BACKGROUND ＆ AIM: To clone the mouse pdx-1 gene and to construct its expression vector. MATERIAL AND METHODS： Mouse pdx-1 gene was amplified from mouse pancreas total RNA and subcloned into the vectors pEGFP-C1 and pcDNA3.0. The expression of pdx-1 was analyzed by RT-PCR and green fluorescence detection after transfection of the SMMC-7721 cell line. RESULTS： The expression vectors of pEGFP-C1 pdx-1 and pcDNA3 pdx-1 were constructed. After transfecting the SMMC-7721 cells with pEGFP-C1 pdx-1, green fluorescence was detected only in the nucleoli. By RT-PCR analysis, pdx-1 mRNA was detected in both pEGFP-C1 pdx-1 and pcDNA3 pdx-1 transfected cells. CONCLUSION： The expression vectors of pdx-1 gene were useful to investigate the development of the pancreas and the proliferation and differentiation of hepatic stem cells.

BACKGROUND ＆ AIM: To identify genes involved in cadmium-induced testicular toxicity. MATERIAL AND METHODS: We used cDNA microarray and real-time quantitative polymerase chain reaction(real-time PCR) technique to analyze the genes expression of testes in rats treated with low cadmium(4 μmol/kg). RESULTS: Our studies for the first time demonstrated that expression of T-kininogen, calmegin, UDP-glucuronyl transferase，heme oxygenase and mismatch repair protein gene were associated with cadmium toxicity. However, as the organelles that produced energy, mitochondrion was not affected in the respiratory chain at low dose cadmium. In addition, vitamin C at the concentration of 400 mg/kg obviously attenuated cadmium-induced toxicity in testes of rats. CONCLUSION: The cadmium-reduced toxicity carcinogenic effects involved energy metabolism, defense mechanisms and DNA repair of the cell. Vitamin C might play essential roles in protecting testes of rats exposed to cadmium.

BACKGROUND ＆ AIM： To investigate the toxic effects of aristolochic acid I,aristolochic acid II and aristolochic acid Ia on renal proximal tubular epithelial cell (cell line HK-2) in vitro. MATERIAL AND METHODS: The morphological changes of HK-2 cell were examined with contrast microscopy and the growth inhibition of HK-2 after treatment with the three aristolochia compounds was studied by[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]assay. The drug treatment group, negative control group and blank control group were set up. The expression of α-smooth muscle actin was defected by immunohistochemistry and cell cycle and apoptosis rate were defermined by flow cytometry. RESULTS: Under the light microscope, HK-2 cells became contracted and rounded in AAI and AAIa groups.The expression of α-smooth muscle actin increased in the AAI,AAII and AAIa groups compared with control; the three compounds all induced the inhibition the growth of HK-2 cells in concentration-and time-dependent manner.The median inhibitory concentration(IC50) revealed that inhibition intensities were AAI>AAIa>AAII. All these compounds could affect cell cycle and even induce cell apoptosis. CONCLUSION: AAI,AAII and AAIa were cytotoxic to various extents which might have resulted from different mechanisms.

BACKGROUND ＆ AIM: To study the changes of pulmonary edema and inflammatory reaction in rats at different times after exposed to phosgene. MATERIAL AND METHODS: 180 male rats were randomly divided into 3 experiments, 6 groups with 10 rats in each experiment. The six groups were: negative group exposed to room air and the positive groups exposed to 38.08 mg/L phosgene for 1 minute. Other five groups of rats exposed to phosgene of the same concentration and for the same time, but rats were killed after 1 hour,3 hours,6 hours,12 hours and 24 hours'. Vascular permeability in lung, wet-to-dry lung weight ratio (W∶D ratio ) , cell count in bronchial alveolar lavage fluid (BALF), TNF-α and IL-10 content in lung and in BALF were determined. RESULTS: With increasing time after the rats were poisoned by phosgene, vascular permeability in lung, W∶D ratio and the cell count in BALF significantly increased (P<0.05); the content of TNF-α significantly increased (P<0.05), at the same time, IL-10 content significantly decreased (P<0.05). CONCLUSION: The time courses of pulmonary edema and inflammatory reaction developing in rats exposed to phosgene were different.

