BACKGROUND ＆ AIM:To explore the possibility of human mesenchymal stem cells (hMSCs) differentiating into neural cells in vivo. MATERIALS AND METHODS: hMSCs labeled with enhanced green fluorescent protein (EGFP) were transplanted into the lateral ventricle of neonatal mouse brain. At 0, 9 and 14 days post-transplantation, mice were sacrificed and their brains were fixed with 4％ paraformaldehyde. The engraftment of transplanted cells in coronal section of the recipient mouse brain was examined under a fluorescent microscope. Indirected immunofluorescent staining was used to detect the expression of neural proteins of these grafted cells. RESULTS: Examination of cryostat section under fluorescent microscopy revealed that hMSCs could engraft into neonatal mouse brain. Neuronal differentiation was confirmed by the expression of β-Ⅲ-Tubulin (Tuj1) and MAP2 of these EGFP-expressing cells, whilst some grafted cells had GFAP staining. CONCLUSION: These results suggest that hMSCs could respond to brain microenvironment and differentiate into neural cells.

BACKGROUND ＆ AIM: To construct sited-directed mutants of human UGRP1 gene promoter. MATERIALS AND METHODS: Mutants were constructed by overlap extention method. The plasmid pGL3-UGRP1(-112G) was used as the template， site-directed mutagenesis was performed by overlap- extention PCR method at －112 in UGRP1 gene promoter,and the expression vector of mutant at －112 was then constructed. RESULTS: By DNA sequencing, human UGRP1 gene promoter was successfully changed from G to A at －112 bp and the expression vector with the site-directed mutants were constructed. CONCLUSION: Overlap-extension PCR method was convenient and efficient for site-directed mutagenesis. Contruction of desired mutant pGL3-UGRP1(－112A) may provide a basis for the research on regulating transcriptional activity of G/A polymorphism at －112 bp of human UGRP1 gene promoter.

BACKGROUND ＆ AIM: To prepare a fusion vaccine with esophageal carcinoma cell (EC109)and dendritic cells(DC), and to study the protective and therapeutic effects against EC109 cells. MATERIALS AND METHODS: Fusion vaccine of EC109-DC(ED) was produced by traditional PEG fusing, cytokine inducing, CD34＋ magnetic microbead marker sorting, and MACS(magnetic cell sorting system). The therapeutic and protective effects of the fusion vaccine against tumor EC109 were analyzed by flow cytometry. RESULTS: The fusion cells ED expressed high levels of FR(folate receptor), CD80, CD83 and CD86. No tumor was found in the spleen, lung and liver of mice after the injection of fusion vaccine ED. In the immuned group, latent period was longer in ED subgroup than in others, and tumor size and tumor weight were also less than those in ED subgroup. In the therapeutic group, tumor size and tumor weight were less in ED subgroup than in control(PBS group), inactivated EC109 and DC group. CONCLUSION: The fusion cells EC109-DC expressed specific antigens. The fusion vaccine didn＇t have oncogenetic effects in vivo, but demonstrated immunologic protection against tumor EC109 and certain effect on inhibiting tumor growth in mice.

BACKGROUND ＆ AIM: To study the anti-hepatofibrotic effect of the Achillea millefolium L.(Aml) extract on experimental hepatic fibrosis and its partial mechanism. MATERIALS AND METHODS: The hepatic fibrosis rat model was developed by subcutaneous injection of 40％CCl 4 (5 ml/kg) and rats were treated with extract of Aml at 3 ml/kg, 6 ml/kg or 12 ml/kg. Alanine aminotransferase(ALT),aspartate aminotransferase(AST)levels were measured and liver tissue examined for pathological changes after HE staining. RESULTS: The extract of Aml could reduce the abnormally high levels of ALT and AST. Examined by light microscope，the degeneration and necrosis level of hepatocytes and the distribution of collagen fibrosis were markedly increased in the model group. The pathological changes and the c ollagen fibrosis associated with hepatic fibrosis decreased significantly in the prophylactically treated groups. CONCLUSION: Extract of Aml could protect hepatocytes from injury and prevent the progression of hepatic fibrosis in rats.

