OBJECTIVE: To design and construct the eukaryotic expression vector of human CtBP1 gene short hairpin RNA(shRNA) then to transfect human endometrial carcinoma cells B-MD-C1(ADR＋/＋), and to investigate the silencing effect of CtBP1 shRNA on the expression of human CtBP1 gene. METHODS: siRNAs were designed by targeting human CtBP1 gene using pGenesil-1 as vector.According to the CtBP1 cDNA sequence in GenBank,2 pairs of oligonucleotides were designed.A hairpin loop with 9 base pairs was added into the interference sequence and inserted into the eukaryotic expression vectors which contain the EGFP gene,kanr gene and U6 promoter.After primer annealing,they were inserted into plasmid pGenesil-1 to construct the shRNA eukaryotic expression vector. The recombinant plasmid were transformed into E.coli DH5α strains,and the positive strains were identified by enzyme digestion and sequence analysis.The recombinant vector were transfected into B-MD-C1(ADR＋/＋)cells with LipofectamineTM 2000. The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by reverse transcription polymerase chain reaction(RT-PCR). RESULTS: The pGenesil-1-CtBP1 shRNA of recombinant plasmid was constructed and confirmed by DNA sequencing.Fluorescence microscope for lots of green fluorescence showed that shRNAs had transfected B-MD-C1(ADR＋/＋) cells(transfection rate was 60％～70％).And the CtBP1 mRNA were effectively inhibited by pGenesil-1-CtBP1-shRNA of recombinant plasmid(inhibition rate was above 50％). CONCLUSION: The eukaryotic expression vectors of CtBP1 shRNA were constructed successfully.The shRNA could effectivelly inhibit the expression of CtBP1 gene, laiding a foundation to further study for its function and reverse the cancer multidrug resistence by blocking CtBP1 gene expression.

OBJECTIVE: To explore the transfection effects and cell toxicity of mock miRNA at different concen- trations, liposome at different concentrations and different transfection times in human bronchial epithelial cells in vitro and optimize transfection conditions. METHODS: 16-HBE cells were incubated with 0, 10, 30 and 50 nmol/L Cy3 labelled mock miRNA and 0, 0.1％, 0.3％ and 0.5％ liposome. The transfection effects in the cells were examined by fluorescence microscopy and were evaluated by fluorescence intensity analysis after 6, 12 and 24 h of transfection. Cell activity was measured after 24 h transfection with different transfection conditions by WST-8 method. RESULTS: Intracellular fluorescence intensity was enhanced with increasing mock miRNA and liposome concentration and transfection time. The transfection effects of 30 and 50 nmol/L mock miRNA transfection groups was significantly higher than 10 nmol/L mock miRNA transfection group(P<0.05). The transfection effects of 0.3％ and 0.5％ liposome groups were obviously higher than 0.1％ liposome groups(P<0.05). Intracellular fluorescence intensity after 24 h transfection was remarkably increased those that after 6 and 12 h transfections(P<0.05). There was no significant difference in cell toxicity between transfection groups and control group(P>0.05). CONCLUSION: 16-HBE cell could be used in the research of liposome-mediated delivery of mock miRNA. Mock miRNA and liposome concentration and transfection time are important influence factors of transfection. Optimal transfection effects could be obtained after 24 h transfection with 30 nmol/L mock miRNA and 0.3％ liposome. There was no effect on the cell activity after transfection.

OBJECTIVE: To construct the report gene expression vector for studying the transdifferentiation potency of liver epithelial progenitor cells (LEPCs). METHODS: The rat insulin Ⅰ promoter(RIP) was amplified by PCR from rat tail genomic DNA and subcloned into promoter-removed pDsRed1-1 vector. By using the reporter gene tagging cells, the phenotype variation of LEPCs transdifferentiate into pancreas-like cells could be evaluated. RESULTS: RIP report gene expression vector could monitor the transdifferentiation of LEPCs into pancreas-like cells in a real-time manner. CONCLUSION: The reporter vector could useful for studying the transdifferentiation of LEPCs into pancreas-like cells.

