Mouse A6-positive hepatic oval cells derived from embryonic stem cells.

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

Fetal alloantigen is responsible for the expansion of the CD4(+)CD25(+) regulatory T cell pool during pregnancy.

Increasing evidence suggests that CD4(+)CD25(+) regulatory T cells (Tregs) participate in the development of maternal tolerance to the fetus during pregnancy; however, the factors controlling the activities of Tregs are poorly understood. In the present study, CD4(+)CD25(+) Tregs were analyzed in syngeneically pregnant mice (BALB/cxBALB/c), allogeneically pregnant mice (BALB/cxC57), ovariectomized mice and pregnant women to investigate the influences of fetal alloantigens and pregnancy-related hormones on the activities of Tregs. It was demonstrated that the frequencies of CD4(+)CD25(+) Tregs increase more in allogeneically than in syngeneically pregnant mice, which contributes to a lowered alloreactivity against paternal antigens in allogeneically compared with syngeneically pregnant mice. The increased Tregs are most likely to be induced in peripheral lymphoid tissues, rather than develop in thymus. Allogeneically mated mice and humans share similar dynamic changes in Treg frequencies, markedly increasing during early pregnancy and progressively decreasing from mid-gestation onwards to return to non-pregnant levels at term. Induction of labor in humans appears to be associated with a decrease of CD4(+)CD25(high) Tregs and increase of CD4(+)CD25(low) T cells. Neither estrogen or progesterone alone, nor their combination, shows an impact on the frequencies of Tregs in ovariectomized mice. These results suggest that fetal alloantigen is responsible for the increase of Tregs during pregnancy, and the expansion of the Treg population is of importance for the allogeneic fetus to evade immune attack from the mother.

[The effect of p38 on the cycloheximide-induced HL-60 cell death through mitochondria pathway].

OBJECTIVE: To study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway. METHODS: Inhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points. RESULTS: The sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01). CONCLUSION: CHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

[Application of vital dye CFDA-SE and SNARF-1 to evaluate mixed lymphocyte reaction].

AIM: To establish a new method for more comprehensive evaluation of responsive intensity in one-way mixed lymphocyte reaction (MLR). METHODS: CFDA-SE labeled lymphocytes from lymph nodes of BALB/cJ mice were applied as responder cells, and SNARF-1 labeled splenocytes from BALB/cJ or C57BL/6 mice which had been pretreated with mitomycin C were applied as stimulator cells to establish mixed lymphocyte culture (MLC). After 96 h of culture, cells were harvested and analyzed by flow cytometry to acquire the proliferative information of SNARF-1 negative and CFSE positive staining responder cells. ModFit software was then used to get more proliferation related index. RESULTS: With increasing division cycles, the CFSE fluorescence was diluted to the extent that the responder cells could not be discriminated from stimulator cells, which was resolved by gating SNARF-1 positive cells out to define responder cells. Several important proliferation related indexes were obtained by ModFit software, such as precursor frequency and proliferation index, etc. CONCLUSION: Our results show that application of vital dye CFDA-SE and SNARF-1, combined with flow cytometry is a powerful tool for evaluation of responsive intensity in one-way MLR.

[Change of mitochondria during apoptosis of HL-60 cells induced by camptothecine].

AIM: To study the changes of mitochondrial mass and membrane potential (deltapsi(m)) during apoptosis of human promyelocytic leukemia cells (HL-60) induced by camptothecine(CPT). METHODS: HL-60 cells were stimulated with 4x10(-6) mol/L CPT. Apoptosis and necrosis of HL-60 cells were detected by annexin V-FITC/PI staining. Mitochondrial mass and membrane potential (deltapsi(m)) were measured by NAO and DiOC(6)(3) stanings, respectively. RESULTS: After 12 hour treatment with 4x10(-6) mol/L CPT, the early apoptotic rate of HL-60 cells was significantly increased compared with the control group (12.75+/-4.61)% vs (2.88+/-2.49)%,(P<0.01), and the necrotic rate was significantly elevated too, (3.48+/-1.67)% and (0.71+/-1.10)%, (P<0.01). The result of PI staining showed that the apoptotic rate of HL-60 cells at late phase (12 h) in CPT group was (3.52+/-1.07)%, whereas that in control group was (0.46+/-1.06)%. At the same time, we observed that the cells in G(2)/M phase were arrested. The percentage of cells in G(2)/M phase in CPT group and the control was (13.45+/-1.91)% and (22.46+/-2.19)% (P<0.01) respectively. The percentage of cells with low mitochondrial mass in CPT and control groups was (25.74+/-2.09)% and (4.53+/-1.26)%, respectively (P<0.01). Mitochondrial membrane potential in CPT group and control group was (17.71+/-5.23)% and (1.64+/-2.00)%, respectively. CONCLUSION: During CPT-induced apoptosis of HL-60 cells, mitochondrial mass and membrane potential drop significantly, but its depolarization heightens.

[The effects of resveratrol on the activation, proliferation and cytokine expression of murine T lymphocytes].

AIM: To study the effects of resveratrol (RSV) on the activation, proliferation and cytokine expression of murine T lymphocytes. METHODS: Murine T lymphocytes were isolated from lymph nodes and cultured in vitro. The lymphocytes were pre-treated with RSV for 1 h prior to activation with PDB plus ionomycin. (3)H-TdR incorporation was used to detect T cell proliferation. IL-2 and IFN-gamma mRNA were detected by RT-PCR, IL-2 and IFN-gamma proteins were detected by intracellular cytokine staining with flow cytometry. RESULTS: RSV can inhibit the proliferation and expression of IL-2 and IFN-gamma of T lymphocytes. CONCLUSION: The immunosuppressive action of RSV may be related with its inhibitory effects on T cell activation, proliferation and cytokine expression.

[Application of vital dye CFDA-SE to study lymphocytic proliferation].

AIM: To explore the application value of vital dye CFDA-SE to study of lymphocytic proliferation. METHODS: CFDA-SE staining, fluorescence antibody labeling, flow cytometry and related software were used to detect the fluorescence intensity and analysis the proliferation kinetics of lymphocytes and their subsets after stimulation with polyclonal stimulators. RESULTS: Lymphocytes divided after stimulation of PDB+ionomycin or ConA for 48 h, manifesting the serial halving of fluorescence intensity. CsA inhibited the proliferative effect of ConA on lymphocytes and no CFSE fluorescence halving were seen. Proliferation of CD4(+) T cells and CD8(+) T cells were asynchronous after ConA stimulation for 48 h, which became more obvious at the time of 72 h. Proliferation-related indexs got ten from ModFit(TM) software showed that the proliferative effect of ConA on CD8(+) T cells was stronger than that on CD4(+) T cells. CONCLUSION: CFDA-SE staining combined with fluorescent antibody labeling and cytometry were powerful tools for analysis of lymphocytic proliferation.