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Objective: To study the effect of gossypol on the expression of connexin 43 (CX43) in Sertoli cells., Methods: TM4 Sertoli cells were cultured and treated with gossypol at the concentrations of 1.25, 2.5, 5 and 10 micromol/L for 6, 12, 24 and 48 hours. The cytotoxicity of gossypol was assessed by CCK-8 assay, and the expression of CX43 in the normal TM4 Sertoli cells and in those treated with different concentrations of gossypol for different times was detected by RT-PCR and immunofluorescence analysis., Results: Semiquantitative RT-PCR and immunofluorescence analysis showed the expression of CX43 in the normal TM4 cells. At 24 hours of exposure to gossypol, the expression began to decrease gradually with the prolonging of time and the increasing concentration of gossypol (P < 0.05)., Conclusion: Gossypol reduces the expression of CX43 in TM4 Sertoli cells, which might underlie the mechanism of its antifertility action.

Aim: (1) To investigate the mRNA expression of the key angiogenic growth factors in the grafts after transplantation. (2) To investigate the potential impact of danshen (Chinese traditional medicine) administration on grafts angiogenesis., Methods: The frozen-thawed ovarian tissue from aborted fetus were xenografted into the renal capsule of the nude mice, recovered 48 h, 7 d and 28 d after respectively. Either danshen or saline (as the control) was administered after transplantation., Results: The mRNA levels of VEGF showed a temporary raise in 48 h after transplantation, then decreased in one week, and no significant difference was fund between the control group and danshen group. Ang-2 was increased in 48 h after transplantation, when Danshen group was significantly higher than the control group (P < 0.05). The microvessel density significantly increased in all the tissues after transplantation. The control group peaked on day 7 after transplantation, while danshen group peaked in 48 h and kept correspondingly steady after that., Conclusion: Early angiogenesis began within 48 h after transplantation of the thawed human fetal ovarian tissue, and its microvessel density peaked within the first week after transplantation. Our results also suggested that the use of danshen injection in conjunction with transplantation could facilitate revascularization of the grafts.

Objective: To establish and improve the method of bisulfite sequencing for methylation status of imprinted genes in single human oocytes., Methods: Single superovulated immature human oocyte was embedded into low melting point agarose, followed by bisulfite treatment and polymerase chain reaction (PCR) amplification of the H19 and MEST genes. The PCR products were then subjected to TA cloning and sequencing to determine the methylation status., Results: With the modified methods of embedding and bisulfite treatment, we achieved a high PCR success rate of 82.46%, with the somatic cell contamination rate as low as 7.14%. The sequencing results showed no non-CpG cytosine and exact conformity to the theoretical sequences., Conclusion: The bisulfite sequencing method we used to determine the methylation status of imprinted genes at the single-cell level was highly efficient and reliable, which can serve as a foundation for the further study of the influences of human assisted reproductive technology on genomic imprinting.

Objective: To assess the accuracy and reliability of the nest-PCR-sequence specific primer(SSP) method in HLA-A site genotyping of single blastomeres retrieved from human pre-implantation embryos., Methods: By nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits., Results: The amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from in vitro fertilization(IVF) surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2%(43/55) and 93.8%(61/65), respectively. And 86.7%(104/120) in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents' homozygosity, the left 63 blastomeres had accurate HLA genotyping. Among these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents(93.6%), 3 blastomeres lost one of parents' alleles(4.8%), and only one blastomere had two more than parents' alleles(1.6%)., Conclusion: The above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in pre-selecting HLA-identical sibling for allogeneic cord blood cell transplantation.

Objective: To investigate the correlation between intercellular adhesion molecule1 (ICAM1) gene K469E polymorphism and coronary heart disease(CHD) in Han Chinese population., Methods: Using the methods of polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP), 173 CHD patients and 141 controls were analyzed for the polymorphism, genotype and allele distribution of ICAM1 gene K469E., Results: The distribution of ICAM1 genotypes was in Hardy-Weinberg equilibrium. The frequency of KK genotype in CHD group was significantly higher than that in control (64.2% vs 48.9%, P<0.01). Similarly, the frequency of K allele in CHD group was significantly higher than that in control (79.2% vs 69.9%, P<0.01). With Logistic Regression Analysis ruling out the influences of age, gender and other CHD risk factor, the homozygous individual with KK genotype was 2.35 folds of KE or EE genotype one suffering from CHD (OR: 2.35, 95%CI: 1.03-5.36, P<0.05)., Conclusion: ICAM1 gene K469E polymorphism is associated with CHD risk of Han Chinese population, the K allele may serve as a genetic risk factor of coronary heart disease.

Objective: To study the antioxidation activity and protective effect of ginger oil on DNA damage., Method: Chemical light assay was used to detect the oxygen radicals scavenging capacity of ginger oil. The erythrocyte oxidation damage was induced by H2O2. The effect of ginger oil on oxidative erythrocyte was observed by the colorimetric analysis assay, and the content of malondialdehyde (MDA) in rabit hepatocyte was measured. The anaylsis of DNA damage was made with single cell gel electrophoresis(SCGE) technique., Result: Ginger oil might decrease light value compared with control group and inhibited erythrocyte oxidation damage. Compared with that in control group, the degress of DNA damage reduced significantly in the protected groups. Ginger oil might decrease the content of MDA remarkably and inhitibition rate was 48.16%., Conclusion: Ginger oil has dominantive protective effect on DNA damage induced by H2O2. Ginger oil might act as a scavenger of oxygen radical and might be used as an antioxidant.

Background & Objective: It has been indicated that fatty acid synthase (FAS) is abnormally overexpressed in human breast cancer compared with normal human tissue. Inhibition of FAS induces apoptosis of human breast cancer cells. The aim of this study was to observe the inhibition of triclosan on FAS from goose uropygial glands for establishing the method and to study the inhibition of triclosan on FAS from human breast cancer SKBr3 cells in vitro., Methods: The goose uropygial glands FAS was purified by ultra-centrifugation and Superdex PG 200 chromatography; the human breast cancer SKBr3 cell FAS was partially purified by ultra-centrifugation. The FAS was interacted with different concentrations of Triclosan with different times before catalyzing. Then the substrates of FAS were added to the reaction system. The inhibitory activities of triclosan against the FAS were investigated using spectrophotometric assays., Results: In the goose uropygial gland group, FAS was purified as a single band at 250kDa with SDS-PAGE. The inhibitory activities of triclosan(12.5 micromol/L) at 0, 5, and 10 minute on FAS were 26.40%, 28.30%, and 43.93%, respectively. The inhibitory activities of triclosan (25.00 micromol/L) at 0, 5, and 10 minute on FAS were 46.22%, 50.28%, and 97.05%, respectively. The inhibitory activities of triclosan (100.00 micromol/L) at 0, 5, and 10 minute on FAS were 98.11%, 97.75%, and 97.37%, respectively. In human SKBr3 breast cancer cell group, the inhibitory activities of triclosan (25, 50, 100, and 200 micromol/L) at 5 minute on FAS were 20.00%, 26.67%, 60.00%, and 100%, respectively., Conclusion: Triclosan inhibits the FAS from goose uropygial glands and human breast cancer SKBr3 cells. The inhibitory activities depended on the concentrations of triclosan and the interaction times between triclosan and FAS before catalyzing.