OBJECTIVE: To construct a system for reverse genetics of minus RNA viruses, the universal reverse genetic vector was constructed. METHODS: Using pcDNA3 as plasmid backbone, non-essential Kanamycin resistant expression cassettes and CMV promoter upstream region were removed. Then the fragment including reverse pol I promoter and multiple clone site were synthesized by PCR and inserted into the reconstructed pcDNA3. The modified pcDNA3 and universal reverse genetic vector were identified by restriction enzyme. RESULTS: Sequencing showed that the universal reverse genetic vector which had CMV and pol I bidirectional promoter was constructed successfully. CONCLUSION: This research established reverse genetics system of minus RNA viruses and provided research basis for further development of minus RNA viruses.

OBJECTIVE: To determine the transfection expression of plasmid pcDNA3.1-IDO in human hepatoma carcinoma cell line hepG2, and to establish a transfection method of hepG2. METHODS: The plasmid pcDNA3.1-IDO was amplified in Escherichia coli. The cultured hepG2 cells were transfected with pcDNA3.1-IDO by lipofectamineTM2000 reagent. The hepG2 cells and hepG2 cells trancfected with blank plasmid pcDNA3.1(pcDNA3.1- hepG2 cell) were used as control group. The transient expression of IDO in hepG2 cells transfected with recombinant plasmid was determined by RT-PCR and Western blot. RESULTS: IDO gene and protein were expressed transiently in hepG2 cells transfected with recombinant plasmid shown by RT-PCR and Western blot,respectively. CONCLUSION: The IDO gene was successfully transfected into human hepatoma carcinoma cell line hepG2 by means of lipofectamineTM2000 reagent, which provides the basis for further studies of IDO gene.

OBJECTIVE: To investigate the function of p53 polymorphic variants, we constructed the p53 codon 72 polymorphic plasmid. METHODS: Primers carrying the appropriate mutation were employed in a two-step PCR amplification. The mutated PCR products were subsequently cloned into pReceiver-M01 expression vector. The p53 protein translated in vitro was used to interact with iASPP to certify its biological activity. RESULTS: The sequence of the recombinant eukaryotic expression vector containing CCC to CGC mutation was proved by DNA sequencing. This p53 polymorphic variant could interact with iASPP, implying its biological activitical. CONCLUSION: The recombinant p53 eukaryotic expression vector containing codon 72 polymorphism was constructed successfully, laying a foundation for further studies on the function of p53.

OBJECTIVE: To study the apoptosis effect of SGC-7901 cells induced by Vitamin E succinate(VES), and explore the involvement and mechanisms of oxidative damage. METHODS: After treatment by 0, 5, 10, 20 μg/ml VES for 24 h, methods of flow cytometry, confocal laser scanning microscopy and western blot were used to measure the mitochondrial oxidative damage and apoptosis induced by VES of SGC-7901 cells. The corresponding detection kits were used to detect the reactive oxygen species and Ca2＋ content. At the same time, SGC-7901 cells were treated with 1 mmol/L of mannitol for 2 h, and then 20 μg/ml VES for 24 h, and the above detection methods were used. RESULTS: With the increasing concentration of VES, apoptosis showed an increasing trend. Compared with the negative control group, ΔΨm of cells in 10, 20 μg/ml VES groups were significantly decreased, while the active oxygen content and the level of Ca2＋ significantly was increased. 1 mmol/L of mannitol could impact the cells of oxygen and Ca2＋ contents significantly, while significantly reduce mitochondrial oxidative damage and apoptosis caused by VES. CONCLUSION: VES could significantly increase SGC-7901 apoptosis, and with increasing doses of VES, the apoptosis rate was higher. Oxidative damage caused by raised active oxygen and Ca2＋ content increasing and ΔΨm depletion in cells may be one of the mechanisms of apoptosis induced by VES.

