BACKGROUND＆AIM：To study the aberrant methylation of the hMSH2 gene promoter and its mRNA expression in the nickel sulfide (NiS) and anti-7,8,-dihydrodiol-9,10-epoxide benzo[a] pyrene(anti-BPDE) transformed 16HBE cells and to explore the possible epigenetic mechanism for NiS and anti-BPDE carcinogenesis. MATERIAL AND METHODS： DNA methylation patterns in the hMSH2 gene promoter were determined by methylation-specific PCR(MSP) assay and mRNA expression was analysed by RT-PCR assay. The results were compared with the non-transformed 16HBE cells which and the aberrant methylation cells were treated with demethylating agent 5-Aza-2′-deoxycytidine. RESULTS： The hypermethylation in CpG island of the hMSH2 gene promoter was indentified in the NiS and anti-BPDE transformed 16HBE cells; comparing with non-transformed cells, hMSH2 gene mRNA expression levels of the NiS and anti-BPDE transformed 16HBE cells were reduced; treatment of the hMSH2 with 5-Azac decreased the methylation. CONCLUSION： Hypermethylation in CpG island of the hMSH2 gene promoter is known to result in mRNA expression reducing and gene silencing probably, it may represent a possible epigenetic mechanism for NiS and anti-BPDE induced cells transformation and carcinogenesis. Reversible methylation offered an important evidence for phenotype inversion and drug treatment.

BACKGROUND ＆ AIM: In order to observe the anti-carcinoma use of lithium carbonate(Li2CO3) and it's mechanism, the histopathological change and the expression of malondialdehyde (MDA) and 4-hydroxynonenal(4-HNE)protein in Aflatoxin B1 (AFB1)-induced rat hepatic putative preneoplastic lesions alleviated by lithium carbonate(Li2CO3). MATERIAL AND METHODS: One hundred and forty-four healthy Wistar rats (155～175 g) were divided randomly into four groups: normal control group (group A), positive control group (group B), group treated with Li2CO3 simultaneously (group C), group pre-treated with Li2CO3 (group D). The rats were killed in batches in the 6th, 9th or 10th week of the experiment. Histopathological and immunohistochemical assay of MDA and 4-HNE protein was made on hepatic. RESULTS: The condition of rats was significantly improved in Group C and Group D, so do the severity of preneoplasm. Coinciding with the histopathological discoveries, a tendency of "Group B>Group C>Group D>Group A" could be found in observation of MDA and 4-HNE protein expressive, which occurred in the early stage (the 6th week) and significantly increased in the 10 th week. CONCLUSION: It suggested that Li2CO3 has obvious against or restrain lipid peroxidation effect on hepatoma induced by chemicals. The immunohistochemistry assay of MDA and 4-HNE can be used as a sensitive and specialize method to reflect oxidative damage，and it may help early finding and monitoring of hepatoma.

BACKGROUND ＆ AIM: To study the mechanism of injury on the fetus and infants in the brain development caused by methly mercury chloride(MMC) and its effects on the transcription activities of three RNA polymerase in the brain nuclei by MMC with experiments in vitro and in vivo. MATERIAL AND METHODS: In vivo，Wistar pregnant rats were administered with 4 mg/kg MMC of body weight by gastric perfused from the 7th to the 9th gestational days. On the 3rd , 7th,14th,21st and 30th days after birth,the rats were killed and the nuclei were harvested from their brain; in vitro, used Dnase I、EcoR I digestion for nick translation to observed the effect of MMC by scintillation counting, of 3H-dAMP. The effect to the activity of three RNA polymerase was also observed by scintillation counting, of 3H-UMP. RESULTS: The 3rd and 7th days after birth 3H-dAMP incorporation of brain nuclei were significantly lower than that of unexposed rats (P<0.01). The activity of three sorts of RNA polymerase was inhibited too. CONCLUSION:It is concluded that MMC could inhibit the translation activity of brain nuclei and the three RNA polymerases, which could explain the mechanism of inhibition to the protein synthesis.

BACKGROUND ＆ AIM： To determine whether an overexpression of IP-10 by transfection experiments in MA-10 cells has any effect on cell growth and progesterone synthesis. MATERIAL AND METHODS： We cloned the complete IP-10 cDNA in a mammalian expression vector with CMV promoter, pcDNA3.1D/V5-His-TOPO and transfected MA-10 cells. We checked the expression with rat IP-10 antibody and V5 antibody using Western blotting. The effects of overexpression of IP-10 gene on cell growth were checked by [3H]thymidine incorporation experiment. The progesterone synthesis was checked by RIA method. RESULTS: Results showed that IP-10 protein secreted in the medium was 30～40 fold more in the IP-10 transfectants over the basal levels as estimated by Western blotting. Transfection of MA-10 cells with IP-10 decreased 8-bromo-cAMP-induced progesterone synthesis from(38.5±1.7) ng/ml (1.5×105 cells/ml) of control cells to (23.2±1.5) ng/ml in transfected cells (P<0.01).8-bromo-cAMP (0.2 mmol/L) induced StAR D1 mRNA and decreased 30 ％～40 ％ by transfection with IP-10. Transfection of IP-10 gene also significantly decreased insulin-like growth factor (IGF-I, 100 ng/ml) induced [3H]thymidine incorporation into DNA. CONCLUSION: IP-10 inhibits StAR D1 expression, decreases progesterone synthesis and inhibits cell proliferation. IP-10 can be used as gene therapy for prostate cancer due to its antiangiogenic effects and its inhibitory effects on steroidogenesis.