BACKGROUND ＆ AIM: Increased motility is the basis of tumor cell invasion and metastasis. The complicated process modulated by multiplegenes. The expression of motility-related-protein-1(MRP-1), a cell- motility- inhibiting protein was reported to be down －regulated and gave rise to the malignant phenotype associated with invasion and metastasis. But the mechanism was unclear. Focal adhesion kinase, as a key component of integrin-stimulated signal transduction pathways, was also reported as a kinase associated with malignant change of cell and differentiation and invasion. But the reports about the expression of these two proteins in lung cancer and the relationship with clinical parameters and their correlation between each other were inconsistent. We hope to explore the role of MRP-1 and FAK proteins in occurrence, development, invasion and metastasis of lung cancer and their value in predicting its prognosis. The expression levels of MRP-1 and FAK proteins in lung cancer, their relationship with each other and with some clinicopathological parameters. MATERIAL AND METHODS: 89 patients with lung cancer and 10 cases of normal lung tissue and 12 with metastatic cancer were analyzed for their MRP-1 and FAK protein levels using immunobiochemical streptavidin peroxidase method (SP). The χ2 test and Spearman analysis were used. RESULTS: The positive rate of MRP-1 was 100.0％ in normal lung tissue, 41.6％ in lung cancer and 8.3％ in metastatic cancer. The difference was significant(P<0.05) . The expression of FAK was opposite. The positive rate of FAK was weak in normal tissue and elevated to 48.3％ in cancer tissue and to 83.3％ in metastatic cancer. The difference of the above three groups were significant(P<0.05). There was a negative relationship between MRP-1 and FAK. The expression of MRP-1 protein in lung cancers had no relationship with ageor gender of the patients and the macroscopic type of cancer. However it was significantly correlated with histological type, degree of tumor differentiation, clinical stage and whether there was lymphoid metastasis(P<0.05). The expression of FAK protein in lung cancer had no relationship with age or gender of the patients and the macroscopic type and histological type of cancer(P<0.05). But it was significantly correlated with the degree of tumor differentiation, clinical stage and lymphoid metastasis. CONCLUSION: The abnormal expression of MRP-1 and FAK proteins may participate in the invasion and metastasis of lung cancer. Monitoring the expression levels of MRP-1 and FAK proteins probably predict the Malignant progression of lung cancer.

BACKGROUND ＆ AIM: To study the polymorphisms of the O6-Methylguanine-DNA methyltransferase (MGMT) gene codon 84(C2740214T), codon 143(A2798995G) and codon 160 (G2799046A)for esophageal cancer and to assess the prevalence of these polymorphisms in Chinese Chaoshan population. MATERIAL AND METHODS: A microarray-based method for genotype analysis of esophageal cancer patients and healthy controls were performed. Amplified PCR products from genomic DNA specimens were spotted and immobilized onto glass slide to fabricate a microarray, then detected by hybridization with dual-color probes.100 patients and 65 healthy controls were tested for codon 84.76 patients and 50 healthy controls were tested for codons 143 and 160. RESULTS: The prevalence of the 2740214 CT in patients was 0.16 and 0.123 in controls .The prevalence of the 2740214 TT was 0.02 in patients but not detected in controls. Heterozygote at codon 143 was found in one healthy individual, but didn't exist in the cancer patients. In all the samples, no mutation was detected at codon 160. CONCLUSION: These results suggested that the polymorphisms of MGMT exist in Chinese Chaoshan population.Homozygous mutant at codon 84 might be correlated with carcinogenesis.

BACKGROUND ＆ AIM: To investigate the differences of gene expression profile among the pulmonary adenocarcinoma and metastatic lymph node tissues by means of cDNA microarray. MATERIAL AND METHODS: Total RNA was extracted from pulmonary adenocarcinoma and metastatic lymph node tissues in order to get purified mRNA which was retro-transcribed into cDNA. As the probes, the cDNA was labeled with Cy5 and Cy3 fluorescence, then hybridized with the whole human gene chip. The signal image was obtained by Scan Array 4000 fluorescence scanner and analyzed with computer software. RESULTS: 8 genes in 3 subjects with pulmonary adenocarcinoma and metastatic lymph node tissues were identified with common differences in gene expression level, including 5 up-regulated and 3 down-regulated genes. CONCLUSION: The expression levels of 8 genes found both in pulmonary adenocarcinoma and metastatic lymph node tissues were probably related to the development and metastasis of lung cancer.