OBJECTIVE: To evaluate the effect of cadmium chloride (CdCl2)on the expression of PKCα mRNA and protein in NRK cells as well as the biological effect of PKC inhibitor(Bisindolymaleimide I) on cells treated with cadmium. METHODS: Real-time fluorescence quantitative PCR assay was applied to test the effects of CdCl2 on the expression of PKCα mRNA in NRK cells exposed to CdCl2 at various concentrations (0, 1.25, 2.5, 5 and 10 μmol/L). Western blot was used to measure the effects of CdCl2 on the expression of PKCα protein in NRK cells exposed to CdCl2 at the same concentrations. Additionally, the expressions of PKCα mRNA and protein were also assessed in NRK cells exposed to CdCl2 after treated by PKC inhibitor. RESULTS:The expressions of PKCα mRNA and protein in NRK cells exposed to CdCl2 at the concentrations between 1.25 and 10 μmol/L were increased, and a dose-effect relationship was found. PKC inhibitor had an obvious effect on the expressions of PKCα mRNA and protein in the cells after CdCl2 treatment. CONCLUSION:CdCl2 could induce an increase in expressions of PKCα mRNA and protein, and the PKC inhibitor showed an obvious inhibitory effect.

OBJECTIVE: To explore the effect of perinatal exposure to bisophenol A(BPA) on reproduction and development of the female offspring of SD rats and the changes of uterine estrogen receptor(ER) expression. METHODS: Pregnant Sprague-Dawley rats received by gavage 0, 5, 50 or 500 mg/(kg•d) of BPA from gestation day (PND) 6 to postnatal day (PND) 21. Uterus and ovarians were weighed and prepared for histological evaluation. The expression of ERα, ERβ, phosphorylated ERK (p-ERK) and total ERK (T-ERK) in uterus were measured. The change of uterine sensitivity to estradiol (E2) was investigated by the modified uterotrophic assay. RESULTS: The toxic symptoms in parental rats and decrease in body weight in female offsprings were observed in 500 mg/(kg•BW) group, but there was an increase in body weight and uterine weight in the 50 mg/(kg•BW) group. No pathological changes were found in BPA- treated group, the thickness of endometrial layer and the ratio to uterus was increased in all dosage groups compared with the control. The protein expressions of ERα and p-ERK but not ERβ in uterus were elevated in all dosage groups. The uterine sensitivity to E2 was increased in all BPA-treated groups. CONCLUSION: Perinatal exposure to Bisphenol A could induce the changes of ER expression in the uterus of female offspring rats and change the uterine physical structure by probably activating the ERK pathway.

OBJECTIVE: To screen the differential expression of proteins in the cerebrum of hens exposed to tri-ortho-cresyl phosphate(TOCP) so as to provide evidence for mass spectroscopy. METHODS:Twelve Roman hens were randomly divided to 2 groups, with six in each group. The hens in experiment group were treated with a single oral dosage of 1000 mg/kg TOCP, the hens in control group received the same volume of sodium chloride. On the 24th day after TOCP administration, hens were sacrificed. Their cerebrum were dissected and homogenized in ice bath. Total proteins of the cerebral tissues were separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The two-dimensional electrophoresis(2-DE) maps were visualized after silver staining and analyzed by ImageMaster2D software. RESULTS: Among the total proteins from the control group and the experiment group, 1 332 and 1 008 protein spots, respectively, were detected. Comparison analysis showed that 77.78％ of the total protein spots were matched between the two groups. Compared with control group, 262 differentially expressed protein spots were detected in experiment group and 43 protein spots showed 4 fold change. CONCLUSION: Proteomic analysis could identify the differentially expressed proteins in the two groups. And some of these protein spots may contribute to the occurrence and development of TOCP-induced delayed neurotoxicity.

