhibit the expressions of MMP-2,MMP-9 mRNA in SGC-7901 cells, and COX-2 might play an important role in mediating the ability of c9,t11-CLA to inhibit the matrix metalloproteinase of tumor cells.

BACKGROUND AND AIM:To analyze whether promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT) gene is associated with the risk for esophageal squamous cell carcinoma(ESCC), and to evaluate the clinical significance in the screening and early diagnosis of ESCC. MATERIALS AND METHODS: 91 patients with newly diagnosed, untreated esophageal cancer were recruited in the present study. Esophageal cancer tissues, tissues adjacent to the tumors and peripheral blood were collected to determine CpG island hypermethylation of the promoter. Methylation specific PCR(MSP) was used to examine the methylation status of MGMT gene, total mRNA was extracted from esophageal cancer tissues and tissues adjacent to the tumors, expression levels of MGMT gene were measured by quantitative real-time reverse transcription-PCR. RESULTS:The risk for ESCC showed significant correlation to promoter hypermethylation of MGMT gene (OR=7.750, 95％CI=2.736～21.955). There was no significant correlation between methylation status and mRNA level. Hypermethylation of MGMT gene in plasma from ESCC patients was significantly correlated to that in tumor tissue(P<0.01). The detection rate of CpG island hypermethylation of MGMT promoter in plasma was moderately correlated with that in tumor tissue(Kappa=0.603, P<0.01). CONCLUSION: This study suggested that CpG island hyper- methylation of MGMT gene was associated with an enhanced risk for ESCC. Detection of the aberrant methylation in the promoter of MGMT gene from ESCC patient plasma might provide valuable information for the screening and early diagnosis of ESCC.

BACKGROUND AND AIM: To explore the molecular mechanisms of resveratrol in inducing apoptosis in human promyelocytic leukemia HL-60 cells. MATERIALS AND METHODS: After treatment with resveratrol at different concentrations for different times, the morphological changes of HL-60 cells were examined by phase contrast microscopy. The proliferation of HL-60 cells and the IC50 were analyzed by methyl thiazolyltetrazolium (MTT)assay.Caspase-3 acticity and expression levels of GADD45α,Annexin A1,Cleaved-caspase 3,Bcl-2 and Bax proteins in HL-60 cell were measured by Caspase-3 activity assay kit and Western blot. RESULTS: After treatment with resveratrol, the HL-60 cells became smaller,less refractive with the appearance of granular materials. The changes were associated with the increase of resveratrol concentration. After treatment with 12.5-200 μmol/L Res, HL-60 cell proliferation was markedly inhibited in a dose and time -dependent manner(P<0.05) , and the IC50 was 76.54 μmol/L. After treatment with Res for 0，12，24，48 h，the Caspase-3 activity in HL-60 cells peaked at 12 h,and then decreased. The expressions of Annexin A1,GADD45α,Bax,Cleaved-Caspase 3 were up-regulated and Bcl-2 was down-regulated in HL-60 cells by resveratrol. CONCLUSION: Resveratrol could inhibit the proliferation and induce apoptosis of HL-60 cells, the effect might be associated with Caspase-3 activity. Expressions of GADD45α and Annexin A1 participated in the apoptotic process modulated by resveratrol.

BACKGROUND AND AIM: To study the effects of the transfection of recO gene in vitro on the oxidation resistance and the secretion of some inflammatory factors of human skin fibroblas (HSF) exposed to UVB. MATERIALS AND METHODS: The recO gene from deinococcus radiodurans was cloned into pcDNA3.1/NT-GFP-TOPO vector, and was introduced into human skin fibroblasts, cells which were then irradiated with 0, 30, 90, 120 mJ/cm2 doses of UVB. The untransfection group and the blank vector transfected group, were also treated with the same doses of UVB as controls. The activity of LDH and SOD, and the content of MDA, IL-6 and TNF-α in each group were detected. RESULTS: After UVB exposure for 24 h, the activity of total SOD decreased, and the activity of LDH and the content of MDA, IL-6 and TNF-α increased with increasing of UVB doses in the two control groups. However, the SOD activity increased and the IL-6 secretion decreased in the recO transfection group exposed to the same dose of UVB. There were significant differences of the SOD activity and IL-6 secretion between the test group and the control groups in the 30-120 mJ/cm2 range(P<0.05). CONCLUSION: To a certain extent, the transfection of recO gene could inhibit the oxidative damage and the generation of some inflammatory factors, which were induced by UVB irradiation.

