OBJECTIVE: Here we report the cloning and characterization of two novel splice variants of DHRS4L2 gene and potential mechanisms regulating the transcription. METHODS: RT_PCR, 3'_RACE and bioinformatics were used to clone and characterize the novel splice variant of DHRS4L2 from human hepatic immortalized cell line HL_7702. Transcription of DHRS4L2 was investigated by RT_PCR in 5_aza_dC treated HL_7702 and Hep_G2 cell lines. RESULTS: we found two novel splice variants DHRS4L2A and DHRS4L2A3，both with alternative first exon Ea. The former lacked exon 1 and the latter, exon 1 and exon 3. After 5_aza_dC treatments, DHRS4L2 transcripts with first exon E1 were increased in normal hepatic cells while the reverse trend was found in malignant hepatic cells. CONCLUSION: We found two novel splice variants of DHRS4 and suggested that the alternative starts of DHRS4L2 transcription may be regulated by methylation. The effect of methylation is different between normal and malignant cell lines and the underlying mechanism remains to be investigated.

OBJECTIVE: To assess the relation of aging and oxidative damage caused by γ_rays． METHODS: 60 mice were divided into 4 groups：group A was control group; mice of group B were treated with intraperitoned D_galactose; mice of group C were irradiated by γ_rays; mice of group D were treated with D_galactose and irradiation. The activity of SOD，contents of MDA and lipofuscin and AGEs in blood serum were determined，the expression in of β_galactosidase by staining was measured at the end of 80 days' experiment． RESULTS: Compared with group A, the activity of SOD was decreased in group B(P<0.05)，the level of AGEs and contents of MDA and lipofuscin increased in group B(P<0.05), also the β_galactosidase activity was positive. The activity of SOD was decreased in group C(P<0.05)，contents of MDA and lipofuscin increased in group C(P<0.05),also the β_galactosidase activity was positive, but no significant difference was found in the level of AGEs in group C(P>0.05). Compared with group B, the activity of SOD was decreased in group D(P<0.05)，the level of AGEs and contents of MDA and lipofuscin were increased in group D(P<0.05), also the β_galactosidase activity was raised. CONCLUSION:Aging was enhanced by oxidative damage caused by γ_rays.

OBJECTIVE: To analyze the DNA methyltransferases (DNMTs) and methyl_CpG binding proteins mRNA expression levels in malignant transformation of human bronchial epithelial cells(16HBE) induced by glycidyl methacrylate(GMA), and to discuss the epigenetic mechanisms of malignant transformation of 16HBE induced by GMA. METHODS: Gene chip was used to screen the differentially expressed genes of DNMTs and methyl_CpG binding proteins, the expressions of DNMT1 and MBD1 mRNA were measured with RT_PCR． RESULTS: The expressions of DNMT1 and MBD1 were up_regulated compared with control group in the transformed 10th_generation(protophase)cells. Further RT_PCR re_examination showed that the levels of DNMT1 and MBD1 mRNA were up_regulated by 80.60％ and 79.10％,respectively. CONCLUSION: Methylation may play an important role during the malignant transformation of 16HBE induced by GMA, DNMT1 and MBD1 could play important roles in the inactivation mechanisms of methylation_related genes in malignant transformation of 16HBE induced by GMA．

OBJECTIVE: To analyze the differential expression of proteins in portal cholangiocellular carcinoma cell line FRH 0201 and normal hepatocellular cell line L02, and to screen potential molecular markers for diagnosis of hilar cholangiocarcinoma. METHODS: Surface enhanced laser desorption/ionization (SELDI) mass spectrometry with Proteinchip IMAC3 and WCX2 was performed to compare the expressed proteins in portal cholangiocellular carcinoma cell line FRH 0201 and hepatocellular cell line L02. Protein profiling was examined by PBSII_C Protein Chip Reader and the proteome database was analyzed by Proteinchip Software 3.0.2. RESULTS: 144 protein peaks in FRH 0201 and L02 cell lines were captured by Proteinchip IMAC3 and WCX2，16 differential proteins were found. Four proteins were up_regulated and 12 proteins down_regulated in portal cholangiocellular carcinoma cell line FRH 0201 as compared with L02 cell line. CONCLUSION: The differential proteins could be candidate biomarkers of portal cholangiocarcinoma. The study may contribute to understanding the mechanisms of development of portal cholangiocarcinoma

