【作者】 罗宏武；

【导师】 刘浔阳；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 在胚胎发育过程中,肝脏的整个发育过程是一个涉及多基因、多环节、多途径的网络调控过程。1965年Farber的研究揭开了对于肝细胞研究的新篇章,他首次提出在肝内存在小上皮细胞,即卵圆细胞。他的发现推动了肝再生及肝细胞肝癌发病机制的深入研究。目前认为肝干细胞（hepaticstem cell,HSC）来源于前肠内胚层,在胚胎发育过程中以未成熟的肝细胞的形式存在,其形态与胆管上皮细胞相似,其生化特征类似于胚胎干细胞。20世纪末,大量研究表明,成体干细胞可以跨越胚层限制分化为肝细胞。美国科学家近年宣布,他们在羊水中发现了和胚胎干细胞一样有效的干细胞,并在实验室中使这种干细胞分化成肌肉、骨头、脂肪、血管、神经和肝脏细胞。和胚胎干细胞相比,这种干细胞更容易分化成各种组织,且不会形成一种名为畸胎瘤的良性肿瘤。来源于人废弃胎盘的羊膜上皮细胞（human amnioticepithelial cells,hAECs）是这样一类极具研究潜力的细胞。目前国内外这方面的研究尚处于起步阶段。本课题对人羊膜上皮细胞完成了体外检测和鉴定,对其向肝细胞样细胞体外分化的条件进行了摸索和优化,在成功诱导生成肝细胞样细胞的基础上,将诱导获得的肝细胞样细胞移植入裸小鼠体内,检测了诱导获得的肝细胞样细胞是否具有生物学功能。本研究分为3个部目的:建立人羊膜上皮细胞的体外分离方法及培养体系,检测人羊膜上皮细胞的生物学特性,检测人羊膜上皮细胞的AAV-EGFP感染效率。方法:使用胰蛋白酶从人羊膜上消化人羊膜上皮细胞进行培养,细胞免疫荧光检测胚胎干细胞表面SSEA-1、SSEA-3、SSEA-4和TRA-1-60、TRA-1-81标记;RT-PCR检测全能性相关基因TERF、THY1、LEFTYA、Rex-1、Sox-2、Oct-4和nanog等的表达;腺相关病毒感染检测人羊膜上皮细胞的AAV-EGFP感染效率。结果:人羊膜上皮细胞表达SSEA3、SSEA4、TRA-1-60、TRA-1-81等胚胎干细胞的特异性标记;表达TERF1、THY、EFTYA、Rex-1、SOX2、Oct-4和Nanog等全能性相关基因;人羊膜上皮细胞的AAV-GFP感染效率在病毒浓度为10^-3pfu/ml时可达58.2%±1.3%。结论:建立了hAECs的细胞分离方法及适合hAECs生长和增殖的体外培养体系;hAECs具干细胞特征;体外培养的hAECs细胞可以被AAV-GFP感染,并表达GFP,且表达效率达50%以上。目的:研究Dex,HGF,IGF等细胞因子联合使用的诱导体系对hAECs向肝细胞样（hepatocyte-like）细胞诱导分化的影响。方法:采用Dex,HGF,IGF等细胞因子联合使用方法诱导培养hAECs向肝细胞样细胞分化,诱导周期为两周。诱导过程中采用RT-PCR鉴定细胞ALB、CYP1A1、CYP1A2、IGFR、c-met等肝细胞相关关键功能基因的表达和HNF3、HNF4和C/EBPa三种转录因子的表达。流式细胞术分析集落细胞表面标记ALB、AFP和CK18的时程变化;为了优化诱导体系,检测了Dex、HGF、IGF等细胞因子的剂量依赖性;比较不同代数的hAECs在诱导向肝细胞样细胞分化的ALB基因表达情况。结果:在诱导两周后的hAECs中可以检测到ALB、CYP1A1、CYP1A2、IGFR、c-met等肝细胞相关关键功能基因的表达,且这些功能基因的表达呈现逐渐递增的趋势。提示hAECs可能已经被成功诱导为肝细胞样细胞。诱导前的hAECs表达HNF1（一种肝细胞转录因子）,不表达HNF3、HNF4和C/EBPa;而诱导后的hAECs不仅可以表达HNF1,还可以表达HNF3,HNF4和C/EBPa,并且HNF1的表达在诱导前后的hAECs中也有所不同,诱导后的hAECs中HNF1的表达水平明显高于没有经过诱导的hAECs。细胞表面标记检测发现:诱导6天时,hAECs主要表达AFP+,约为15.1±2.1%;随着诱导时间的延长,10天时hAECs表达AFP+/ALB+,约为6.5±1.4%;而诱导14天时,hAECs基本上只表达ALB+,为13.9±2.3%。诱导10天时的hAECs开始表达ALB+/CKl8+;约为2.5±1.4%;诱导14天时,细胞依然表达ALB+/CK18+,且这种双阳性的细胞量有明显增加,为18.9±3.1%;同时CK18+细胞数并没有明显减少,诱导10天时,CK18+细胞数为16.1±1.2%;诱导14天时,CK18+细胞数为21.3±4.6%。提示,诱导过程中的hAECs随着诱导时间的延长有一个逐渐成熟的过程。剂量依赖性实验结果显示,IGF、HGF均存在剂量依赖性,而Dex的剂量依赖性实验结果不具有统计学意义,认为不存在剂量依赖性。对不同代数的hAECs在诱导向肝细胞样细胞分化的ALB基因表达情况进行比较,结果显示,传代三代的hAECs诱导后均能表达ALB基因,且在表达强度上没有明显区别结论:hAECs确实可以在体外向肝细胞样细胞进行诱导分化,并能被成功诱导分化为有功能的肝细胞样细胞;hAECs向肝细胞样细胞的诱导过程中,对于HGF和IGF具有剂量依赖性,而对于Dex的这种剂量依赖性并不明显;传代三代内的hAECs诱导后,在肝细胞样细胞特异性功能基因ALB的表达上没有明显区别。目的:观察人羊膜上皮细胞（hAECs）在肝脏受损裸鼠脾内移植后是否具有肝细胞样功能的表达并探讨其治疗肝功能损伤的可行性。方法: 40只裸小鼠,随机分为三组,A组:20只,行2/3肝叶切除后,自脾下极移植5×10⁶绿色荧光蛋白标记的hAECs;B组:10只,不行肝叶切除,自脾下极移植5×10⁶绿色荧光蛋白标记的hAECs;C组:10只,行2/3肝叶切除后,自脾下极注射生理盐水0.2 mL。荧光显微镜hAECs体内示踪并观察其是否表达肝细胞特异性蛋白;术后2周A、B组各10只裸小鼠采血定量检测裸鼠血中人白蛋白及肝功能,术后4周A组另10只及C组10只裸小鼠做以上相同检测。结果: A组镜下肝脏和脾脏组织中均发现绿色荧光信号的hAECs,大部分表达人白蛋白;B、C组镜下均未见绿色荧光细胞。A组血中可检测到人白蛋白且术后4周明显高于术后2（3.9±1.34vs0.37±0.14,P<0.01）;术后4周A组肝功能（ALB、ALT、AST）明显优于C组（p<0.05）,A组肝功能术后4周亦优于术后2周（p＜0.05）。结论:肝损伤可以有效诱导hAECs向肝迁移增殖,大部分hAECs在肝细胞损伤的微环境下有肝细胞样功能表达,hAECs移植可以在一定程度上修复受损肝功能。更多还原