BACKGROUND AND AIM: To explore the effect on the function of 249 Arg to Ser mutation in the p53 gene in mice. MATERIALS AND METHODS: Arg to Ser mutation was introduced into the 249 position of the p53 gene by knock-in method. These ES cells with this mutation were selected according to the homologues-recombination with PCR and Southern blot. The positive ES cells without a selection marker were injected into blastocysts recovered from Hprt-/- mice, which were derived from Hprt-deficient ES cells. The injected blastocysts then were implanted into pseudopregnant females. At embryonic day 14, MEFs were recovered from the embryos and cultured under the selection of HAT (0.016 mg of hypoxanthine/ml, 0.01 mmol/L aminopterin, 0.0048 mg of thymidine thymidine/ml)，and the cell cycle, apoptosis, and the relative protein expressions were analyzed by flow cytometry and Western blot. RESULTS: The ES cells with 249 Arg to Ser mutation were more resistant to the IR-induced cell cycle arrest compared to the wild type ES cell(P<0.05).The ES and EF cell with 249 Arg to Ser mutation were more resistant to the IR- or UV-induced apoptosis compared to the wild type ES and EF cell(P<0.05). The mutation did not affect the expression of p53 after IR or UV, but decrease the expression bax and p21 after IR or UV. CONCLUSION: The p53 249 Arg to Ser mutation could disable the function of p53, but did not change the expression of p53.

K562； 凋亡； Bcl-2/Bax； 细胞周期BACKGROUND AND AIM: To investigate the apoptotic effect and mechanism of K562 cells exposed to cerium chloride(CeCl3) and CeCl3 combined with gamma radiation. MATERIALS AND METHODS: Apoptosis and cell cycle of K562 cells were examined using flow cytometry and expressions of Bcl-2 and Bax were assessed with Western blot. RESULTS: Compared with control,CeCl3 exposure for 26 h ,gamma irradiation,and both combined could induce apoptosis of K562 cells(P<0.05).The cells in S phase were reduced in CeCl3 group(P<0.05), in irradiation and combined treatment group also(P<0.01). The cell cycle was blocked in G2/M phase in irradiation group and combined treatment group compared with control. The Bcl-2/Bax ratio in all 3 groups were lower than control. CONCLUSION: CeCl3 and CeCl3 combined with gamma radiation induced K562 cells apoptosis via mitochondrial-mediated apoptosis pathway. The apoptosis of the combined treatment group was perhaps related to the block of cell cycle in G2/M phase.

BACKGROUND AND AIM: To study the antioxidative activities of different concentrations of ethanol and water extracts of Acanthopanax in several free radical models in vitro and their dose-effect relationships. MATERIALS AND METHODS: The DPPH system model was built to study the inhibitive rates of DPPH radicals by different concentrations of Acanthopanax extracts. Two chemiluminescence system models for determination of ·OH and O2 were set up to examine the inhibitive rates of luminous intensity by different concentrations of Acanthopanax extracts. The lipid microsomal peroxidation model stimulated by CHP, VC／Fe2＋ and CC14／NADP were used to assess the inhibitive rates of MDA by different concentrations of Acanthopanax extracts. RESULTS: In the DPPH system, the inhibitive rates of DPPH radicals by the two Acanthopanax extracts were significantly higher than that in control group which showed an evident dose-effect relationship. The IC50 of DPPH radicals by the ethanol and water extracts of Acanthopanax was 0.18 mg/ml and 0.09 mg/ml, respectively. In the two chemiluminescence systems, except the ethanol extract of 0.625 mg/ml (P=0.102), the inhibitive rates of ·OH and O2 by different concentrations of Acanthopanax extracts were significantly higher than that in control group and showed an evident dose-effect relationship. The IC50 of O2 and ·OH by ethanol extracts of Acanthopanax were 0.488 mg/ml and 1.29 mg/ml, respectively, and the IC50 were 0.24 mg/ml and 0.37 mg/ml, respectively, by the water extracts. In the CHP model, the water extracts of Acanthopanax showed an activity of promoting oxidation when the concentration was 1.25 mg/ml. But the variance didn't reach statistical significance. Compared with that in control group, the content of MDA in the three LPO models decreased significantly(P<0.05) at different concentrations and had a dose-effect relationship. CONCLUSION: In several in vitro models described above, the ethanol and water extracts of Acanthopanax demonstrated effective antioxidative effects. In DPPH and two chemiluminescence systems, the antioxidative activities of the water extracts were stronger than that of the ethanol extracts; whilst in the three LPO models, the results were opposite.

BACKGROUND AND AIM: To locate and monitor Asialoglycoprotein receptor(ASGP-R) on the sperm surface using FITC-labeled asialofetuin(FITC-ASF). MATERIALS AND METHODS: Asialofetuin was labeled with FITC to produce FITC-ASF, which was used in the investigation of binding between foreign protein and ASGP-R on the sperm surface using immunofluorescence and flow cytometry. RESULTS: FITC-ASF was able to label ASGP-R on sperm surface well and successfully applied to assess the binding of foreign protein to the receptor. CONCLUSION: The study provided a new potential and convenient method to monitor ASGP-R.