OBJECTIVE: To study the effects of human apolipoprotein B mRNA_editing enzyme catalytic_polypeptide (APOBEC)_3F (hA3F) and _3G (hA3G) on the replication and mutation of influenza viruses. METHODS: A549 and MDCK were transfected using hA3F and hA3G plasmids, and the expression of recombinant proteins was detected using Western blotting and immunofluorescence assay. The titers of influenza virus, which replicated on the hA3F and hA3G expressing and control cells, were measured using TCID50 and plaque assay. The viral growth curves were drawn. The expression levels of hA3F and hA3G in influenza infected cells were detected using Western blotting. The mutation frequency of influenza viruses was detected using RT_PCR and DNA sequencing. RESULTS: The viral titers and viral growth curves indicated that there were no significant differences in replication of influenza virus between APOBEC proteins expressing cells and the control cells. Western blotting indicated that hA3F and hA3G were both over_expressed during the infection of influenza viral. DNA sequencing showed that there was no hypermutation in influenza viral genome induced by hA3F or hA3G. CONCLUSION: hA3F and hA3G have no obvious effects on the replication and mutation of influenza virus in A549 and MDCK cells.

OBJECTIVE: To investigate the changes of GDF15 mRNA expression in lymphoblastoid cells induced by ionizing radiation, in order to provide scientific evidence for gene expression as a biomarker in radiobiodosimetry. METHODS: Lymphoblastoid cells were irradiated by 60Co γ_rays with doses ranging from 0 to 15 Gy, and cultivated for different periods from 4 to 72 hours. The GDF15 mRNA levels in lymphoblastoid cells were analyzed with real_time PCR. RESULTS: After irradiation, the GDF15 mRNA level was enhanced gradually to peak at some dose, and then decreased in all time groups except the 12 h group. In the 12 h group, the GDF mRNA level increased significantly; the dose_effect fitted a linear model: y=1.046 9x＋0.527 8(R2=0.945 7，P<0.05). The changes of GDF15 mRNA levels varied at different time_points. After irradiation of 8 Gy, GDF15 mRNA level rose significantly and peaked at 12 h. CONCLUSION: The enhanced GDF15 mRNA level induced by ionizing radiation depended on the exposed dose and duration.

OBJECTIVE: To investigate the effects of thioglycolic acid(TGA) on calcium ionophore A23187_ induced parthenogenetic activation of xenopus oocytes and its possible mechanism. METHODS: Xenopus oocytes were pretreated with TGA in vitro at the doses of 0, 5, 25, 125 μg/ml, and then activated by A23187. During this process, the activation rates were noted. Oocytes were collected 1h after the addition of A23187 and stained with Hoechst 33258 to check the formation of pronucleus. RESULTS: Following TGA treatment, the black spot appeared on the animal pole, known as fertilization coat, which was not in good condition. TGA significantly inhibited the rate of parthenogenetic activation(P<0.05) and the formation of pronucleus. Through Western Blotting analysis, TGA inhibited the degradation of Mos, p_ERK1 and CyclinB2. CONCLUSION:TGA treatment could inhibit the parthenogenetic activation of xenopus oocytes induced by A23187, possibly due to the down_regulation of MPF and MAPK degradation.