BACKGROUND ＆ AIM: To investigate the anti-tumor activity of extract from Periploca Sepium Bge (PSBE) and to study its probable mechanisms. MATERIALS AND METHODS: The growth-inhibitory effect of ethanol extract from Periploca sepium bge (PSBEE) and water extract from Periploca sepium Bge (PSBWE) on MCF-7,TE-13,QG56,SMMC7721,T24,Hela,K562 tumor cell lines were examined by using MTT assay, the change of cell cycle and the induction of apoptosis were analyzed with flow cytometry. RESULTS: Both PSBEE and PSBWE could significantly inhibit the proliferation of tumor cell lines, the effect was dose-dependent, PSBEE (IC50<10 μg/ml) was more effective than PSBWE (IC50<40 μg/ml). PSBEE could arrest the cell cycle at G0/G1 phase and induce apoptosis in MCF-7 cells in a time-dependent manner. The proportion of tumor cells in the G0/G1 phase increased after treatment with 10 μg/ml PSBEE for 24 hours, the apoptotic rate reached 13.54％±2.12％ after 48 h. CONCLUSION: Cell growth in vitro was inhibited significanttly by extract from Periploca sepium Bge, PSBEE might exert its anti-tumor activity by blocking the cell cycle and inducing apoptosis of tumor cells.

BACKGROUND ＆ AIM: To investigate the antioxidative action of the ethanol and water extract of Rhodiola and their dose relationship. MATERIAL AND METHODS: Sprague-Dawley rats were killed and the livers were removed to isolate the microsomes by calcium ion precipitation. The ethanol and water of Rhodiola were extracted by recirculating and boiling. Four LPO models were built which were stimulated by Vc/Fe2＋,CHP, CCl4/NADP and NADPH-ADP／Fe2＋. Two different extracts were added into four models and titrated to final concentrations of 25 mg/ml,12.5 mg/ml,6.25 mg/ml,3.13 mg/ml and 1.56 mg/ml. Then the contents of malondialdehyde (MDA) were measured to assess the antioxidative action of Rhodiola extracts in the first three models.The electrode oxygraph was used to determine the inhibition rate of oxygen consumption in NADPH-ADP／Fe2＋ model. RESULTS: In CHP model, compared with control group, the content of MDA in group 6.25,12.5 and 25.00 mg/ml of water extract decreased significantly (P<0.05). In the Vc/Fe2＋、CHP and CCl4/NADP models, the content of MDA in those groups with different dosages of the two extracts decreased significantly(P<0.01)，and there was dose-dependent relationship between the final concentration and the inhibition rate of MDA to some extent.In NADPH-ADP／Fe2＋ model，the inhibition rates of the highest concentration of the ethanol and water extracts reached 76％ and 43％,respectively. CONCLUSION: The two extracts of Rhodiola had good antioxidative effects. The ethanol extract had comparatively stronger effect in the models containing enzymes.

BACKGROUND ＆ AIM: To investigate genotoxic effect of lanthanide ions on prokaryote by observing the effects of lanthanide nitrate on genomic DNA of E.coli. MATERIALS AND METHODS: TreatmentⅠ: E.coli was cultured in LB medium with different concentrations of lanthanide nitrate (10,40,80,160,320 mg/L), LB medium without lanthanide nitrate was the control. Genomic DNA was extracted at 10, 15, and 20 h. Then Agarose gel electrophoresis was performed Treat mentⅡ: E.coli was cultwred in LB medium with different concentrations of lanthanide nitrate (50,200,400,800,1 600 mg/L) LB medium without lanthanide nitrate was the control. Genomic DNA was extracted at 30 h. Then Agarose gel electrophoresis was performed RESULTS: DNA degradation or cross-linkage was produced by high concentrations of lanthanide ions. CONCLUSION: Lanthanide ions affected degradation and crosslinking on genomic DNA of E.coli.

BACKGROUND ＆ AIM: N-terminal ubiquitination may play a role in p21Cip1/WAF1 degradation via proteasome-dependent proteolytic pathway. We studied the influence of adding a N-terminal flag tag on the stability of p21 protein. MATERIAL AND METHODS: NIH3T3 cell stably expressing flag-p21 was set up by retrovirus pWB3flag-p21 infection followed by Blasticidin selection. Half-life of p21 protein was tested by western blot with rabbit polyclonal anti-p21 antibody, and proteasome inhibitor MG-132 was employed to test the role of proteasome pathway on the degradation of p21 protein. RESULTS: Half-life of endogenous p21 in NIH3T3 cells was about 30 minutes, while the half-life of exogenous flag-p21 fusion protein expressed in the same tested cells was obviously increased. Although MG-132 significantly increased the amount of endogenous p21 protein, there was no detectable change of flag-p21 expression under the same condition. CONCLUSION: N-terminal ubiquitination gvas necessary for effective p21 proteasome-dependent degradation. That adding flag tag at the amino acid termini may stabilize p21 protein should be considered.