BACKGROUND AND AIM: To study the expression differences of O6-methulguanine-DNA methyltransferase (MGMT) and mutant P53 protein between primary and secondary glioblastomas(GBM) and the biological implications. MATERIALS AND METHODS: Immunohistochemistry(IHC) methods was used to measure the expressions of MGMT and mutant P53 protein in 39 cases of GBM(13 primary and 26 secondary),and the correlation with prognosis. The relationship between these gene expressions in primary and secondary GBM were analyzed. RESULTS: The differences of positive rate and expression intensity of mutant P53 protein expression were statistically signifiant between primary and secondary GBM (P<0.01；P<0.01 ,respectively). Moreover, there was an inverse correlation between MGMT and mutant P53 protein expression intensity in secondary GBM(r=-0.602, P<0.01).but in primary GBM, there is no correlation between MGMT and mutant P53 protein expression(P>0.05).Kaplan-Meier analysis revealed that primary GBM and high expression of MGMT were significantly related to short survival(Log-rank test，P<0.05,P<0.01, respectively).Cox multivariate analysis revealed that subtype of GBM and MGMT expression were prognosticators for GBM(P<0.05, P<0.01, respectively). The survival period of GBM patients was not associated with age,gender,tumour size or mutant P53 protein expression. CONCLUSION: P53 gene mutations was frequent in tumorigenesis of secondary GBM,and the strong mutant P53 protein expression might be related to the weak MGMT expression. But in primary GBM, there is no correlation between MGMT and mutant P53 protein expression.It implicates different genetic pathways of developing in primary and secondary GBM. The subtype of GBM and MGMT expression were prognosticators for GBM.

BACKGROUND AND AIM: To study the correlations between p16 gene methylation,Her-2 expression and serum CA125 level in endometrial carcinoma(EC)and clinicopathological characteristics. MATERIALS AND METHODS: A total of 38 EC tissues and 20 normal controls (NC) were analyzed.We studied the p16 gene methylation by methylation specific PCR (MSP)and Her-2 expression by Envision method, and the preoperative serum CA125 levels were checked by radio-immunity in all cases. The correlations of expression levels and EC clinicopathological characteristic were studied. RESULTS: The positive rates of p16 methylation , Her-2 expression and serum CA125 level were 57.9％(22/38),60.5％(23/38)and 23.7％(9/38),respectively, in 38 cases of EC, but were not detected in 20 normal controls, the difference was significant (P<0.05). Methylation of p16 gene, Her-2 expression and serum CA125 level were all statistically related to clinical stage of EC in test group(P<0.05), methylation of p16 gene and the level was statistically related to histological grade(P<0.05). Serum CA125 levels were statistically related to pelvic lymph node mestastasis(P<0.05),the 3 indexes were all statistically related to the depth of tumor invasion(P<0.05). Spearman correlation revealed there was a statistically positive relationship between Her-2 and serum CA125 level(P=0.048)and negative correlation between methylation of p16 gene and Her-2 expression(P=0.026). CONCLUSION: The measurements of p16 methylation, Her-2 expression and serum CA125 level may reflect different behaviors of genes and tumor markers at different stages of tumor pathogenesis. They may be useful tools for providing information about the malignant degree,prognosis, and may guide postoperative treatment for patients with EC.

BACKGROUND AND AIM: To study the clinicopathological significance of p53 gene expression and telomerase activity in circulating tumor cells of patients with colorectal carcinoma. MATERIALS AND METHODS: Mononuclear cells containing circulating tumor cells were isolated by Ficoll-Hypaque gradient centrifugation in peripheral blood from 113 preoperative patients with colorecatal carcinomas,13 patients with benign colorectal disease, and 28 healthy volunteers. FCM and TRAP-ELASA were used to evaluate p53 gene expression and telomerase activity,respectively. Experimental data were analyzed by SPSS 11.0 software and χ2 test. RESULTS: Among 105 informative patients with colorectal carcinoma, positive rate of p53 expression and telomerse activity were 74.3％(78/105) and 61.0％(64/105),respectively. Aberrant expression levels of both p53 gene and telomerase in patients with colorectal cancer were significantly correlated with lymph node metastasis (P<0.05) and their simultaneous measurement could raise the prediction accuracy of metastasis. Expression of p53 gene was also significantly correlated with differentiation degree of colorectal carcinoma (P=0.003). CONCLUSION: Aberrant p53 gene expression and telomerase activity in circulating tumor cells might be malignant biomarkers. Both were closely related and could predict nodal involvement.

BACKGROUND AND AIM: Mig-2 protein is an important moleculer in the process of tumor formation, and is a target for tumor therapy. To search new effective ways to diagnose and treat malignant tumors, we prepared the McAbs of Mig-2 protein. MATERIALS AND METHODS: BALB/c mice were immunized with recombinant plasmid of Mig-2(the vector is P3XFLAG-CMV-10).By cell fusion and cell cloning, the McAbs of Mig-2 protein were prepared. RESULTS: Three strains of hybridoma cells 3C4,4H2 and 1F8 all secreting a subclass of IgM, anti-mig-2 antibody,were obtained after fusion followed by three or four screenings. The a scitic fluid McAbs were prepared by injecting the hybridoma cells into mice abdomen. The indirect ELISA results showed that ascitic McAbs revealed high affinity with the Mig-2 protein at the titer of 1∶2.4×104-1∶4.8×104,and the titer of cell supernatant was 1∶32-1∶512. CONCLUSION: Prepared McAbs of Mig-2 were specific and stable agent for tumor therapy research.