BACKGROUND ＆ AIM: To construct the fluorescence protein expression vector as a reporter for studying the transdifferentiation potency of liver epithelial progenitor cells (LEPCs) to pancreas-like cells. MATERIAL AND METHODS: The Pancreatic duodenal homeobox-1(PDX-1) promoter was amplified by PCR from mouse tail genomic DNA and subcloned into promoter-removed pEGFP N1 vector. By using the reporter gene tagging cells, the phenotype variation of LEPCs transdifferentiated into pancreas-like cells could be evaluated. RESULTS: EGFP expression vector driven by PDX-1 promoter could monitor the transdifferentiation of LEPCs into pancreas-like cells in a real-time manner. CONCLUSION: The reporter vector is useful for studying the transdifferentiation of LEPCs into pancreas-like cells.

BACKGROUND ＆ AIM: To evaluate the mechanisms involved in acquiring the TRAIL drug-resistance gene in human colon cancer DLD1 cell line. MATERIAL AND METHODS: DLD1 cells were repeatedly treated with adenovirus-mediated TRAIL gene(Ad/gTRAIL), and DLD1-TRAIL/R cells resistant to Ad/gTRAIL were obtained. The cell growth was measured by MTT method. The expression of proteins involved in TRAIL-mediated apoptosis pathway was also showed by western blot analysis. Then the possible mechanisms involved in acquiring resistance were analyzed. RESULTS： DLD1-TRAIL/R cells resistant to TRAIL were obtained from TRAIL-sensitive DLD1 parent cells. But they were still sensitive to adenovirus-mediated Bax gene therapy. Further studies demonstrated that expression of Bcl-XL was dramatically increased whereas caspase-8 was significantly decreased in DLD1-TRAIL/R cells. Simultaneously, there was no cleaved caspase-8 found in the DLD1-TRAIL/R cells. CONCLUSION: After repeated treatment, DLD1 cells could acquire the specific resistance to TRAIL gene. The involved mechanisms may involve up-regulation of Bcl-XL and down-regulation of caspase-8.

BACKGROUND ＆ AIM: To study the aberrant DNA methylation of genomic DNA in lung cancer tissues as a possible epigenetic mechanism in the development of lung cancer. MATERIAL AND METHODS: Genomic DNA isolated from lung cancer and its adjacent tissue was restriction digested with Mse1 (methylation non-sensitive) alone or with Mse1 and BstU1 (methylation sensitive). The resulting DNA was analyzed for aberrant methylation using a PCR-based technique- Methylation-sensitive Restriction Fingerprinting (MSRF). Several DNA fragments differentially methylated in the transformed cells compared with the non-transformed cells were identified by MSRF. These fragments were subcloned, sequenced and compared with GenBank. At the same time, the DNA methylation was analyzed according to age, sex and smoking status. RESULTS: As compared with the control tissues, 84.5％ (49/58) of lung cancer tissues were found to have aberrant DNA methylation, but the methylation rate did not differ significantly among the squamous carcinoma (82.1％), and adenocarcinoma (88.5％) and other cancer types (75.0％)(χ2=0.073, P>0.05). Among the fragments methylated, 83％ was hypermethylated and 17％ was hypomethylated. We have identified the fragments encoding for human cyclin C (CCNC) and Wilms tumor (WT-1). When smoking, age and sex were considered as factors, no significant differences were observed. CONCLUSION: The aberrant DNA methylation, especially hypermethylation, seemed to play an important role in the development of lung cancer. It appeared to be a possible epigenetic mechanism for lung cancer.

BACKGROUND ＆ AIM: Inhibitory effects of 5-azacytidine on the proliferation of human breast cancer cell line Bcap-37 and its mechanism were investigated. MATERIAL AND METHODS： After treatment of Bcap-37 cells with 5-azacytidine, cell proliferation was analyzed by cell counting . The methylation and unmethylation status of RASSF1A gene were detected by methylation special PCR(MSP)and RASSF1A mRNA expression was detected by RT-PCR. RESULTS: After treatment with 5-azacytidine(>5 μmol/L),the Bcap-37 cells growth rate was decreased in a dose-dependent manner. Demethylation of RASSF1A gene occurred and RASSF1A mRNA expression was increased. CONCLUSION： 5-Azacytidine could effectively enhance RASSF1A gene demethylation and enhance the expression of RASSF1A mRNA and inhibit cell growth.