BACKGROUND ＆ AIM： To investigate the effects of haloperidol (Hal) on oocyte chromosomes in oogenesis. MATERIAL AND METHODS: Forty healthy female mice, 12 weeks-old, were randomly divided into four groups. The animals of different groups were exposed to Hal by intra-peritoneal injection of 0, 0.05, 0.50 and 5.00 mg/(kg•d) of Hal for successive 8 days, respectively. Oocytes were collected by superovulation and the metaphase were prepared for karyotypic analysis. RESULTS: For 0, 0.05, 0.50 and 5.00 mg/(kg•d) Hal-groups, the frequencies of hyperhaploid complements were 3.8 ％,4.4 ％,7.3 ％,18 ％，the frequencies of aneuploid complements were 7.5 ％,8.8 ％,14.5 ％,36.0 ％, the frequencies of degenerative oocytes were 1.3 ％,1.4 ％,1.8 ％,10.0 ％ and the mean number of splitting chromosomes were 0.013,0.014,0.018,0.100，respectively. Comparing all the experimental parameters of the 0.05 and 0.50 mg/(kg•d) Hal-groups with those of the control, no significant differences were observed (P>0.05). All the experimental parameters of 5.00 mg/(kg•d) Hal-group were higher than those of the control, and the differences of the parameters between two groups were statistically significant. CONCLUSION: A large-dose of Hal might be able to induce aneuploidy and degeneration of oocytes in oogenesis.

BACKGROUND ＆ AIM: To compare the allelic variants of NAT1 genes the wild-type alleles between lung cancer population and the normal populations of Yunnan province. MATERIAL AND METHODS: Polymerase chain reaction restriction-fragment length polymorphism (RCR-RFLP) and allele specific-PCR(AS-PCR) methods was emplyed to justify genotype of the aim population. RESULTS: The results show that there was no significant difference between and genotype frequencies between case and control population. CONCLUSION: Our date suggest that there wasn't relationship between the genotype and lung cancer in lung cancer population and the normal populations of Yunnan province.

BACKGROUND ＆ AIM： To analyze the expressions and their sequences of cytochrome P4502D(CYP2D) subfamily gene in rat heptocarcinoma induced by dimethylaminoazobenzene(DAB). MATERIAL AND METHODS： The rats were induced by azo dye DAB ,the mRNA expression of three isoforms(CYP2D1、CYP2D2 and CYP2D4)were detected by quantitative RT-PCR,then PCR products were sequenced and analyzed. RESULTS: The expression in CYP2D2 and CYP2D4 mRNA decreased dramatically both in hepatocarcinoma and paracancerous tissues(P<0.01),and mRNA of the two isoforms in hepatocarcinoma tissues was lower than in paracancerous tissues(P<0.01). Though CYP2D1 mRNA had a trend of decrease in hepatocarcinoma and paracancerous tissues, no significant difference was found between them. Sequence analysis of those PCR products showed that only a single-site base mutation happened in CYP2D2 from hepatocarcinoma tissues. CONCLUSION: the expression of three isoforms(CYP2D1,CYP2D2 and CYP2D4) mRNA were reduced differently by oza dye DAB, DAB could result in CYP2D2 gene mutation, these indicate that the variants of CYP2D genes may contribute to the progression of hepatocarcinoma. 　　

BACKGROUND ＆ AIM: To investigate the effects of α-tocopheryl succinate(α-TOS) on S180 sarcoma and immune functions in tumor-bearing mice. MATERIAL AND METHODS: The experimental animals were divided into 5 groups, negative control, tumor model, α-TOS treatment at dose 40 mg/kg and 80 mg/kg, and cyclophosphamide positive group.α-TOS was injected intramuscly into the transplanted S180 sarcoma on mice for 5 days. The inhibitory rates of α-TOS on tumor and subsequent changes in hemogram were observed;nature killer(NK) cell cytotoxicity and the proportion of T lymphocyte subsets were detected by lactate dehydrogenase method and Flow Cytometer, respectively. RESULTS: The inhibitory rate of α-TOS at dose 40 mg/kg and 80 mg/kg on S180 sarcoma were 32.38 ％ and 38.92 ％, respectively; the amounts of leucocytes in α-TOS treatment groups were much higher than positive control (P<0.01); the NK cytotoxicity and the proportions of CD3＋ became much higher in α-TOS treatment groups compared with the tumor model(P<0.05 and P<0.01), CD4＋/CD8＋ ratios were closed to the negative control. CONCLUSION: α-TOS could be a potent cancer chemotherapeutic agent through inhibiting the growth of S180 sarcoma and improving the immunal function in tumor-bearing mice, without interfering the number of leucocytes.