OBJECTIVE: To investigate the effects of triterpenes compound of cortex periplocae (TCCP) on CD4＋ CD25＋ regulatory T cells in peripherial blood in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats. METHODS: 40 male F344 rats (5-6 weeks of age) were randomly divided into four groups: rats were treated with 0.5 mg/kg NMBA only as model group and rats received NMBA 0.5 mg/kg s.c. plus TCCP 10 mg/kg i.m. as TCCP treatment groups and those treated with soya oil 1 ml/kg intramuscularily(i.m.) acted as soya oil control and normal control groups. 10 rats in each group were treated thrice per week for 5 weeks. Then the numbers of CD4＋ and CD4＋CD25＋ regulatory T cells in peripheral blood were counted by flow cytometry at weeks 9 and 15. The levels of IL-2,IL-10 and TGF-β1 in peripheral blood were detected with ELISA. RESULTS: After 0.5 mg/kg NMBA treatment for 9 weeks, CD4＋ T cells in peripheral blood of NMBA group rats were decreased significantly whilst CD4＋CD25＋ regulatory T cells were significantly increased compared with the control group (P<0.05). At weeks 9 and 15, TCCP treatment decreased the proportions of CD4＋CD25＋ regulatory T cells, while the proportions of CD4＋ cells were significantly increased (P<0.05), compared with NMBA group. At week 9 after NMBA treatment, the protein levels of TGF-β1 and IL-10 in peripheral blood were significantly higher than those in normal control group and soya control group. However the protein levels of IL-2 were significantly decreased, as compared with normal control. TCCP could decrease the expression of IL-10 and TGF-β1, but increase the expression of IL-2. CONCLUSION： In rats the expression of CD4＋CD25＋ regulatory T cells in peripheral blood were enhanced after treatment with NMBA, while the triterpenes compound of Cortex Periplocae effectively inhibited the expression, thus it could improve the immune dysfunction in experimental esophageal tumorigenesis in F344 rats.

OBJECTIVE: To compare the difference on the frequency of apoptosis induced by choline(CC) and folic acid(FA) deficiency in lymphoblast cell lines from breast cancer patient carrying BRCA1 genetic mutation and healthy control. METHODS: Lymphoblast cell lines from breast cancer patient carrying BRCA1 genetic mutation and healthy control were cultured under different media with 24 concentration combination of CC(0～21.5 μmol/L) and FA(30～240 nmol/L) for 9 days. Apoptosis(APO) frequencies of two cell lines was analysed by cytokinesis-block micronucleus cytome assay (CBMN Cyt) . RESULTS: The APO frequencies of two cell lines were significantly decreased at and over 6 μmol/L CC or 120 nmol/L FA (P<0.01 or P<0.05). The APO frequencies in cell line of case group were lower than the control cell line in all combinations . After the genetic damage background was reduced, there was no statistical significance(P>0.05) in APO frequencies difference of two cell lines. CONCLUSION: Apoptosis of lymphoblast cell lines from breast cancer patients was not more sensitive to CC and FA than control group.

OBJECTIVE: To investigate the expression of estrogen receptor alpha(ESR1) in thyroid cancer tissues and explore its clinical value. METHODS: The paraffin-embedded tissue sections of 64 thyroid cancer patients, 21 cases of thyroid adenoma, 13 cases of Hashimoto's thyroiditis, 6 cases of nodular goiter and 6 cases from the peri-tumor tissue were immunohistochemically examined for the expression of ESR1. The association between ESR1 expression and clinico- pathological parameters were analyzed. RESULTS: The positive rate of ESR1 in thyroid cancer tissues was significantly higher than those in thyroid adenoma, Hashimoto's thyroiditis, nodular goiter and peritumor tissues(P<0.01). The positive expression of ESR1 correlated significantly with tissue stage, clinical stage , lymph node metastasis and prognosis(P<0.05). CONCLUSION: ESR1 could play an important role in the occurrence and development of differentiated thyroid cancer, so to prevent the function of ESR1 may provide a new strategey to cure the thyroid cancer.