BACKGROUND ＆ AIM: To study the antitumor activity and immunoregulatory activity of Chinese medicinal herb Rhizoma Menipermi extracts in vivo. MATERIAL AND METHODS: The chemical composition of Rhizoma Menipermi extract was separated with water, ethanol extraction and distillation methods. The suppressive effect of Rhizoma Menipermi extracts on the proliferation of tumor cells were assayed in vitro using MTT colorimetric method. The effect of Rhizoma Menipermi extracts on lymphocytes and the effect on the phagocytosis of mouse peritoneal macrophages were studied using MTT colorimetric method and neutral red method. The tumor bearing mouse model was constructed by injecting tumor cells subcutaneously. Then PE2 from Rhizoma Menipermi extract was given to the mouse orally to observe its antitumor activity and immunoregulatory activity in vivo. RESULTS: Rhizoma Menipermi extracts RMW and RME markedly inhibited the proliferation of tumor cells such as K562. The effect of RME was stronger than RMW.The PE2 constituent purified from RME might be the main antitumor active part of Rhizoma Menipermi as shown by antitumor test in vitro. After treatment with PE2, the general condition of the test mice was much better than that of control mice, with tumor growing more slowly and life prolonged. CONCLUSION: PE2 might be the main antitumor active part of Rhizoma Menipermi. PE2 also had antitumor effect in vivo. PE2 could exert antitumor effect in vivo by enhancing the phagocytosis of macrophages and by enhancing NK cells activity.

BACKGROUND＆AIM：To investigate the effect of an anti-CD44 antibody,HI44a,in inducing differentiation and apoptosis of freshly isolated acute myeloid leukemic cells. MATERIAL AND METHODS：The effect of HI44a on the differentiation and apoptosis of fresh leukemia cell from 31 acute myeloid leukemia patients were studied in vitro. Cell morphology, nitroblue tetrazolium(NBT) reduction and expression of CD11b, CD14 and CD15 were measured. Early apoptotic cells were detected by annexin-Ⅴassay. Expressions of G-CSF, M-CSF and c-myc transcript were investigated by RT-PCR. RESULTS：In the presence of HI44a, the primary leukemia cells became more mature in morphology, the percentage of NBT-positive cells increased to 31％ (control 9％), 55％ (control 10％), 25％ (control 12％) and 32％(control 11％) in all the four subtypes, there was a significant difference between the groups (P<0.01). The expression of CD11b, CD14 and CD15 also increased to 19.29％,40.60％and 66.82％, respectively, whereas that in untreated control cells was 9.65％，27.40％ and 57.38％. In addition, HI44a could efficiently induce leukemia cell to undergo apoptosis, the mean percentage of early apoptotic cells was significantly increased compared to the untreated control AML cells (41.18％ vs 26.21％). This effect was associated with enhanced M-CSF transcript expression and decreased c-myc transcript expression. CONCLUSION： HI44a could effectively induce differentiation and apoptosis of leukemia cells and thus warrants further studies as a therapy for acute myeloid leukemia.