BACKGROUND ＆ AIM: To determine the differentiation of myoepithelial cell (MEC) in the development of salivary glands and its pleomorphic adenomas; and the relation between the differentiation and the biological behavior of these adenomas. MATERIAL AND METHODS: 24 embryonic salivary gland samples in different stages and 31 salivary gland pleomorphic adenomas samples were collected. The development of embryonic salivary gland and pleomorphic adenomas were examined by H＆E and CK14, CK19, α-SMA and P63 markers by immunohistochemistry. The relation between myoepithelial cell differentiation and the biological behavior of the pleomorphic adenomaswas was also studied. RESULTS: Luminal cell of secretory and excretory duct appeared to have been derived from luminal cell lineage because they mostly expressed CK19, and less extensively CK14, but never α-SMA. Basal layer of intercalated duct and acinar cell appeared to have been derived from MEC lineage because expressed CK14, α-SMA, and P63. In pleomorphic adenomas, the markers of MEC were positive in spindle cells, some of the epithelioid cells and also the mucoid or chondroid components.Therefore, they were likely to be derived from MEC lineage. The marker of MEC was negative in plasmacytoid cells, clear cells, and the tubular structure of pleomorphic adenomas. Therefore, they were likely to be derived from luminal cell lineage. CONCLUSION： Acinar cell and intercalated duct appeared to have been derived from MEC lineage, and the structure of duct appeared to have been derived from luminal cell lineage.MEC neoplasms have low-grade invasiveness.

BACKGROUND ＆ AIM: To develope the two-dimensional gel electrophoresis (2-DE) profiles in the proteome research of HaCaT cell line. MATERIAL AND METHODS: HaCaT cells were cultured in RPMI1640 culture medium containing 10％ fetal bovine serum, then harvested after digestion by trypsin, and lysed in lysis buffer. After centrifugation, supernatant was separated by isoelectric focusing (IEF), equilibrated and run on SDS polyacrylamide gel electrophoresis (SDS-PAGE), stained by Coomassie Brilliant Blue then followed by image scanning and analyzsis. RESULTS: 700±23 protein spots were detected in the 2-DE profiles on 17 cm IPG strip (pH 3-10 L) of HaCaT cell proteome separated by 2-D PAGE. A match rate of 72.2％ of the spots between gels was obtained. CONCLUSION: The 2-DE profiles of proteome from HaCaT cells was established and lay foundations for the proteome research HaCaT cells.

BACKGROUND ＆ AIM： To explore the expression and significance of metallothionein(MT) and Cu/Zn-SOD in buccal mucosa squamous cell carcinoma. MATERIAL AND METHODS： The localizations of MT and Cu/Zn-SOD expressions in 8 samples of buccal mucosa squamous cell carcinoma were studied by immunohistochemical methods and analyzed quantitatively. RESULTS: In the well-differentiated squamous carcinoma, the MT and CuZn-SOD were expressed mainly at peri-tumor area, but less or not expressed at the centre and in interstitial tissue. While in poorly differentiated carcinoma, the expression was scattered. There was no significant difference between the expressions of MT and Cu/Zn-SOD among the 8 samples (P=0.554). CONCLUSION: MT and Cu/Zn-SOD were expressed mainly in the poorly differentiated and actively growing tumor cells. Their similar expression and the association with tumor development, progression and prognosis should be further studied.

BACKGROUND ＆ AIM： To explore the relationship between the free radical toxicity and genotoxicity of cumene hydroperoxide(CHP). MATERIAL AND METHODS： 36 mice were divided into four groups randomly. Their average body weight was (38.3±7.8) g. The CHP exposure doses were 2 000，500，125 and 0 μg/kg by gavage daily，five days in a week for 40 days. The change of the marrow micronucleus in polychromatic erythrocytes(PCE)and sperm deformity ratio were detected. The contents of malondialdehyde(MDA)，total superoxide dismutase(T-SOD) and reduced glutathion(GSH) in homogenate of testis were tested. RESULTS: The body weight of mice exposed to CHP had decreased to different degrees. With the increasing dose of CHP, the contents of MDA in testis homogenate were increased and the contents of T-SOD and GSH were gradually decreased. The MDA in testis homogenate of mice exposed to 2 000 μg/kg CHP was significantly higher than that of control group(P<0.01). The GSH and the T-SOD in testis homogenate of mice exposed to 500 μg/kg CHP were markedly lower than that of control group(P<0.05). The change of the marrow micronucleus in PCE and sperm deformity ratio were increased. Mice exposed to 2 000 μg/kg CHP had obvious change compared with that of control group(P<0.01). CONCLUSION: CHP twed strong free radical toxicity and genotoxicity on mice, the oxidative injury caused by free radical of CHP might induce its genotoxicity.