BACKGROUND ＆ AIM: We made anti-human diamine oxidase (DAO) monoclonal antibody, analyzed the expression of diamine oxidase in colorectal tissue to assess its significance in pathologic diagnosis and its correlation with clinicopathologic parameters. MATERIALS AND METHODS: We used immunohistochemical technique to study the expressions of DAO in different colorectal tissues.The expression was compared with clinicopathological features of colorectal neoplasia. RESULTS: DAO positive expression was found in cell cytoplasm. There were significant differences among the colorectal carcinomas, tous adenomatous polyps and mucosa adjacent to cancer. DAO positive expression rate in villous adenoma was significantly higher than that of tubulo adenoma(P<0.05). DAO was expressed in dysplatic tissues, and frequency increased with the progression of the dysplasia. CONCLUSION: DAO may be associated with the growth of colorectal neoplasia. It could be an early biomarker in human colorectal carcinomas and could predict growth of tumor at an early stage.

BACKGROUND ＆AIM: To evaluate the effects of hydroxyurea(HU) on embryonic development, effects of hydroxyurea on proliferation and differentiation were studied by using rat embryo midbrain(central nervous system, CNS) cell micromass culture. MATERIALS AND METHODS: Midbrain cells from 13-day rat embryos were harvested. After exposed to different doses of hydroxyurea in the culture medium for 5 days, proliferation and differentiation of neurons from midbrain cells were examined. RESULTS: Hydroxyurea could significantly inhibit the proliferation and differentiation of midbrain cells in a dose dependent manner, and could arrest neurogenesis and colony formation. The numbers of differentiated foci decreased with increasing concentration of HU, and nerve fiber bundle reduced during the differentiated foci,showing a typical dose-effect relationship. The IcV50 value of hydroxyurea was 0.078 mmol/L(5.9 μg/ml)and the IcD50 value was 0.018 mmol/L(1.37 μg/ml). The ratio of IcV50 and IcD50 was 4.33. CONCLUSION: Hydroxyurea is a non-specific inhibitor of cell proliferation and differentiation; it could potentially result in teratogenicity in vitro.

BACKGROUND ＆ AIM: Hyaluronan(HA), a kind of polysaccharide with high molecular weight, is present extensively in the mesenchyme of colon carcinoma. The objective of this study was to investigate the effects of HA on proliferation, adhesion and invasion abilities of human colon carcinoma cell SW480 in vitro. MATERIALS AND METHODS: cultured SW480 cells were divided into three groups: control group (C): cultured with serum-free medium(SFM); HA1 and HA2 treatment groups: cultured with SFM containing different concentrations of HA (HA1: 10 μg/ml, HA2: 20 μg/ml). After some time, proliferation ability was examined by MTT assay and soft agar clone formation assay, while adhesive and invasive abilities were assessed by plate adhesion model and Boyden chamber transwell assay. RESULTS: Compared with that in C group，in HA1 and HA2 treatment groups, the number of proliferative cells，soft agar cell clone formation, cell adhesion on plate and septum permeation of Boyden chamber increased significantly in HA1 and HA2 treatment groups in a HA dose-dependent manner(P<0.05). CONCLUSION: HA could enhance the proliferation, adhesion and invasion abilities of human colon carcinoma cell SW480 in vitro.

BACKGROUND ＆ AIM： To evaluate the effects of green tea (GT) on Microcystin-LR (MC-LR)-induced hepatocelluar apoptosis, Bcl-2 expression and micronucleus test so as to explore antagonistic mechanism of GT. MATERIALS AND METHODS： 50 male mice were randomly divided into five groups. Mice in GT pretreated groups were given green tea as free drink at doses of 2 g/(L·d) and 12 g/(L·d) prior to MC-LR intoxication, consecutively for 18 days. The toxin treatment mice in group MC-LR control received continual intraperitoneal injections of MC-LR at dose of 10 μg/(kg·d) for 13 days from day 6 till sacrifice. CP control group was treated with intraperitoneal cyclophosphamide twice at dose of 50 mg/(kg·d) 24 h interval on days 17 and 18. Mice were sacrificed and immediately subjected to autopsy. Hepatocellular apoptosis, Bcl-2 protein expression and micronucleus frequencies of bone marrow were evaluated immediately. RESULTS： (1) MC-LR induced obvious hepatocellular apoptosis. High dose of GT pretreatment significantly inhibited MC-LR-induced hepatocellular apoptosis(P<0.05).(2) There was no significant change of Bcl-2 protein expression in MC-LR control compared with control. As compared with only MC-LR, the expression of Bcl-2 protein was significantly increased in GT pretreatment groups(P<0.01).(3) No significant difference in micronucleus frequencies was found in either MC-LR control or GT pretreatment groups compared with control (P>0.05). CONCLUSION： GT could increase Bcl-2 protein expression and inhibit hepatocellular apoptosis，MC-LR and GT pretreatment did not cause damage to mice chromosome.