BACKGROUND ＆ AIM: To study the effect of lead acetate on the apoptosis and the expression of fos, jun, P53 and NOS and to provide some scientific basis for further delineation of the neurotoxic mechanisms of lead. MATERIAL AND METHODS: Mature and healthy Sprague-Dawley rats were randomly divided into four groups. Lead acetate was injected intraperitoneally. The determination of apoptosis in hippocampus and cerebral cortex was made by terminal-deoxynucleotidyl transferase mediated d-UTP nick and labeling(TUNEL). The expression of fos, jun, P53 genes and nNOS, iNOS in hippocampus and cerebral cortex were measured by using immunohistochemical method. RESULTS: ① TUNEL showed that lead acetate induced apoptosis of cells from hippocampus, cerebral cortex in every treatment group (P<0.05), and there was a significant dose-response relationship.② The expression of fos, jun, P53 and iNOS increased in neural cells from hippocampus, cerebral cortex in every lead acetate treatment group compared with the control.③ The expression of nNOS significantly increased in hippocampus in every treatment group compared with the control.However the expression of nNOS in cerebral cortex showed no significant difference between the treatment groups and the control group.④ Correlation analysis demonstrated that apoptosis correlated positively with the expression of fos, jun, P53 and iNOS. CONCLUSION: The overexpression of P53, which was caused by damage of DNA elicited by the excessive amount of NO from the overexpression of NOS, induced apoptosis of neural cells. Lead acetate also induced the prolonged expression of fos and jun which may initiate the expression of P53 followed by apoptosis of neural cells.

BACKGROUND ＆ AIM： To investigate the effects of the untreated water and chlorinated drinking water extracts of the Han River on DNA damage and the expression of the gadd153 promoter and mRNA. MATERIAL AND METHODS： The DNA damage was assessed by the alkaline comet assay. The plasmid(pGADD153-Luc)containing DNA damage and repair inducible gene 153 (gadd153) promoter and luciferase reporter gene were constructed. The activity of gadd153 promoter was represented by the luciferase activity, and the inducible luciferase activity was detected by bioluminescence. The expression of gadd153 mRNA was detected by RT-PCR. RESULTS： The Olive Tail Moment(OTM) induced by the untreated water and chlorinated drinking water extracts was increased at the dose of 10, 100ml/ml medium (P<0.01), compared with control. There was a good dose-response relationship (r=0.882, P<0.05; r=0.940, P<0.05); The OTM induced by the chlorinated drinking water extracts was higher than that of untreated water (P<0.05). The luciferase activity was significantly increased in each treatment group at each dose (P<0.01) and there were a good dose-response relationship (r=0.814, P<0.05; r=0.921, P<0.05). There were positive correlations between the OTM and the luciferase activities (r=0.980, P<0.01, r=0.995, P<0.01); The high expression of gadd153 mRNA was induced by the water extracts at dose of 100 ml/ml medium (P<0.05). There werepositive correlation between the OTM and the expressions of gadd153 mRNA (r=0.864, P<0.05; r=0.897, P<0.05). CONCLUSION： The untreated water and chlorinated drinking water extracts of Han River could induce DNA damage and further activated the gadd153 promoter which regulated the expression of gadd153 mRNA.

BACKGROUD ＆ AIM: To explore the malignant transformation effects of Yunnan tin mine dust on human bronchial epithelial cells, further investigate the cause and mechanism of lung cancer in Yunnan tin miners. MATERIAL AND METHODS: The immortalized human bronchial epithelial cells BEAS-2B were treated with tin mine dust at the concentrations of 200 μg/ml and 50 μg/ml for 72 hours on every other generation and was stopped in the 9 th generation, including a negative control group. The characteristics of the cellular biology and the malignant transformation phenotype of cells were identified through observing serum resistance, anchorage independent growth, etc. RESULTS: Each group of cells in the 20 th generation didn't show serum resistance. For the high mine dust concentration group cells in the 25 th generation, the multiplication time, the serum resistance and the aberration of chromosome were increased, with the formation of small cell colonies in soft agar in the 30th generation. The anchorage independent growth appeared in high and low concentration groups in the 40th generation. CONCLUSONS: Tin mine dust in Yunnan province could induce malignant transformation of BEAS-2B cells.By passing generation and selection in soft agar medium we established the pre-cancerous transformation cells that could be cultured long term in vitro.

