猕猴胚胎干细胞向肝细胞分化及猕猴肝前体细胞向肌成纤维细胞分化的研究
文献类型：学位论文
| 作者 | 王喜宏 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 季维智 |
| 关键词 | 猕猴 胚胎干细胞 肝前体细胞 上皮-间充质转化 PTEN |
| 其他题名 | Hepatocytic differentiation of rhesus monkey embryonic stem cells and generation of myofibroblasts-like cells from liver epithelial |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 细胞替代治疗作为终末期肝病的治疗方法,给无数患者带来了新的希望。胚胎干细胞和成体肝前体细胞因其多能性成为理想的治疗用细胞来源。猕猴作为一种重要的实验动物，因为与人的高度相似性，成为临床前研究的理想的替代模型。本文就猕猴胚胎干细胞向肝细胞分化中的调控因子和猕猴成体肝前体干细胞的多能性进行了研究。 首先我们以猕猴胚胎干细胞向肝细胞分化为模型，采用分步诱导法研究细胞基质和生长因子对分化的影响。经activin A诱导后，约80%胚胎干细胞分化为限定性内胚层细胞。得到的限定性内胚层细胞在一型胶原或者鼠尾胶原上继续分化。在一型胶原上的分化细胞呈现未成熟肝细胞特征，而在鼠尾胶原上产生高达68%±7% AFP/CK18双阳性细胞，且分化的细胞具有典型的肝细胞样结构，表达成熟肝细胞相关的基因和功能。而在不添加生长因子的鼠尾胶原体系中，只检测到未成熟肝细胞基因的表达。本文建立猕猴胚胎干细胞高效定向分化为肝细胞的体系，并揭示鼠尾胶原和生长因子共同作用促进肝细胞的发生。 另外我们报道了猕猴肝脏上皮前体细胞可以分化为肌成纤维细胞。TGF-β诱导以后，部分成体肝脏上皮前体细胞克隆转化为成纤维样细胞。所得成纤维样表达间充质细胞标记但失去了上皮细胞标记的表达。成纤维样细胞可以培养长达40代，并表达肌成纤维细胞标记。此外,延时视频观察细也观察到成纤维样细胞的运动增加。我们的结果表明,猕猴肝脏上皮前体细胞,通过上皮-成纤维转化转化为肌成纤维细胞。这项发现将有助于理解肝纤维化中肌成纤维细胞的起源。 PI3K信号通路对胚胎的生存和发育都有着重要的作用。PTEN是该通路中的负调节因子，但是PTEN在早期胚胎发育中的存在和分布以及作用都还未见报道。通过免疫荧光，我们发现PTEN从一细胞到囊胚都有表达，而活化的PTEN主要分布在致密桑椹胚的卵裂球表面，这一结果暗示PTEN可能在致密化中有某种作用。而在培养中添加PTEN抑制剂bpV，可以显著降低胚胎发育率甚至引起二细胞阻滞。我们的结果证明PTEN在小鼠着床前胚胎发育中有重要作用。 |
| 英文摘要 | Cell replacement strategies have proven to be effective in the treatment of a variety of life threatening liver diseases in recent years. Due to their pluripotency, the embryonic stem cells and adult hepatic progrnitor cells (HPCs) both provide ideal cell source for cell therapy. Rhesus monkey has similar characteristics to human, and might be useful as a substitute model for preclinical research. So in this study we use rhesus monkey ES cells （rES）to value the factors that control the differentiation we also use monkey HPCs to study their polirepotency. Here, we analyzed the effect of collagen gels on rES cells differentiation into hepatocytes by stepwise protocols. About 80% of definitive endoderm (DE) cells were generated from rES cells after treated with activin A. The DE cells were then plated on collagen gels or type I collagen-coated wells with growth factors to induce hepatocytes differentiation. In type I collagen systems, characteristics of immature hepatocytes were observed. In collagen gel culture, differentiated cells exhibited typical hepatocyte morphology and expressed adult liver specific genes. Compared to type I collagen systems, significantly higher AFP/CK18 double-positive cells (68%±7%) were produced in collagen gel culture. Furthermore, some differentiated cells acquired the hepatocytic function of glycogen storage. However, only immature hepatic genes were observed in collagen gel systems if growth factors were absent. Thus, collagen gels combined with hepatocyte-inducing growth factors efficiently promoted differentiation of hepatocytes from rES. Also, we reported rhesus monkey liver epithelial progenitor cells (mLEPCs) could differentiate into myofibroblasts. After cultured with TGF-β and FBS, some cells in adult mLEPCs colonies converted to fibroblasts-like cells. Immunostaining and RT-PCR showed that fibroblasts-like cells had acquired the expression of mesenchymal markers but lost the expression of epithelial markers. Fibroblasts-like cells were maintained in culture for up to 40 passages and express myofibroblastic-related proteins. Furthermore, increased cell motility was also detected in these fibroblasts-like cells by time-lapse video observation. Our results demonstrate that hepatic epithelial progenitor cells, mLEPCs, transform to myofibroblast-like cells via epithelial-mesenchymal transition. This finding will facilitate understanding of the origin of myofibroblasts in liver fibrosis. The PI3K/Akt signal transduction pathway is a well-known mediator of growth promoting and cell survival signals. And recently it was also found critical in murine preimplantation embryos. PTEN known as a tumor suppressor gene is a major negative regulator of PI3K. But currently, there is no evidence demonstrating either the presence or function of PTEN in murine preimplantation embryos. We found, using confocal immunofluorescent microscopy, that the PTEN were expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. The activated form of PTEN especially located near the blastomere surface in compact morula stage. This result suggested PTEN may function in embryonic compaction. In addition, PTEN inhibitor bpV(pic) reduced the embryonic development rate, even caused the ‘2-cell block’. Taken together, these data are the first to demonstrate the presence and function of the PTEN in mammalian preimplantation embryos. |
| 语种 | 中文 |
| 公开日期 | 2011-09-19 |
| 源URL | [http://159.226.149.42:8088/handle/353002/6791]  |
| 专题 | 昆明动物研究所_生殖与发育生物学 |
| 推荐引用方式 GB/T 7714 | 王喜宏. 猕猴胚胎干细胞向肝细胞分化及猕猴肝前体细胞向肌成纤维细胞分化的研究[D]. 北京. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：昆明动物研究所
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