BACKGROUND ＆ AIM: To study on the susceptibility of two human-derived hepatic cell lines in evaluating DNA damage induced by 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone(MX). MATERIALS AND METHODS: Using single cell gel electrophoresis assay, to evaluate the DNA damage induced by MX in human hepatocellular carcinoma cell lines(HepG2) and human fetal hepatocytes (L-02). RESULTS: MX could induce HepG2 and L-02 DNA single strand breaks at the concentrations of 30 and 100 μmol/L, respectively. The length of DNA migration induced by MX increased significantly in comparison to solvent control (DMSO) in a dose-response manner(P<0.05). Moderate or greater DNA damages induced by MX in HepG2 were more serious than that of L-02(χ2=20.985；8.0；11.97， P<0.05). CONCLUSION: HepG2 was more susceptible than L-02 cells to DNA damage induced by MX evaluated by using single cell gel electrophoresis assay.

BACKGROUND ＆ AIM: Human somatic cells have species superiority over animal cells in genotoxicity assay. This study aimed to demonstrate the performance of specific gene mutation in two human lymphoblastoid cell lines with different p53 status for its further use in genotoxicity assay. MATERIALS AND METHODS: TK and HPRT mutation was induced by four chemical mutagens, including EMS (ethyl methanesulfonate), MMS (methyl methanesulfonate), GLY (glyoxal), OPD (o-phenylenediamine). Mutants were selected by microwell method. RESULTS： TK6-E6 was more tolerant to cytotoxic agents than TK6. All 4 agents induced positive mutation responses at two loci in two cell lines. The most sensitive mutation was at TK locus in TK6-E6. The RSC (rate of slow growth colonies) in TK6 was much lower than that in TK6-E6, and there was little variation in RSC to those mutagens. CONCLUSION: TK and HPRT mutation test in two cell lines could be used to assay genotoxicity. TK6-E6 was superior to TK6 in view of higher sensitivity and higher dosage treatment allowance.

BACKGROUND ＆ AIM: To study the potential carcinogenic risk of siritch oil fume condensate (SOFC) to human. MATERIALS AND METHODS: Different concentrations of SOFC were co-incubated with human embryo lung diploid cells. The degree of malignant transformation was observed by assessing the biological characteristics of the cells. RESULTS: The concentration of SOFC within the dose range of the experiment(25～400 μg/ml) could induce malignant transformation of the human embryo lung diploid cell, and with an obvious dose-response relationship. Transformed cells exhibited many characteristics associated with malignant transformation, such as losing contact-dependent inhibition, saturated density angmentation, growing at low serum concentration, agglutinating by different concentrations of ConA, losing anchorage dependence, forming tumor at inoculation site of athymic mouse. CONCLUSION: SOFC could induce human embryo lung diploid cell malignant transformation and showed potential carcinogenicity to human.

BACKGROUND ＆ AIM: To investigate the relationship between the toxins of Alternaria alternata and the incidence of esoghageal cancer in Linxian. MATERIALS AND METHODS: Alternaria alternata was isolated from grains in Linxian and its toxins from grains and urine from residents in Linxian were examined by the thin-layer chromatography, and results were compared with previous measured levels. RESULTS: ①the rate of contamination by Alternaria alternata in wheat was less than that in 1981 (χ2=359.283，P<0.01). ② the positive rate of AOH in wheat in 2004 were lower than those in 1995 (χ2=5.152，P<0.05). ③the positive rate of AOH in the wheat flour was lower than those in grain (χ2=5.128，P<0.05). ④ 4 out of 99 samples of urine contained AOH ,and 1 of these had AME in 2004. The results were all lower than those in 1992, (χ2=7.082，P<0.01; χ2=14.282,P<0.01 respectively). CONCLUSION: There is an association between the decrease in exposure to the toxins of Alternaria alternata and the lowered incidence of esophageal cancer in local residents.

BACKGROUND ＆ AIM: To investigate the acute toxicity and the inheritable toxicity of 1,2-dimethyl-3- hydroxypyrid-4-one(DHPO) in mice. MATERIALS AND METHODS: LD50 test was performed ,then Kun Ming mice were given different oral doses of the DHPO to carry out the micronucleus test under low(1/8 LD50), mid (1/4 LD50), high(1/2 LD50) doses. RESULTS: LD50 of DHPO was 562.34 mg/kg peroral in mice. 1/2 LD50 group was different from the negative control in the micronucleus test(P<0.05), whereas no significant differences were observed among 1/4 LD50 , 1/8 LD50 groups and negative control(P>0.05). CONCLUSION: Our results suggested that DHPO had low toxicity but had inheritable toxicity to a certain extent.