BACKGROUND ＆ AIM: To study the inhibiting effects of α-tocopheryl succinate(α-TOS) on the growth of Ehrlich ascitesocarcinoma in vivo and explore the mechanisms. MATERIAL AND METHODS: The mice loaded with Ehrlich ascitesocarcinoma were used as tumor model, all the experimental animals were divided into negative control, tumor model, α-TOS treatment at dose 20, 40 and 80 mg/kg and cyclophosphamide positive groups. The survival ratio of tumor-bearing mice and subsequent hematological changes were measured, NK cytotoxicity and the proportion of T lymphocyte subsets were detected by lactate dehydrogenase method and Flow Cytometer, respectively. Inducibility of Ehrlich cell apoptosis was examined by DNA agarose gel electrophoresis. RESULTS: The survival ratio in α-TOS treatment group at dose 80 mg/kg was 33.09％. The white blood cell counts in α-TOS treatment groups were much higher than in positive control, the platelet counts were much lower than in tumor model. The NK cytotoxicity became much higher in α-TOS treatment groups at dose 40 and 80 mg/kg compared with the tumor model(P<0.05 and P<0.01). The T lymphocyte subset counts were close to negative control in α-TOS treatment groups. The DNA ladder was found in Ehrlich ascitesocarcinoma cells at dose 80 mg/kg. CONCLUSION: α-TOS was capable of prolonging the survival time of mice with Ehrlich ascitesocarcinoma; could alleviate hyperviscosity caused by the increase in the number of platelets, without interfering with the number of leucocytes; was able to activate immune system in the process ofinhibiting the growth of tumor due to improved immune function. The result of DNA agarose gel electrophoresis demonstrated that α-TOS induced apoptosis of Ehrlich ascitesocarcinoma cells at a dose of 80mg/kg.

BACKGROUND ＆ AIM: To investigate the inhibition of grape proanthocyanidins(GPC) on MMP-2 protein of breast cancer tissue. MATERIAL AND METHODS: Forty famale BALB/c mice inoculated with EMT-6 breast cancer cells were divided into tumor-control group and 10、100 and 200mg/kg GPC groups. GPC experimental groups were given different dose of GPC by mouth once a day for two weeks.Mice in control group were given equal amount of nomal salt solution only.MMP-2 protein in tumor tissues was assessed by Western blot and immunohistochemistry .The proliferation of cancer cells were evaluated by MTT assay, and the weights of the tumor in each group were compared also. RESULTS: 100、200 mg/kg GPC could inhibit the expression of MMP-2 protein and the proliferation of breast cancer cells. The expression of MMP-2 protein in the 100、 200 mg/kg GPC groups were 0.73±0.04 and 0.69±0.04 respectively, by western bolt and the proliferation of cancer cells were 0.81±0.21 and 0.71±0.11 respectively, by MTT assay . Compared with the control group, the differences were significanly(P<0.05). There was no difference between 10 mg/kg GPC group and the tumor control group.CONCLUSION: GPC could inhibit the expression of MMP-2 protein and proliferation of breast cancer cells.

BACKGROUND ＆ AIM: To investigate the effects of Gynostemma pentaphyllum (GP) extract on DNA oxidative and alkylating damages. MATERIAL AND METHODS: Adult Wistar rats received 100 mg/kg D-galactose subcutaneous injection each day to build the aging model. There was also a control group. These groups of rats were fed with fully compounded diet which was supplemented with different doses of Gynostemma pentaphyllum extract or vitamin E, C(VE＋C) for 8 weeks. Whole blood was collected at the end of the trial.Spontaneous and induced oxidative DNA damages were analyzed by SCGE. Urine was collected before the end of the trial. Urinary O6-MeG was analyzed by high performance capillary zone electrophoresis. RESULTS: There was no statistical difference in spontaneous DNA damage among 6 groups(P>0.05). But less oxidative DNA damage induced by 5, 10, 25 μmol/L H2O2 were found in the groups supplemented with Gynostemma pentaphyllum extract and the VE＋C group than in the aging group(P<0.05). When supplemented with higher dose of Gynostemma pentaphyllum extract, DNA damage showed no significant differences compared with the control group and VE＋C group. The level of urinary O6-MeG in the aging group was not higher than that in the other groups (P>0.05). CONCLUSION: D-galactose could induce DNA oxidative damage while it had no effect on DNA alkylating damage. When Gynostemma pentaphyllum extract was given to aging rats, the oxidative DNA damage induced by H2O2 could be effectively decreased and the alkylating DNA damage showed no change.