BACKGROUND ＆ AIM: To observe the effects of arsenic trioxide (As2O3) on the growth of human bladder cancer cells EJ and illustrate its mechanism. MATERAIL AND METHODS: The cell proliferation inhibition was measured by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromid,MTT) colorimetric assay. Morphological changes of EJ cells were observed by fluorescence staining of Hoechst 33258. Cell cycle and apoptosis were analyzed by flow cytometry (FCM). The expression of caspase-3 in EJ cells was observed by immunocytochemistry staining. RESULTS：There was obviously a concentration-dependent relationship between As2O3 and the inhibition of EJ cells proliferation IC50 was 22.51 μmol/L. Morphological observation by fluorescence staining and FCM indicated that As2O3 induced apoptosis of EJ cells. The apoptosis rate was increased from 3.37 ％ to 19.07 ％. Immunocytochemistry demonstrated that the expression of caspase-3 in EJ cells was enhanced after treated by As2O3. CONCLUSION： The results suggest that As2O3 can inhibits human bladder cancer cells EJ growth by means of cell proliferation inhibition, expression of caspase-3 and apoptosis.

BACKGROUND ＆ AIM: To investigate the relationship among the endothelin－1(ET-1) content, microvessel density, the grading and prognosis of astrocytomas. MATERIAL AND METHODS: Seventy-four cases of astrocytoma with definite follow-up data who underwent surgical treatment in recent 8 years in our hospital were collected. Image cytometry(ICM), and Immunohistochemistry Streptavidin-Peroxidase method were carried out to study the microvessel density(MVD), the positive expression rates of ET-1 and ET-1 content. The incidence of the MVD, the positive rates of ET-1, ET-1 content, survival time and grading were evaluated with correlation analysis. RESULTS: ① The results showed that the positive rates of ET-1 and ET-1 content were both significantly different between normal astrocyte and astrocytomas, and high grade(Ⅳ-Ⅲ)astrocytomas and low grade(Ⅱ-Ⅰ) astrocytomas. But there was no significant difference between grade Ⅰand grade Ⅱ.② The results showed that MVD in high grade astrocytomas was much higher than that in low grade astrocytomas, and the survival time was significantly different among each grade (P<0.05). ③ The correlativity of the grading, ET-1 content, MVD and the survivial time show that ET-1 content have positive correlation with the grading, and MVD,but negative correlation with survival time of astrocytoma. CONCLUSION: ①It is suggested that ET-1 may act on the growth and angiogenesis of astrocytoma. ② It is suggested that ET-1 content,and MVD may be an useful method for diagnosis, grading,prognosis of astrocytoma.

BACKGROUND ＆ AIM: To investigate the mutation and methylation status of mismatch repair gene human mutl homolog l gene(hMLH1) in sporadic colorectal cancer. MATERIAL AND METHODS: Genomic DNA extracted from 50 sporadic colorectal cancer tissues was subjected to the mutation analysis of exon3,8,12,13,15,16 in hMLH1 gene by capillary electrophoresis-single strand conformation polymorphism(CE-SSCP) followed by DNA sequencing technology,and methylation of hMLH1 promotor was measured by HapⅡ,MspⅠenzyme and reverse transcriptase-polymerase chain reaction(RT-PCR) technology. RESULTS: ① Among the 50 cases,3 cases showed the missense mutation in exon 12(GTT→GAT，Val384Asp),and the mutation rate was 6 ％.② 9 cases showed methylation of hMLH1 promotor,and methylation rate was 18 ％. CONCLUSION: Mutation of hMLH1 gene in sporadic colorectal cancer may be casual events;people with the missense mutation in exon 12 of hMLH1 gene may be subject to sporadic colorectal cancer;there is no significant correlationship between hMLH1 gene methylation and clinicopathology in sporadic colorectal cancer.