OBJECTIVE: To investigate the effect of olaquindox on the antioxidant system in human hepatoma G2(HepG2) cells. METHODS: HepG2 cells were treated with 0, 200, 400 and 800 μg/ml olaquindox in 24 h. The activities of superoxide dismutase(SOD) and catalase(CAT), the level of malondialdehyde(MDA), the activities of glutathione (GSH) and glutathione peroxidase (GSH_PX), Na＋K＋_ATPase and Ca2＋Mg2＋－ATPase were measured. SOD or CAT, and the combination of SOD＋CAT were incubated for 3 h with olaquindox_treated HepG2 cells. The levels of reactive oxygen species (ROS) were evaluated by 2, 7_dichlorodihydrofluorescein diacetate(DCFDA). RESULTS: Olaquindox decreased the levels of T_ATPase, Na＋K＋－ATPase and Ca2＋Mg2＋－ATPase, SOD,GSH ,GSH_PX, in HepG2 cells(P<0.05 or P<0.01), with a dose_response relationship. After HepG2 cells were treated with olaquindox, the activity of CAT was decreased when compared with the control(P<0.01). Olaquindox led to a dose_dependent increase in the level of MDA(P<0.05 or P<0.01). While HepG2 cells were treated with olaquindox (800 μg/ml), CAT,SOD and SOD＋CAT reduced ROS about 80％ ,40％and 95％, respectively(P<0.01). CONCLUSION: Olaquindox disrupted the antioxidant system in HepG2 cells. Our results indicated that olaquindox_induced HepG2 cells predominantly generated H2O2, which could be scavenged by CAT.

　　【ABSTRACT】 OBJECTIVE: To investigate the amplification and overexpression of PIN1 gene in human laryngeal squamous cell carcinoma. METHODS: PIN1 gene amplification(increase of gene copy number) was detected by differential PCR and Karyotype analysis. PIN1 mRNA was evaluated by RT_PCR. Furthermore, immunohistochemistry and Western Blot were employed to detect the PIN1 protein. RESULTS: Compared with nomal control, PIN1 gene amplification rate was 22.5％(9/40), while 17.65％(3/17) was found to have an increase of chromosome 19; the rate of mRNA overexpression was 67.5％(27/40). PIN1 protein was obviously upregulated, the ratio was 65％(26/40)(P<0.05). CONCLUSION: PIN1 gene copy increased, with mRNA and protein overexpression in laryngeal carcinoma. This fact may be relevant to laryngeal cancer development.

OBJECTIVE: To study the expression of 4_1BB and its biological function in different human gastric cancer cell lines. METHODS: The expression of 4_1BB in different human gastric cancer cell lines was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT_PCR). The killing activity of lymphocytes against gastric cancer cell lines was detected by MTT, whilst IL_2,IL_10,TNF_α were tested in the supernatant by ELISA. RESULTS: RT_PCR analysis showed that the 4_1BB mRNA expressions were MGC_803 (77.30％),BGC_823 (71.68％),SGC_7901(40.06％) and MKN_28(37.65％) in decreasing order. The 4_1BB protein expressions were MGC_803, BGC_823, SGC_7901 and MKN_28 in decreasing order by cytoimmunochemistry staining. MTT analysis showed that the killing activities from high to low were MKN_28, SGC_7901, BGC_823 and MGC_803. ELISA analysis showed that IL_2 and TNF_α levels were MKN_28, SGC_7901, BGC_823 and MGC_803 and IL_10 level was MGC_803,BGC_823, SGC_7901 and MKN_28, from high to low. CONCLUSION: The four different gastric cancer cell lines all expressed 4_1BB at mRNA and protein levels. The 4_1BB expression level related to gastric cancer cell lines to different degrees. The killing activity of lymphocytes against gastric cancer cell lines increased with decreasing 4_1BB expression. 4_1BB could regulate tumor immunity by inhibiting cell factors TNF_α and IL_2 and promoting cell factor IL_10.

