ABSTRACT
Objective To explore the effect and mechanism of the bone morphologenetic protein 4 (BMP4)/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes .Methods Three-day-old Kunming mouse ovarine tissues were digested by the two-step enzymatic method to extract and purify oocytes .The cultured oocytes were divided into three groups: the normal culture medium (Con group), the medium with BMP4 (BMP4 group), and the medium with BMP4 and BMP4 inhibitor ( BMP4+inhibitor group ) .TUNEL was used to examine the effects of BMP 4 on the survival of the primordial follicle oocyte;Immunohistochemical staining and Real-time quantitative PCR were performed to investigate the expressions of p-Smad1/5/8, sohlh2, c-kit and foxo3a;siRNA interference, sohlh2 plasmid transfection and LY294002 treatments were performed to explore the mechanism of the BMP 4/Smad signaling pathway on the apoptosis of oocytes . Results TUNEL results demonstrated that the ratio of apoptotic oocytes in BMP 4 group was significantly lower than that in the Con group ( P <0.05 ) and the BMP4 +inhibitor group ( P <0.05 ); BMP4 significantly promoted the nuclear translocation of Smad and inhibited the nuclear translocation of foxo 3a, the mRNA and protein levels of sohlh2 and c-kit remarkably increased in BMP4 group.The effect of BMP4 on the oocyte survival was significantly repressed after sohlh 2 siRNA transfection.Sohlh2 overexpression up-regulated the expression of p-foxo3a, and this activity was abolished by LY 294002.Conclusion BMP4/Smad signaling pathway may inhibit primordial follicle oocyte apoptosis , via up-regulation of the expression of sohlh2 and c-kit, and then down-regulation of the nuclear translocation of foxo 3a.
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Objective To explore the optimal condition of direct differentiation into dopaminergic neurons of embryonic stem cells in serum-free and feeder layer cell free medium. Methods We used the method of phase induction to culture embryonic stem cells. At first, embryonic stem cells were cultured in the serum-free medium with bFGF and LIF so as to realize the direct differentiation from embryonic stem cells to neural precursors. Differentiated cells were determined by nestin immunocytochemical staining. On this basis, we transferred embryonic stem cells to the B27 serum-free medium with IL-1 so as to realize the direct differentiation from neural precursors to dopaminergic neurons. Differentiated cells were determined by TH immunocytochemical staining. Results Approximately 85 percent of cell masses were nestin immuno-positive. The differentiation ratio of dopaminergic neurons was 13%, which increased significantly in comparison with natural differentiation ratio of dopaminergic neurons.Conclusion Without serum and feeder layer cell, we can induce embryonic stem cells to differentiate into dopaminergic neurons effectively by adding different growth factors at different phases, which makes the inductive processes more easily.
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Objective To investigate the milieu-dependent differentiation of primordial germ cells(PEGs) in the acute damaged liver microenviroment. Methods After PGCs were cultured and proliferated,these cells were labelled with 5-bromo-2-deoxyuridine(BrdU),then transplanted into the acute damaged liver by CCl_4 through tail vein.Two and four weeks later,the liver was extracted and 10?m-cryostat continuous sections were obtained.The existing and differentiation of the transplanted cells were identified by immunohistochemistry,immunofluorescence double staining and histochemistry for BrdU and hepatic-specific ALB,and the glycogen. Results Transplanted PGCs were found to be incorporated into the acute damaged liver and differentiated into hepatocytes,compensating for acute liver failure.Conclusion PGCs can be induced to differentiate into hepatocytes in the acute damaged liver microenvironment,and can be used for cellular hepatoplasty to treat severe liver disease.
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Objective To explore the inductive effect of striatal tissue on mouse embryonic stem cells and further analyse the cell source and inductive pattern of this inductive effect. Methods We employed striatal extracts、conditioned medium of striatal astrocytes and conditioned medium of striatal neurons to induce embryonic stem cell to differentiate directly. The differentiated cells were evaluated by morphological observation and TH immunocytochemical staining method. We also performed a quantitative analysis of the results. Results Striatal extracts and conditioned medium of striatal astrocytes had obvious inductive effect on embryonic stem cell.Percentages of three groups were 15%,15.2% and 3% respectively. Conclusions The astrocytes in striatum might have an inductive effect on dopaminergic neuronal differentiation of embryonic stem cells.The determination of inductive factor will be our next research aim.;
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Objective To carry out construction of chimeras from embryonic stem cells of outbred KM mice. Methods We isolated embryonic stem cells from inner cell mass of KM mice blastocysts. Then we transferred embryonic stem cells into blastocoele of C57BL/6J inbred mice by microinjection in order to construct chimeric mice. Results The cells isolated from inner cell mass have typical characteristics,i e positive alkaline phophatase staining,normal karyotype,forming embryoid body. Now,we have constructed one chimeric mice successfully. Conclusion Embryonic stem cells isolated from inner cell mass can be used for the chimeras production successfully,which forms the substantial base of transgenic animal model by the way of using embryonic stem cells.