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猕猴多潜能成体肝前体细胞及猕猴ES 定向分化为胆管上皮细胞的研究
文献类型：学位论文
| 作者 | 金立方 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-07 |
| 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 肝前体细胞分离 多潜能 胚胎干细胞 限定性内胚层细胞 胆管上皮细胞 |
| 其他题名 | Isolation and characterization of multi-potent liver epithelial progenitor cells from normal adult rhesus monkeys and direct differentiation of rES cell into cholangiocytes |
| 学位专业 | 动物学 |
| 中文摘要 | 为解决供体器官的不足，以细胞移植为基础的替代疗法已成为治疗不可逆肝脏疾病新的希望。 肝前体（干）细胞（Hepatic progenitor cellS，HPCs）和胚胎干细胞（embryoic stem cells, ES）由于其特殊的细胞特性已成为细胞替代治疗理想的种源细胞。 然而一方面包括人在内的灵长类动物的正常成体肝来源的HPCs的分离依然是很困难的；另一方面，ES细胞来源的肝细胞和胆管细胞的生成效率依旧很低。因此有必要建立稳定的高效的灵长类动物HPCs细胞分离培养体系及ES细胞的肝细胞或胆管细胞分化体系以满足供体细胞的不足；这种体系的建立还有利于研究肝细胞生物学如分化机制、自我更新机制等方面的重要基础问题。 本研究以猕猴为实验模型，研究了正常成体肝来源的猕猴HPCs分离、纯化的条件，系统地鉴定了猕猴HPCs的细胞特性和体内、外分化潜能，并评价了体内移植效果。 同时以rES为材料，建立了rES高效分化为限定性内胚层（definitive endoderm cells, DE）和胆管上皮细胞的分化体系。主要实验结果包括：1）： FBS、EGF、HGF及rat tail collagen (鼠尾胶原)是分离培养正常成体猕猴来源的肝上皮前体细胞(rhesus monkey liver epithelial progenitor cells, mLEPCs)所必需的，mLEPCs在此培养体系中至少可以扩增20代或5个月以上，并仍然保持原有的细胞特性；mLEPCs呈现典型的上皮细胞形态，并表达HPCs细胞特有的表达模式即同时表达肝细胞和胆管细胞相关基因（ALB，APOH，CX43，IB4）或蛋白（CK7，CK8，CK18）；在适宜的分化体系下，mLEPCs可分化为功能性的肝细胞，形成具有胆管上皮细胞的胆管样结构，并能转分化形成肌肉样细胞、肌样成纤维细胞及少突样细胞；移植入肝损伤的免疫抑制的小鼠体内后，mLEPCs能参与受体肝组织的再生，并能分化成ALB阳性的肝细胞；体内定位发现mLEPCs与胆管区的细胞有相似的免疫原性，提示mLEPCs可能来源于胆管区。2）：rES在高浓度的acitvin A（100ng/ml）和低浓度的血清(1%)单层诱导体系下可定向分化得到高比率的限定性内胚层细胞（definitive endoderm cells，DE细胞）(约80%)； 高比率的DE细胞的得到还与rES细胞的接种密度相关；BMP4和FGF1可诱导DE细胞高效向胆管上皮细胞分化（约90%），但并不能得到肝细胞；而Notch信号通路可维持DE细胞的存活，并决定着DE细胞向胆管细胞分化，在Notch信号通路失活的情形下，即使存在BMP4和FGF1都不能促使DE细胞向胆管细胞分化。 本实验首次成功建立了正常猕猴成体肝HPCs分离培养体系，证实了分离得到的猕猴肝上皮前体细胞不但具有正常HPCs的增殖活力和参与受体肝组织的再生能力，而且还具有三个胚层的分化潜能，这一结果将为以HPCs为基础的细胞替代治疗人类肝脏疾病的实现提供了可能，并首次证明了HPCs也可以像某些少数成体干细胞一样具有三个胚层得分化潜能。 此外，本研究建立了rES高效定向分化为DE细胞和胆管细胞的分化体系，这一方法的建立将促进灵长类动物的DE细胞的发育机制研究，同时也可为高比率的内胚层功能细胞（如胰岛细胞、肝细胞、肺细胞）的获得提供丰富的种源细胞和平台。 |
| 英文摘要 | Due to the lack of donor organs for orthotopic liver transplantation, cell based transplantation is emerging as a potential way for therapy of irreversible human liver diseases. Among of these cells, hepatic progenitor cells (HPCs) and embryonic stem cells (ES) may be ideal source cells because of their specific cell properties. However, it remains difficult to isolate the HPCs from normal adult liver from primates including human; on the other hand, the efficiency of hepatocytic and cholangiocytic differentiation of ES is low. Thus, it is necessary to establish an efficient and stable system for isolation of HPCs from primates and to improve the efficiency of hepatocytic and cholangiocytic differentiation of ES. Establishment of these systems will also be valuable for study of basic hepatocytes biology such as mechanism of differentiation and self-renew. In the present study, we selected the suitable condition for isolation of HPCs from normal adult monkey liver, systematically characterized the properties and differentiation capacity of HPCs,, and evaluated the effect of HPCs transplantation. Additionally, we established a system for efficient differentiation of monkey ES cells to definitive endoderm and cholangiocyic cells. The main results were as follows: 1) FBS，EGF，HGF and rat tail collagen were essential for isolation of rhesus monkey liver epithelial progenitor cells ( mLEPCs) from normal adult liver, and mLEPCs could be passaged up to 20X or maintained more than 5 months without lost of their properties; mLEPCs represented typical epithelial morpha and expressed hepatobilialry specific genes （ALB，APOH，CX43，IB4）and proteins (CK7，CK8，CK18）; In proper differentiation mediums, mLEPCs could differentiate into functional hepatocytes, form bile duct-like structure with cholangiocytes cells, and transdifferentiate into muscle-like cells, myofibroblasts and oligodendrocytes; after transplantation into liver injured mice, the mLEPCs formed cell clusters in host hepatic parenchyma and differentiated into hepatocytes; immunohistochemistry revealed that mLEPCs may originate from bile duct regions. 2）Monkey ES could efficiently give rise to definitive endoderm cells (>80%) in condition of high concentration of activin A (100ng/ml) and low serum (1%), and their efficient induction was cell density related; BMP-4 and FGF-1 could induce definitive endoderm cells to differentiate into bile duct cells（90%）but not hepatocytes; Notch signal pathway was essential for survival and differentiation of DE cells to cholangiocytes, restrain of Notch signal pathway resulted to failure to induce DE cells to cholangiocytes despite in the presence of BMP-4 and FGF-1. Here, we first report that monkey epithelial cells function as HPCs could be derived from normal adult liver, and confirmed that the isolated HPCs possessed of multiple differentiation capacity and repopulation capacity in receipt tissue. These results provided convincing evidence for plasticity of adult stem cells, and indicated that mLEPCs may be served as ideal source for cell therapy for human liver disease based on the transplantation of hepatic progenitors. In addition, we established an efficient system for differentiation of monkey ES into DE and cholangiocytes cells. This method will facilitate the study for developmental mechanism of DE and provide resource cells to obtain high ratio of functional endoderm cells. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6111]  |
| 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 | 金立方. 猕猴多潜能成体肝前体细胞及猕猴ES 定向分化为胆管上皮细胞的研究[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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