OBJECTIVE: To investigate the relationship between single nucleotide polymorphisms of DNA repair genes ERCC1 C118T and XPD Lys751Gln and sensitivity to platinum_based chemotherapy in non_small_cell lung cancer(NSCLC). METHODS: A total of 73 patients with NSCLC were analyzed. All the patients were treated with platinum_based chemotherapy and the DNA of their peripheral blood was obtained before the therapy. ERCC1 and XPD genotypes were evaluated by DNA sequence and the polymerase chain reaction_restrictive fragment length polymorphism (PCR_RFLP) method. RESULTS: The clinical benefit rates to therapy among the patients with ERCC1 C118T C/C,C/T and T/T genotypes were 94.9％,71.4％and 83.3％, respectively . The clinical benefit rate of C/C genotype was significantly higher than C/T and T/T genotypes. There were significant differences in clinical benefit rates to platinum_based chemotherapy (P<0.05).The clinical benefit rates to therapy among the patients with XPD Lys751Gln Lys/Lys and Lys/Gln genotypes were 80.3％and 75.0％, respectively. There were no significant differences in clinical benefits to platinum_based chemotherapy(P=0.702).The XPD Gln/Gln genotype was not detected. There were no significant differences between the genetic polymorphisms and clinical benefits to platinum_based chemotherapy on ERCC1 C118T and XPD Lys751Gln(P=0.134 and P=0.236). CONCLUSION: Single nucleotide polymorphisms of ERCC1 C118T was associated with clinical response to platinum_based chemotherapy in NSCLC patients, suggesting the ERCC1 C118T SNP may be one of predictors for NSCLC patients who would be responsive to platinum agents.

OBJECTIVE: To study mRNA expression of apoptosis genes Bcl_2, BAX, BAD in spleen and peripheral blood of guinea pigs after treatment with trichloroethylene (TCE). METHODS: Guinea pig maximization test (GPMT) was applied in this study, 18 guinea pigs were randomly divided into 3 groups, namely negative control, positive control and TCE treatment. Animals of the 3 groups were treated with olive oil, 2,4_dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. Spleens and peripheral blood were collected from the animals for mRNA expression assay, Bcl_2, BAX, BAD in lymphocytes of spleen and peripheral blood were measured by real_time fluorescent PCR assay. RESULTS: Obvious skin damage was observed in the groups of positive control and TCE treatment, the skin damage included erythema and edema. The levels of Bcl_2, BAX and BAD mRNA expression increased significantly in spleen lymphocytes in positive control and TCE treatment groups. However, the levels of Bcl_2, BAX and BAD mRNA did not elevate significantly in peripheral blood lymphocytes in positive control and TCE treatment groups, when compared with negative control. CONCLUSION: Trichloroethylene could induce skin allergic reaction in guinea pigs，it also had effects on mRNA expressions of Bcl_2, BAX, BAD in spleen lymphocytes of guinea pigs.

OBJECTIVE: To evaluate clinical significance of application of fluorescence in situ hybridization in preimplantation genetic diagnosis (PGD) for carriers of chromosomal abnormalities. METHODS: according to the categories of chromosomal abnormalities, we selected the appropriate sub_telomeric probes and centromere probes, or sex chromosome probe, hybridized once or twice so the 7 carriers received preimplantation genetic diagnosis. RESULTS: Preimplantation genetic diagnosis were performed in 7 cycles of 7 couples. A total of 131 oocytes were retrived, 77 embryos were available for biopsy, 87 blastomeres detected, 20 embryos transplanted, 4 clinical pregnancies obtained, and 2 healthy babies had been. CONCLUSION: Application of fluorescence in situ hybridization technique in preimplantation embryo genetic diagnosis for carriers of chromosomal abnormalities was an effective way.