BACKGROUND ＆ AIM: To clone human MDA7 cDNA gene, construct pEgr-MDA7 plasmid, and measure its mRNA level in esophageal squamous EC9706 cells induced by irradiation. MATERIAL AND METHODS: Human MDA7 cDNA gene was isolated from peripheral blood and was then ligated to downstream of Egr-1 promoter to construct pEgr-MDA7 plasmid. The recombinant plasmids were transfected into EC9706 cells with liposome, then MDA7 mRNA level of after different doses of X-ray irradiation was quantified by PCR. RESULTS: Sequencing and restriction enzyme digestion were performed ensuring that MDA7 cDNA gene cloning and construction of pEgr-MDA7 were correct. MDA7 mRNA level in cells transfected with pEgr-MDA7 induced by different doses of irradiation was higher than that of sham-irradiation group. CONCLUSION: X-ray could induce and enhance expression of pEgr-MDA7 gene in EC9706 cells.

BACKGROUND ＆ AIM: To study the expressions of survivin and Bcl-2 in nasopharyngeal carcinoma(NPC) and the association between their expressions and NPC. MATERIAL AND METHODS: Using immunohistochemical staining, the expressions of survivin and Bcl-2 were assessed in 68 cases of NPC and 45 cases of nasopharyngeal chronic inflammation tissues. Using RT-PCR, the expressions of survivin and Bcl-2 was measured in 24 cases of NPC and 18 cases of nasopharyngeal chronic inflammation tissues. RESULTS: Analyzed by immunohistochemical staining, the expressions of survivin and Bcl-2 were 69.1％(47/68) and 79.4％(54/68),respectively,in NPC, statistically different(P<0.01)when compared with 15.6％(7/45) and 13.3％(6/45),respectively,in nasopharyngeal chronic inflammation tissues. Analyzed by RT-PCR, the expressions of survivin and Bcl-2 were,79.2％(19/24) and 87.5％(21/24),respectively, in NPC, statistically different(P<0.01)when compared with that 22.2％(4/18) and 27.8％(5/18),respectively, in nasopharyngeal chronic inflammation tissues. CONCLUSION: The expressions of survivin and Bcl-2 might play important roles in the development of Nasopharyngeal carcinoma.

BACKGROUND＆AIM: To study the anti-tumor effects of Sarcodonin G, extracted from Sarcodon scabrosus karst by dichlorolomethane on HeLa cell in vitro and explore the possible mechanisms. MATERIAL AND METHODS: The effect of Sarcodonin G on HeLa cell line was determined by MTT assay in vitro. The apoptosis induced by Sarcodonin G was studied by FCM. The morphological changes of HeLa cells were assessed by transmission electron microscope. RESULTS：Sarcodonin G dose dependently inhibited the proliferation of cultured HeLa cells, the IC50 was 7.19 μmol/L. Compared with the control group, the apoptosis rate and the expression of P53 in HeLa cells were markedly higher in the three Sarcodonin G group(P<0.01). The morphological changes of HeLa cells incubated with Sarcodonin G showed chromatin condensation, marginal and nuclear fragmentation. CONCLUSION: Sarcodonin G could inhibit the proliferation of HeLa cells in vitro. Its anti-tumor effect might be associated with up-regulating the expression of P53 and inducing apoptosis.

BACKGROUND ＆ AIM: To explore the antioxidant effects of snow frog oil capsule. MATERIAL AND METHODS: The mice were divided into four groups,study groups were given snow frog oil at a dose of 2.5，0.5，0.1 g/kg,while distilled water was given in control group.The levels of SOD and GSH_Px in serum and the level of MDA in the liver tissue were evaluated. RESULTS: Snow frog oil capsule could increase the levels of SOD and GSH-Px in serum and decrease the level of MDA in liver tissue. CONCLUSION: Snow frog oil capsule antioxidant effects.