BACKGROUND ＆ AIM： Human lymphoblastoid cell line TK6 was used to investigate the in vitro genotoxicity of Microcystin-LR. MATERIAL AND METHODS： Cytotoxicity response, micronucleus(MN) and mutation frequency at tk locus induced by MCLR after 4 h or 24 h treatment were detected. RERULTS： Treatment with MCLR for 4 h did not induce a significant cytotoxic response at less than 80 μg/ml. Exposure to MCLR for 24h decreased relative survival(RS), induced both MN and TK mutation in a concentration-dependent manner. The maximum induction of MN and TK mutation were 4.8 and 5.1 times those of the control,respectively. Two distinct phenotypic colonies of TK mutants were generated , namely tk-NG and tk-SG mutant colonies but the latter dominated. CONCLUSION： MCLR was clastogenic in TK6 human lymphoblastoid cells .

BACKGROUND ＆ AIM: To explore the feasibility of hepatitis B virus (HBV) vertical transmission via male germ line, the replication and expression of sperm-mediated HBV DNA in mouse embryo were studied. MATERIAL AND METHODS: Young adult male mice were injected with HBV DNA-liposome complex. These males were serially mated with superovulated females 2 days after injection. Fluorescence in situ hybridization (FISH) was carried out to confirm the integration of HBV DNA into male pronucleus and its replication with cell division in embryonic development.(Reverse transcriptase polymerase chain reaction,RT-PCR) and immunofluoresence assay were performed to observe the expression of the HBV gene in two-cell stage. RESULTS: FISH demonstrated that the male pronuclei in some one-cell embryos and the each nucleus in some two-cell embryos presented positive signals. RT-PCR showed the specific bands of cDNA of HBx DNA in some two-cell embryos. Immunofluorescence assay presented the positive signals for HBsAg in some two-cell embryos. CONCLUSION: Our results provided the direct evidence for that HBV DNA are able to transmit vertically to next generation via male germ line.

BACKGROUND ＆ AIM: To study whether hepatitis B virus DNA carried by oocyte can express and replicate in mouse embryo, then provide evidence for that HBV can transmit vertically via oocytes. MATERIAL AND METHODS: Ovaries of young adult female mice were injected with HBV DNA. These females were superovulated after an estrual cycle and serially mated to males. Two-cell embryos were collected for Fluorescence in situ hybridization (FISH) to detect the integration of HBV DNA into nuclei and its replication with cell division in embryonic development. Reverse transcriptase polymerase chain reaction(RT-PCR) and immunofluorescence assay were performed to observe the expression of the HBV gene in two-cell stage. RESULTS: FISH demonstrated that the each nucleus in some two-cell embryos presented positive signals for HBx DNA.RT-PCR showed the specific bands of cDNA of HBx DNA in some two-cell embryos. Immunofluoresence assay presented the positive signals for HBsAg in some two-cell embryos. CONCLUSION: HBV DNA integrated into oocyte genome can be brought into embryo by fertilization with normal spermatozoon. The replication and expression of HBV genes in embryo provide the direct evidence for that HBV DNA are able to transmit vertically to next generation via female germ line.

BACKGROUND ＆ AIM： To observe whether there are embryonic and teratogenic toxicity on rats disposed by different dosages of recombinant human glucagon_like peptide(rhGLP) continuously during the period of organ formation. MATERIAL AND METHODS： The SD rats were used in this experiment. The pregnant rats were divided into five groups, three dosage groups (260,130 and 65 μg/kg), one positive and one negative control groups. There were more than 16 pregnant rats in every group. The treated groups and the negative control (normal physiological saline 1.0 ml/kg) group were injected continuously via hypodermic of back neck every day for ten days during the period of organ formation, while the positive control group was disposed via mouth by 10.0 ml/kg of aspirin. The numbers of luteal, nidation, alive_embryo, forepart fetal death, advanced stage fetal death were counted after the pregnant rats were sacrificed by disjointed cervical vertebrae at the twentieth day. After observed the sex, appearance and weight of embryonic rats, they were divided into two groups. In one group, the bone development was observed after ethanol fixing, potassium hydroxide melting, Alizarin Red staining transparencing. In another group, the viscera development was observed after Bouins solution fixing under and glycerin big_inspect_instrument. RESULTS： There were no obvious development abnormality in embryonic rats' appearances, bones and viscera in every disposed dosage groups. CONCLUSION： Recombinant glucagonant_like peptide has no embryonic and teratogenic toxicity under the dosage of 260 μg/kg.

BACKGROUND ＆ AIM: To check the mutagenicity of the matter extracted from the Pulp of cornus by ethanol. MATERIAL AND METHODS: Chromosome aberration test, micronucleus test of bone marrow cell and sperm shape abnormality test in mice were used in this study. RESULTS: The test result shows that: there have no significant difference between the matter extracted from the pulp of cornus by ethanol and negative control, but has significant difference to positive control. CONCLUSION: The matters extracted has no damage effect on the chromosome and cell levels. It provides science proof on exploiting functional health food of cornus of ficinalis sieb.et Zucc.