BACKGROUND ＆ AIM： To design and fabricate a kind of mouse toxicology gene chip to be used in toxicology research. MATERIAL AND METHODS： Some genes related to different biological functions were chosen according to the requirement of environmental and toxicology research. NIA mouse cDNA clone sets were applied to copy and amplify the gene fragments which were dotted on the slide, and the Picogreen fluorescence staining was used to detect the quality of chip. RESULTS： We chose 1 796 genes involved in eight biological functions for the construction of the mouse toxicology gene chip. 95.12％ of the PCR products had strong and single band, and the slides selected from each batch had good images with Picogreen dye detection. CONCLUSION： We have designed and fabricate the mouse toxicology. gene chip successfully. This could be applied in toxicology-related research.

BACKGROUND ＆ AIM： To establish the techniques of gene chip hybridization, and to verify the reliability of the gene chip data. MATERIAL AND METHODS： Several methods were applied to test the reliability of data, including data normalization treatment, analysis of reference spots, self-comparison test and differential expression experiment. RESULTS： The localized mean normalization method could be used to treat the original data effectively. There was no nonspecific hybridization in the negative control spots, and good reproducibility could be achieved among the repetitive genes in a chip. Meanwhile different batches of chips had good quality and reproducibility, and fluorescein swap labeling had advantages in reducing the staining errors. CONCLUSION： The results proved the validity of the chip hybridization and detection technology established in our lab.

BACKGROUND ＆ AIM:To search for the differentially expressed genes related to lipid metabolism in the liver of Balb/c mouse treated by tetracycline hydrochloride using mouse toxicological microarray. MATERIAL AND METHODS: The mouse toxicological microarray was developed. The model of tetracycline hydrochloride damaging mouse liver ,which included two doses (200 mg/(kg·d) and 50 mg/(kg·d))and three time phases,was established.The total RNA of mouse liver was isolated for hybridization experiment of the microarray. RESULTS: 28 genes differentially expressed in all treated groups were found. These were involved in lipid synthesis，catabolisim and transport in the liver,preliminary analyses of these gene functions and their relationship were performed. CONCLUSION： Multiple genes were markedly changed in the liver of Balb/c mouse treated by tetracycline hydrochloride.This set the stage for further research on the molecular mechanisms of the hepatoxicity of tetracycline.

BACKGROUND ＆ AIM: We investigated the gene expression profiles of primary mouse hepatocyte culture induced by 13 chemicals by using DNA microarray. We then used clustered analysis on the gene expression profiles, and then tried to classify chemicals based on the level of the genome response, so that a relation could be made between the classification of chemicals and its gene expression profiles. MATERIAL AND METHODS: Primary mouse hepatocyte culture was treated with 30％ of the lethal dose of 13 chemicals. 24 hours after treatment, RNA was extracted and reverse transcribed to cDNA. The cDNA were detected by DNA microarray. RESULTS: By using average- linkage hierarchical clustered analysis, 13 chemicals were classified into 3 groups. CONCLUSION: Classifying chemicals by gene expression profiles, so as to classify them relative to their toxic action then to study their toxicity mechanism and to predict the toxicity of unknown chemicals is a worthwhile and promising research direction.

BACKGROUND ＆ AIM: To explore an economic,quick and valid method for counting foci in vitro micromass cell cultures,and to provide a useful procedure relevant to this short-term test. MATERIAL AND METHODS: Midbrain cells and limb bud cells were collected from pregnant female rats at 13 days after conception and cultured constantly for 5 days in culture medium.The foci was counted by the new method introduced:the light microscopy field was divided into several compartments, moving across the compartments regularly and counting the numbers of foci.This paper also evaluated the accuracy and sensitivity of this procedure by testing reproducibility. RESULTS: The validity of test data was obtained via Grubbs test.The relative error of midbrain and limb bud cells were 0.776％ and 0.213％;systerm standard deviations were 0.933 9 and 2.883 9; the coefficients of variation(CV) were 1.24％ and 2.05％, respectively. CONCLUSION: High accuracy and seusitivity was obtained by the use of new parallel section mensuration method.The procedure could be used as aroutine method in micromass embryo cell cultures.