OBJECTIVE: To investigate the expressions of EGFR(endothelial growth factor receptor), and VEGFR1/ Flt_1(vascular endothelial growth factor receptor 1/Flt_1) in esophageal squamous cell carcinoma tissues with or without radiotherapy, and evaluate the correlation between the expressions and the sensitivity of preoperative radiotherapy. METHODS: We used immunohistochemical method(EnVision) to assess the expressions of EGFR and VEGFR1/Flt_1 in 40 squamous cell carcinomas with radiotherapy, 40 without radiotherapy, and 20 normal tissues at the proximal end of resection. RESULTS: For EGFR, there was no expression in normal esophageal tissues, but 70％ and 77.5％expression in esophageal squamous cell carcinoma with and without radiotherapy, respectively (P<0.05). For VEGFR1/Flt_1, 80％ expression in normal tissue was observed, while 80％ and 62.5％ in esophageal squamous cell carcinoma with and without radiotherapy, respectively, were found (P<0.05). CONCLUSION: EGFR, VEGFR1/Flt_1 could be used as marker for esophageal squamous cell carcinoma radiotherapy sensitivity preoperatively.

OBJECTIVE: To explore the effect of herbicide paraquat on the expression of peroxidase(POD) and esterase(EST) in Carassius auratus serum. METHODS: The 0.5,1.0,1.5 and 2.0 ml/L concentrations of paraquat were prepared according its 24 h LC50， Carassius auratus were treated with 0.1 ml/g dose of each concentration. SDS_polyarylamide gel were used to test the expressions of POD and EST. RESULTS: Compared with the control group, the four treated groups showed significant variety. The activities of POD and EST increased or decreased at low concentration，absent or increased at high paraquat concentration,concentration and activity had dose_effect relationship. CONCLUSION: The toxicity of paraquat on POD and EST varied in Carassius auratus.

OBJECTIVE: To explore the mechanism of spermatogenesis inhibition induced by chronic alcohol consumption, and to examine the apotosis and proliferation of spermatogenic cells. METHODS: 40 healthy Sprague_Dawley adult male rats were randomly divided into four groups. Different doses of alcohol(0,2.7,4.5 and 7.5 g/kg) were administrated to the rats for 13 weeks by a gastric tube. The histological changes of rat testicular tissue were examined by light microscopy. The apoptosis and count of germ cells were evaluated by Flow Cytometry(FCM) and TUNEL, respectively.The expression of Caspase_3 and proliferating cell nuclear antigen (PCNA) in testes were detected by western blot. RESULTS: Light microscopic evaluation of testes demonstrated typical morphological changes of germ cell apoptosis including nuclear degeneration, condensation, and increased sloughed cellular element in tubular lumina especially in 7.5 g/kg alcohol_treated rats. Furthermore, the degree of testicular injury was associated with the dose of alcohol. Compared with control group , the apoptosis index in alcohol 7.5 g/kg group was higher(P<0.05). There was a marked decrease in haploid and tetraploid cells and significantly down_regulated expression of PCNA in alcohol 7.5 g/kg group(P<0.05). Western blot analysis showed that the expression of Caspase_3 increased markedly and the expression of PCNA significantly decreased in alcohol_treated group(P<0.05). CONCLUSION: Chronic alcohol intake could lead to an enhancement of germ cell apoptosis and a reduction of cell proliferation in rat testes, which may at least partly contribute to spermatogenesis inhibition.

OBJECTIVE: To study the mutagenicity of nitenpyram, and provide basis for safety in production and application. METHODS: Micronucleus test of polychromatic erythrocytes(PCE) in mice bone marrow, Ames test, chromosomal aberration test of Chinese hamster lung cell (CHL) and TK gene mutation assay of mouse lymphoma cell (L5178Y) were taken to examine the mutagenicity of nitenpyram. RESULTS: Nitenpyram didn't cause the increase of micronucleus rates (P>0.05). The number of revertant colonies was no more than twice the spontaneous colonies for each dosage group．Compared with the control group，there was no significant difference for chromosomal aberration rates and mutation frequency of TK gene in each dosage group(P>0.05). CONCLUSION: Mutagenic effects of nitenpyram were not detected under these test condition.