【Abstract】 In the process of embryonic development, the development of the liver is involved in a multi-gene, multi-link, multi-channel network and the hepatic stem cells play a very important role in this process. This kind of stem cells was developed from the foregut. it is similar to bile duct epithelial cell in morphology and to embryonic stem cells in biochemical.In the end of the 20th century, numerous studies show that trandifferentiation could happen when adult stem cells were cultured in special condition. In recent years, American scientists announced that they found some stem cells in the amniotic fluid as effective as embryonic stem cells, and they found that those stem cells can differentiated into muscle, bone, fat, blood vessels, nerves and liver cells in the laboratory. Compared to embryonic stem cells, they could differentiate into various organizations more easily, and could not formate teratomas. Human amniotic epithelial cells, extracted from the abandoned placenta is such a great research potentiality cells. Current domestic and international research in this area is still in its infancy. The aim of theasis was to discuss the method of isolation and identification of the human amniotic epithelial cells, to invstigate the posibility of its transdifferentiation into hepatocyte-like cells and the Intrasplenic Transplantation. This study is divided into three parts, the main research methods and results are as follows: Objectives: To isolate hAECs from amnion and establish a culture system for hAECs in vitro. To identify the stem cell related characteristics of these isolated hAECs . To test the AAV transfection efficiency of hAECs.Methods: Trypsin-EDTA was used to digest the amnions we collected. The cell sample of P1 passage was collected and then semi-quantity RT-PCR was performed to examine the pluripotency-related genes expression, such as TERF、THY1、LEFTYA、Rex-1、Sox-2、Oct-4 and nanog. Immunocytofluorescental analysis was taken to test the expression of stem cell related markers, such as SSEA-K SSEA-3、SSEA-4 and TRA-1-60、TRA-1-81. The AAV transfection efficiency to hAECs was analyzed with AAV-GFP in vitro.Results: RT-PCR showed that TERF、THY1、LEFTYA、Rex-1、Sox-2、Oct-4 and nanog were expressed in P1 hAECs. Immunocytofluorescent analysis showed that most of hAECs were SSEA-3⁺,SSEA-4⁺,TRA-1-60⁺ and TRA-1-81⁺. The transfection efficiency of hAECs estimated by EGFP staining was about 58.2%±1.3 % with AAV concentration of 10^-3 pfu/ml. Conclusions: We isolated hAECs from amnions successfully and stablished a culture system for hAECs in vitro. hAECs was a kind of stem cells. hAECs cultured in vitro can express GFP and its expression efficiency is more than 50 percent. Objective: To establish a optimizing culture system of the inducing process and study the contribution of the three factors in the system: Dex, HGF, IGF. To discuss detecting index of the hepatic transdiffernetion of HAECs.Methods: The induction cycle lasted for two weeks, in the end of induction functional genes and transcriptional factors were selected to detect the hepatic fate commitment. For example, the expression of albumin, CYP1A1, CYP1A2, IGFR, c-met, and