Identification and functional study of immortalized mouse thymic epithelial cells.

As the key cells in a three-dimensional scaffold within the thymus, Thymic epithelial cells (TECs) play critical roles in the homing, migration and differentiation of T cell precursors through adhesive interactions and the release of various cytokines. In this study, primary cultures of mouse TECs were isolated and identified with TEC-specific antibodies CK5 and CK8. These TECs were immortalized by retroviral transduction of simian virus (SV) 40 large T antigen. We then compared the functions of TECs and immortalized TECs (iTECs). Cell morphology and the proliferative capacity of TECs and iTECs were observed by inverted microscope photography and crystal violet assay after passage. A soft agar assay was then performed to observe their clone formation ability. The expression levels of epithelial cell related factors, such as IL-7, Lptin, Pax-9, Sema3A and et al., were detected by IF and qPCR. TECs were co-cultured with human acute monocytic leukemia cells (THP-1), and the effect of TECs on promoting THP-1 proliferation was observed with flow cytometry and CFSE labeling. Senescence-associated ß-galactosidase assay was measured to detect the anti-aging capabilities of the cells. Cell cycle distribution was analyzed by propidium iodide (PI) staining, and paclitaxel (PTX)-induced apoptosis was detected by Annexin V-PI staining to evaluate the anti-apoptotic ability of the cells. Throughout, we found that the immortalized TECs still retain the characteristics of primary TECs, such as the morphology, function and epithelial characteristics; however, iTECs have stronger capabilities in proliferation and anti-aging. Our research suggests that the iTECs were successfully immortalized by SV40 large T antigen, and that the biological characteristics and functions of iTECs were similar to the original TECs. This immortalized cell can be used as an efficient cell model in functional research of the thymus substituting primary TECs with iTECs.

miRNA-101 inhibits ovarian cancer cells proliferation and invasion by down-regulating expression of SOCS-2.

OBJECTIVE: To investigate the expression of miRNA-101 in normal and malignant ovarian tissues and cells as well as its impact on the proliferation and invasion of human ovarian cancer H08910 and SKOV3 cell lines. METHODS: Real time polymerase chain reaction (RT-PCR) was employed to detect the miR-101 and SOCS-2 expression in 20 separate ovarian cancer tissues and para-carcinoma tissues, human ovarian cancer cells (H08910 and SKOV3) and normal human ovarian epithelial cells (HUM-CELL-0088). After H08910 and SKOV3 ovarian cancer cells were respectively transfected with miR-NC (H08910/NC and SKOV3/NC) and miR-101 (H08910/miR-101 and SKOV3/miR-101), Western Blot was employed to detect the SOCS-2 expression in transfected cells. CCK-8 and clone formation and Transwell assays were employed to determine the proliferation and invasion ability of wild type and transfected ovarian cancer cells. RESULTS: The expression of miR-101 in ovarian cancer tissues and cells was significantly lower than that in para-carcinoma tissues (t=19.12, P=0.002) and normal human ovarian epithelial cells (HUM-CELL-0088) (F=14.37, P=0.000), respectively. In contrast, the SOCS-2 expression in ovarian cancer tissues and cells was significantly higher than that in para-carcinoma tissues (t=25.03, P=0.000) and HUM-CELL-0088 cells (F=14.9, P=0.000) by Western Blotting analysis, respectively. Compared with wild type and empty vector transfected cells, the expression of SOCS-2 was significantly decreased in miR-101 transfected H08910 (t=10.9, P=0.001) and SKOV3 cells (t=21.03, P=0.000). The results of CCK-8, clone formation and Transwell assays revealed that the proliferation and invasion ability of ovarian cancer cells was markedly inhibited by the transfection of miR-101. CONCLUSION: MiR-101 was validated to be reduced and SOCS-2 expression increased in ovarian cancer tissues and cells. The over expression of miR-101 can remarkably reduce the in vitro proliferation and invasion ability of ovarian cancer cells through the down-regulation of SOCS-2.

Optimization of technology for dietary fiber extraction from Maixiansan by response surface methodology.

BACKGROUND: Our study aims to determine whether response surface methodology can optimize the extraction of dietary fiber from Maixiansan. METHODS: A Box-Behnken design was employed to optimize the extraction parameters, including α-amylase concentration (X1: 0.3 - 0.5%), enzymolysis time (X2: 30 - 60 min) and NaOH content (X3: 1.0 - 5.0%), of dietary fiber from Maixiansan using an enzyme-alkali extraction technique. RESULTS: The optimal technological conditions were as follows: α-amylase concentration: 0.4%; enzymolysis time: 45 min; NaOH content: 4.0%. Under these conditions, the extraction yield reached 57.14%, which was well consistent with the predicted models with a coefficient of determination (R2) of 0.9818. An evaluation of the anti-inflammatory activity indicated that Maixiansan was able to significantly inhibit dextran sodium sulfate-induced ulcerative colitis in rats by increasing the concentration of short-chain fatty acids (acetate, propionate and butyrate), among which the butyrate content was significantly higher in the Maixiansan group than in the other groups. CONCLUSION: Our experiments showed that response surface methodology can optimize the extraction of dietary fiber from Maixiansan. Maixiansan could be explored as an anti-ulcerative colitis agent.