BACKGROUND ＆AIM: To study the relationship between polymorphisms of methylenetetrahydrofolate reductase C677T and hyperuricemia in males . MATERLALS AND METHODS: A case-control study was conducted with 91 males with hyperuricemia and 81 males as control. Anthropometric, blood pressure and biochemical variables, including serum lipids, glucose,serum uric acid, creatinine and urea nitrogen were measured. MTHFR genotypes were detected by DNA microarray technology. RESULTS: The frequency of MTHFR T allele among the cases(50.5％) was significantly higher than the controls(38.8％)(P=0.028),the odds ratios for hyperuricemia was 1.615. Uric acid , triglyceride concentrations and body mass index were markedly higher in subjects with TT genotype than in subjects with CC genotype(P<0.05), homozygotes for the MTHFR mutation had a significantly higher mean uric acid level (458.69±128.51 μmol/L)than those without mutation(392.77±118.90 μmol/L,P<0.05), whereas heterozygotes had an intermediate value (411.28±118.34 μmol/L). CONCLUSION: MTHFR C677T mutation may be considered a risk factor for hyperuricemia in Chinese males.

BACKGROUND ＆ AIM： To investigate the role of Bcl-2，Bcl-xL and Bax protein in colorectal carcinomatous tumorigenesis by detecting their expression in cancerous and adjacent tissues(>5cm from the cancerous tissue). MATERIALS AND METHODS： The expression of Bcl-2，Bcl-xL and Bax protein by S-P staining were detected in 39 colorectal cancerous tissues and adjacent tissues. RESULTS：The expression of Bcl-2 protein were often absent or weak，and no significant difference between cancerous tissue and adjacent tissue was found(Z=0.072,P>0.05).On the contrary，the immunostaining intensity of Bcl-xL protein was higher and stronger in cancerous tissues than adjacent tissues(Z=3.157,P<0.05), The immunostaining intensity of Bax protein was the strongest，but showed no significant difference between cancerous and adjacent tissues(Z=1.707,P>0.05). Only the expression of Bcl-2 protein demonstrated a negative correlation with Dukes and TNM stages(rs=－0.389,－0.396,P<0.05), all others had no relationship with clinicopathological parameters(P>0.05). CONCLUSION： Bcl-2 gene might play an important role in the early stage of colorectal carcinomas，however, Bcl-xL gene might be more important in the later stage. On the other hand, Bcl-2 protein expression may correlate with prognosis.

BACKGROUND ＆ AIM： To assess the effects of selenium on MMC-induced DNA damage in human peripheral lymphocytes. MATERIALS AND METHODS： The effects of selenium on the DNA damage and chromosome damage induced by MMC were evaluated using single cell gel electrophoresis(SCGE)，sister chromatid exchange(SCE) and chromosome aberrations (CA) in human peripheral lymphocytes. RESULTS: The tail length ,SCE frequencies and the chromosomal aberration rate of three doses of selenium were significantly lower than that of the positive control. Furthermore, the extent of DNA damage of medium dose group of selenium(6 μmol/L) was lower than the other groups. CONCLUSION: To some extent, selenium could protect DNA damage induced by MMC. The protective effect of medium dose of selenium (6 μmol/L) was stronger than the other doses.

BACKGROUND ＆ AIM: To study the effects and mechanisms of pleurotas sapidus(PS) extract on function of 5-fluorouracil(5-Fu) in chemotherapy. MATERIALS AND METHODS： Nude mice transplanted with human cervical carcinoma were divided into four groups randomly，model group , 5-Fu group , PS group and PS＋5-Fu group. After 14 days the markers in nude mice were assessed. RESULTS： The weight and volume of tumor in model group were higher than that in other three groups. The weight and volume of tumor in PS＋5-Fu group(0.19±0.03) g and(0.134±0.016) cm3,respectively, were lower than that in 5-Fu group(0.30±0.03) g and (0.210±0.018) cm3,respectively.The number of leukocytes in peripheral blood in 5-Fu group(3.52±0.43)×10 9/L was lower than that in PS＋5-Fu group(5.00±0.74)×10 9/L and model group(5.51±0.50)×10 9/L. Significantly higher expressions of Bax and caspase-3 protein of tumour tissues were observed in nude mice exposed to combination of 5-Fu plus PS compared with the nude mice exposed to 5-Fu alone. CONCLUSION： The extract from pleurotas sapidus had the synergistic antitumor effect and increased the number of leukocytes in 5-Fu chemotherapy，which were related to the upregulation of Bax and caspase-3 protein expressions.