OBJECTIVE: To study the expressions of STAT3 and VEGF gene and to explore their roles in the process of esophageal cancer in the Kazaks. METHODS: Using RT-PCR to detect STAT3, VEGF in 30 cases of esophageal cancer tissues and their corresponding adjacent tissues. RESULTS: The positive rate of STAT3,VEGF mRNA expressions were 73.33％(22/30) and 76.67％(23/30),respectively,in the esophageal cancers, and 43.33％(13/30)in adjacent tissues. There was significant difference in the expressions of STAT3, VEGF mRNA level between tumor tissue and adjacent tissues. CONCLUSION: STAT3, VEGF gene could have important roles in carcinogenesis of esophageal cancer in the Kazaks. The combined action of STAT3 and VEGF genes could promote carcinogenesis and development of esophageal cancer.

OBJECTIVE: To study the effects of NJY lyophilized powder on physical tolerance and its immune- regulating, antineoplastic effects. METHODS: Loaded-swimming test, hypoxia tolerance test, delayed hypersensitive reaction(DHR) experiment, and macrophage phagocytosis experiments were conducted to investigate the effects of NJY on immune function. By transplanting S180 (1.0×107/ml) sarcoma at the axilla of Kunming mice, the inhibitory effect of NJY on its growth was observed, promotion of the antineoplastic effect of cyclophosphamide, and the possibility of extending mice survival were examined. RESULTS: NJY 16 mg/kg and 64 mg/kg exerted an obvious anti-fatigue effect on mice, with significant difference(P<0.05) from the control group; the survival time of mice with hypoxia in NJY 32 mg/kg group and 64 mg/kg group was significantly increased, with significant difference(P<0.05) from the control group. DHR: swelling extent of mice ear in NJY 32 mg/kg and 64 mg/kg groups was significantly increased compared with the model control group, with significant difference(P<0.05). Macrophage phagocytosis experiment: the phagocytic index of NJY dose groups had no significant difference(P>0.05). The inhibiton rates of NJY dose groups had no significant difference(P>0.05), compared with control group. The life extension rates of NJY dose groups were not prolonged, without significant difference(P>0.05). Combined administration of NJY with cyclophosphamide significantly increased the antineoplastic effect of cyclophosphamide. The inhibition rates were significantly increased compared with the control group after combined administration of cyclophosphamide 60 mg/kg and NJY 16, 32 and 64 mg/kg dose, with significant difference (P<0.05).CONCLUSION:NJY lyophilized powder has immune enhancing effect; it can enhance the anti-fatigue ability of mice and anti-anoxia capacity; markedly enhanced anti-tumor effect of cyclophosphamide.

OBJECTIVE: The effects of fructus schisandrae chinensis decoction on dams and embryo-fetal development were examined using Sprague-Dawley rats. METHODS: Fructus schisandrae chinensis decoction was administered at dose of 15 g/kg(herbal raw material) per day orally to groups consisting of 20 mated animals, once daily for 12 days from day 6 through day 17 of gestation. The clinical signs of rats, as well as abortions, premature deliveries, body weights, and feed intake, were monitored. Caesarean section and autopsy were performed on GD 20. Uterine content were evaluated for number and distribution of implantations, live and dead fetuses. The number of corpora lutea in each ovary was also recorded. Fetuses were weighed and examined for gender and appearance, visceral and skeletal alterations. RESULTS: There were no significant differences in any parameter of dams and fetuses. CONCLUSION: No maternal toxicity and embryo-fetal toxicity were found when fructus schisandrae chinensis decoction administered orally during the period from implantation to closure of the hard palate.

OBJECTIVE:To analyze quantitatively the analgesic effect of scopoletin and hesperetin from Ipomoea Stolonifera in combination using the method of uniform design. METHODS: According to the uniform design method, 5 different doses at 5 different proportions were administered (intra-gastric) to observe the analgesic effect to mice by writhing test. The optimal doses and proportions in combination were obtained from experimental data, by mathematical analysis and professional knowledge. RESULTS: The combination of hesperetin at 194.5 mg/kg and scopoletin at 15.36 mg/kg was appropriate. CONCLUSION: The uniform design method could be effectively applied to analyze the multidrug effects under some limited conditions, but the results must be verified by further experiment.