人羊膜上皮细胞移植治疗小鼠肝损伤

发布时间：2018-05-01 21:29

本文选题：人羊膜上皮细胞 + 诱导分化　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：背景:近年来,关于干细胞治疗的研究越来越火热,而在临床治疗的应用上,对众多干细胞的选择是其关键问题。人羊膜组织作为生产的附属品,来源丰富。人羊膜来源的干细胞,尤其是人羊膜上皮细胞(human Amnion Epithelial Cells,hAECs)具有向三胚层分化的潜能,表达多种胚胎干细胞标记物,并且hAECs具有无致瘤性、来源丰富、容易获取、免疫原性低、无医学伦理限制等优点,很多优势是胚胎干细胞和诸多成体干细胞所不具备的。肝脏是人体重要的代谢、排毒、免疫器官,对人体至关重要,但严重的肝脏疾病目前还没有可以完全治愈的治疗措施。因此hAECs有望成为今后临床移植治疗的重要种子细胞。方法:使用酶消化法从人羊膜中分离得到hAECs,观察细胞形态,并绘制第2代和第5代hAECs的生长曲线。免疫细胞化学法检测hAECs角蛋白19的表达。RT-PCR鉴定hAECs表达干细胞相关基因。用四氯化碳(CC14)腹腔注射诱导小鼠肝损伤。将荧光染料羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记的hAECs通过尾静脉注射移植到肝损伤小鼠体内,检测细胞移植后小鼠肝功、组织病理学变化。结果:hAECs核大、形态呈铺路石状,可在体外连续传代培养。免疫细胞化学检测显示hAECs表达上皮细胞标志角蛋白19。RT-PCR检测显示hAECs表达干细胞标志基因REX1、Oct-4和Nanog。体外可成功诱导为脂肪细胞、神经细胞和肝样细胞,并在诱导后表达相应细胞的标志基因PPARγ2、MAP-2和albumin。冰冻切片免疫荧光显示hAECs可以归巢到受损肝脏。hAECs移植后对肝损伤小鼠的肝功、组织病理学都有修复作用,治疗效果显著。结论:人羊膜上皮细胞可以用于异种移植。移植到四氯化碳诱导肝损伤模型小鼠体内,对于损伤肝脏恢复具有促进作用。
[Abstract]:Background: in recent years, the research on stem cell therapy has become more and more hot, and the selection of stem cells is the key issue in clinical treatment. Human amniotic tissue, as a subsidiary of production, is rich in sources. Stem cells derived from human amniotic membrane, especially human amniotic epithelial cells (HMECs), have the potential to differentiate into the tridermal layer and express a variety of embryonic stem cell markers. Moreover, hAECs has non-tumorigenic properties, rich sources, easy to obtain, and low immunogenicity. Without medical ethics restrictions, many advantages are not available to embryonic stem cells and adult stem cells. Liver is an important metabolism, detoxification and immune organ, which is very important to human body. However, there is no cure for serious liver diseases. Therefore, hAECs is expected to be an important seed cell for clinical transplantation in the future. Methods: hAECs were isolated from human amniotic membrane by enzyme digestion. The morphology of the cells was observed and the growth curves of the second and fifth generation hAECs were plotted. The expression of hAECs keratin 19 was detected by immunocytochemistry. RT-PCR was used to identify the stem cell related genes expressed by hAECs. CC14) was used to induce liver injury in mice. HAECs labeled with fluorescein hydroxy fluorescein diacetate succinimide (CFSE) was transplanted into mice with liver injury by tail vein injection. The liver function and histopathological changes were detected after transplantation. Results the nucleus of 1% hAECs was large and its morphology was paving stone, which could be subcultured continuously in vitro. Immunocytochemistry showed that hAECs expressed epithelial cell marker keratin 19.RT-PCR and hAECs expressed stem cell marker REX1 Oct-4 and Nanog4. Adipocytes, nerve cells and hepatoid cells were successfully induced in vitro, and the marker genes PPAR 纬 2 MAP-2 and Albumin were expressed after induction. The immunofluorescence of frozen sections showed that hAECs could homing to the injured liver. After transplantation, the liver function and histopathology of the injured mice were repaired, and the therapeutic effect was remarkable. Conclusion: human amniotic epithelial cells can be used in xenotransplantation. Transplantation into carbon tetrachloride induced liver injury model mice can promote the recovery of injured liver.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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