BACKGROUND ＆ AIM: To study the embryonic and teratogenic toxicities of chitosan in rats during the period of organ formation. MATERIALS AND METHODS: Ninety pregnant SD rats were randomly divided into five groups with 18 rats in each, three chitosan dosage groups (50, 250, 1 000 mg/kg), one negative and one positive control group. Chitosan and normal saline was given by gavage to the treated groups and the negative control group for ten days during the period of organ formation (the 6th to 15th day of gestation),respectively. Rats in the positive control group received intraperitoneal cyclophosphamide (CP) on the 11th to 13th day of gestation at a daily dose of 7 mg/kg. The rats were sacrificed to examine the fetuses on the 20th day. RESULTS: The results showed that chitosan didn't produce significant effects on health, behavior, weight gain and reproductive parameters of pregnant rats and fetal growth, and didn't induce any teratogenic effect on the appearance, bones and bowels of the fetuses. CONCLUSION: Chitosan has no embryonic and teratogenic toxicity when used under 1 000 mg/kg.

BACKGROUND ＆ AIM: To study the anti-tumor effect of [pulsatilla chinensis(Bunge)regel,PCR] extracts. MATERAIALS AND METHODS: The effect of PCR extracts on the proliferation of tumor cell lines were assayed in vitro using 3-[4,5-di methylthiazol-2-yl]-2,5 diphenyltetrazoliumbromide colorimetry method.The anti-tumor active component of PCR extracts was separated and purified using three steps methods. RESULTS: PCR extracts markedly inhibited the proliferation of tumor cell lines and in a dose-dependent manner. The anti-tumor active component was indentified. CONCLUSION: PCR awaits further development as an anti-tumor drug.

BACKGROUND ＆ AIM: To study the toxicity attenuation and antimutagenic effects of health-preserving cigarettes made from Chinese herbal medicine. MATERLALS AND METHODS: The health-preserving cigarettes were made by certain Chinese herbal medicine such as piper sarmentosum Roxb. We made these cigarettes into a smoke liquid of normal saline solution as an experimental material to do an acute toxicity test in mice.A micronucleus test of mice bone marrow polychromatophilic erythroblast(PCE) and an Ames test were performed. RESULTS: The LD50 of mice in the acute toxicity test was four to five times more than the control group. The micronucleus test of PCE in the mice bone marrow was 6‰～10‰, obviously lower than the original control group(16‰～28‰) with a dose-effect relationship(r=0.9554). In the Ames test，the number of the bacterial mutagenesis could be reduced 69％～83％ of that in the control group,also with a dose-effect relationship(r=0.9843). CONCLUSION: Health-preserving cigarettes made from Chinese herbal medicine could greatly lower the toxicity of cigarettes，and have obvious antimutagenic effect.

BACKGROUND ＆ AIM: To investigate p16 gene alterations in breast cancer. MATERIALS AND METHODS: 33 fresh tumor specimens taken from breast cancer patients were analyzed for p16 gene deletion and mutation by polymerase chain reaction (PCR) and DNA sequencing. RESULTS: No homozygous deletion of p16 gene exon 1, 2 and 3 was observed in any of the 33 cases and no point mutation of p16 gene exon 2 was detected in 10 specimens. CONCLUSION: The finding suggested that there was little or no relationship between the homozygous deletion and point mutation of p16 gene and breast carcinogenesis.

BACKGROUND ＆ AIM: To prepare green fluorescent protein(GFP) polyclonal antibody. MATERIALS AND METHODS: Muscle from one GFP-transgenic mouse was taken as antigen, mixed with adjuvant sufficiently,then immunized homogeneous mice. Blood was collected 4 weeks later and the serum was used for immunocytochemistry, immunohistochemistry and western blot. RESULTS: The serum we collected showed strong positive response when diluted at 1∶200. CONCLUSION: Green fluorescent protein polyclonal antibody could be prepared directly with muscle of GFP-transgenic mouse, with which we efficiently detected GFP positive cells transplanted in the liver.