| 其他摘要 | Before clinical applications of embryonic stem cells (ESCs) are begun, extensive preclinical studies must be completed in suitable animal models for assessing safety, efficacy, and long-term survival of transplanted ESC-derived phenotypes. So, establishment of available ESC lines from other animal species will be requisite. ESCs self-renew indefinitely and give rise to derivatives of all three primary germ layers, but the signaling pathways that govern the pluripotency of ES cells remain largely unknown and have species difference. In this thesis, the progress in establishment of ES cell lines, improvements on culture system, and self-renewal mechanism of ESCs have been reviewed, and some research findings have been made in the following: 1) We described the derivation of four stable pluripotent rabbit embryonic stem cell (ESC) lines, one (RF) from in vivo fertilized and in vitro cultured blastocysts and three (RP01, RP02, and RP03) from parthenogenetic blastocysts. These ESC lines have been cultivated for extended periods (RF >1 year, RP01>8 months, RP02>8 months, RP03>6 months) in vitro while maintaining expression of pluripotent embryonic stem cell markers and a normal XY or XX karyotype. The ESCs from all lines expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, SSEA-4), and the tumor-related antigens (TRA-1-60, and TRA-1-81). Similar to human and mouse ESCs, rabbit ESCs expressed pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and signaling pathway genes (FGF, WNT, and TGF pathway). Morphologically rabbit ESCs resembled primate ESCs whereas their proliferation characteristics were more like those seen in mouse ESCs. Rabbit ESCs were induced to differentiate into many cell types in vitro and formed teratomas with derivatives of the three major germ layers in vivo when injected into SCID mice. Our results showed that pluripotent, stable ESC lines could be derived from fertilized and parthenote derived rabbit embryos. 2) Our findings from a combination of growth factor addition and receptor inhibition experiments provide substantial evidence to support the hypothesis that FGF and TGFβ signalling plays key roles in maintaining pluripotency of rabbit ESCs, which is similar to human ESCs. Our findings also show that there is a regulatory network among FGF, WNT, and TGFβ pathway to regulate the pluripotency of rabbit ESCs. Inhibition of FGF and TGFβ pathway result in decrease of rabbit ESCs proliferation rate. Long-term maintenance of rabbit ESCs pluripotency can be achieved with the combination of Activin A or TGFβ1 plus FGF2 and Noggin in the absence of feeder-cell layer and serum. However, in recent report, inhibition of Activin-Nodal-TGFβsignaling by Smad7 or SB-431542 dramatically decreased ESC proliferation without decreasing ESC pluripotency in mouse, which suggests that rabbit ESCs may be a good model for some specific human diseases in preclinical trials. 3) The four homologous feeder cell lines (MESF, MOF, MFG and CMESF) can be used to support the undifferentiated growth and maintenance of pluripotency in rES cells, but MFGE can not. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. These findings showed that the abilities variation of the feeders might be related with some genes expression difference. |
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