BACKGROUND ＆ AIM: This study was designed to elucidate Rhubarb and its anti-tumor and immune modulation functions. MATERIAL AND METHODS: The stem of Rhubarb was cooked in distilled water. It was prepared in three concentrations(100 mg/ml、50 mg/ml and 10 mg/ml)to observe its inhibitory effect on esophageal carcinoma cell and peripheral blood lymphocytes(PBL)in patients by MTT assay. RESULTS: There were different inhibitory effects of various rhubarb doses and serum concentration.Proliferation of Esophageal carcinoma cell was stronly inhibited with 0.1 mg/ml of Rhubarb. Inhibition rate was 51.9％; while, its effects on esophageal carcinoma cell was weaken at 37.9％.of Inhibition rate of patient PBL was 30.1％. The proliferation of PBL was induced with 5 mg/kg and 10 mg/ml of Rhuland. The inhibitory rate(36.7％) of the 1 000 mg/kg serum on carcinoma cell TE13 was much higher than that the 100 mg/kg serum(P<0.01). The inhibition rate(33.0％)of the 1 000 mg/kg serum for PBL was significantly different compared with negative and positive control(P<0.05). CONCLUSION: Rhubarb and its serum content inhibited esophageal carcinoma cell. This suggestsed rhubarb had anticancer and immune modulation effect. The effects were related to rhubarb concentration.

BACKGROUND ＆ AIM: To investigate and evaluate the usefulness of P16 expression in the diagnosis of cervical intraepithelial neoplasia. MATERIAL AND METHODS: Samples from 40 patients with chronic cervicitis associated with squamous metaplasia, 40 with condyloma acuminata, 30 with CINⅠ, 25 with CINⅡ, 20 with CINⅢ and 30 patients with invasive cervical squamous carcinoma were immunohistochemically stained. RESULTS： 38 of 40 chronic cervicitis with squamous metaplasia did not stain, 34 of 40 condyloma acuminata stained focally, all CIN and invasive squamous carcinoma cases, except for 3 CINⅠcases, stained diffusely. CONCLUSION： The staining pattern of P16 was useful to differentiate CIN with chronic cervicitis with squamous metaplasia and cervical condyloma acuminatum. The expression of P16 could be helpful to diagnose CIN in small fragmented tissues or thin epithelium.

BACKGROUND＆AIM: To study the mutagenicity of laxative tea bags. MATERIAL AND METHODS： Micronucleus test of bone marrow PCE cell in mice,sperm shape abnormality test of mice, Ames test were used. RESULTS： The results of all tests were negative,including micronucleus test ,sperm shape abnormality test and Ames test. CONCLUSION： Laxative tea bags had no mutagenic effcts in our study.

BACKGROUND ＆ AIM： This article exploved a modified method of comet assay from the Singh's alkaline comet assay and discussed the reliability of this method and the CASP software used to measure the DNA damage induced by UV light． MATERIAL AND METHODS： DNA damage of K562 cells induced by UV for different times (3 s,5 s,10 s,40 s,60 s,120 s,180 s,240 s) was measured by comet assay．The results were assayed by CASP． RESULTS： UV( 0.3 mW/cm2)induced a significant DNA damage (P<0.01) when the results were assayed by the four parameters (tail length，comet length，tail moment，olive tail moment)． CONCLUSION： The results suggested that the four parameters indicated a good time_dependent effect.The method which we developed and the four parameters were reliable．

BACKGROUND ＆ AIM: Optimizing the protocol of two-dimensional electrophoresis by Mini Zoom IPG Runner System to analyze plasma proteins. MATERIAL AND MATHODS: Using the immobile IPG strips, ZOOMTM Gels for 2D Electrophoresis and buffer solution system purchased from Invitrogen,two dimensional electrophoresis of plasma proteins was performed.The effect of changing the condition of isoelectric focusing and pretreating the samples,on the two dimensional profiles was analyzed so as to optimize the protocol of the system. RESULTS: Compared with the recommended condition, the profiles were improved by enhancing the voltage of isoelectric focusing, extending the time and desalinizing the samples. CONCLUSION: The protocol of two-dimensional electrophoresis by Mini Zoom IPG Runner System for the analysis of plasma proteins should be optimized by increasing the voltage of isoelectric focusing, extending the time and desalinizing samples.