RESUMO
The present study aimed to investigate the effects of the WW domain-containing oxidoreductase (WWOX) gene on the stem cell properties of human ovarian cancer stem cells. A eukaryotic expression vector containing the WWOX gene was transfected into human ovarian cancer stem cells and Western blotting was used to assess the expression of WWOX protein in the transfected cells compared with the control cells (untransfected cells and cells transfected with the empty vector). The self-renewal abilities of these three types of stem cells was also assessed in vitro. To monitor changes in their differentiation potential, cells were cultured in medium supplemented with serum, and the expression of specific stem cell markers was determined. Drug-sensitivity tests were used to measure the sensitivity of the stem cells to cisplatin, doxorubicin, and mitoxantrone. The cells were also transplanted into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice to determine the changes in their tumorigenicity in vivo. Cells transfected with the WWOX-expressing plasmid stably expressed WWOX protein, while no WWOX protein was detected in control cells. Compared with the two types of control cells, WWOX-expressing stem cells manifested significantly reduced self-renewal ability. Compared with control cells, the expression levels of stem cell markers, including CD133, CD117, ATP-binding cassette sub-family G member 2, Nanog, octamer-binding transcription factor 4 and breast cancer resistance protein, were significantly lower in WWOX-expressing cells, while the level of the differentiation marker E-cadherin was significantly higher in WWOX-expressing cells. Furthermore, WWOX-expressing cells were more sensitive to treatment with cisplatin, doxorubicin and mitoxantrone. In NOD/SCID mice, the tumorigenicity of WWOX-expressing cells was significantly lower compared with that of control cells. The results indicate that the tumor suppressor WWOX suppresses stem cell properties in cancer stem cells, including self-renewal ability, differentiation potential, in vivo tumorigenic capability, high-level expression of stem cell genes and multidrug resistance.
RESUMO
The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 µg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.