HNF3, HNF4 and C/EBP were examined by the RT-PCR. Then flow cytometric analysis were performed to test the cell surface markers such as ALB, AFP and CK18 to explore the hepatocyte-like cell maturation; In order to optimize the induction system, detected the dose-dependent of the Dex, HGF, IGF cytokines; Albumin gene expression of different generation’s amniotic epithelial cells were examed in the processes of the amniotic epithelial cells differentiated into liver-like cell.Results: Two weeks after the induction, the expression of albumin, CYP1A1, CYP1A2, IGFR, c-met and other liver cell-related key functional gene’s can be detected in the human amniotic epithelial cells. Further study found that these gene’s expression level showed the trend of gradual increasement which suggested that hAECs may have successfully be induced into hepatocyte-like cells. After inducement, HAECs expressed HNF3, HNF4 and C/ EBPa which were not detected before the induction; and the expression level of HNF1 is significantly higher than that not been induced. The detection of cell surface markers found that: 6 days after be induced, hAECs mainly were stained AFP+, the positive rate was about 15.1±2.1% ; with the time extension, 10 days after be induced, part of the hAECs showed AFP+/ALB+, which was about 6.5±1.4%; and on 14 days, hAECs basically only showed ALB+, the rate was about 13.9±2.3%; the positive rate of the ALB+ cells increasing indicated a grandual founctional maturation from the hAECs to hepatocyte-like cells. Similaritly, the CK18+ cells in the whole population were also increased in the number, for example on the 10 day, the rate was 16.1±1.2%; on the 14^th day, 21.3±4.6%, which proved the above hypothesis of the trandifferentiation. Experimental results of the dose-dependent study show that: IGF, HGF were dose dependent,while Dex’s dose-dependent experimental results was not statistically significant, suggested that dose-dependent manner does not exist. Three passages of HAECs were compared in the gene expression of albumin during the induction. It showed that all the third generation cells which were induced, could all expressed the albumin gene and the expression had no obvious distinction.Conclusion: hAECs could indeed been induced to differentiate into hepatocyte-like cells in vitro, and could be successfully induced to form special functional hepatocyte-like cells; In the induction process, had a dose-dependent on HGF and IGF, whereas this dose-dependent is not obvious on Dex; In the passage of three generations, the expression of albumin,which was the hepatocyte -like-cells-specific gene, had not significantly difference. Objective To observe whether human Amniotic Epithelial Cells （hAECs） have the potential to possess hepatocyte-like function after intrasplenic transplantation in nude mice with liver injury as well as the therapeutic feasibility to repair damaged liver.Methods 40 nude mice were randomly divided into three groups: Group A （ n=20） : 5×10⁶hAECs labeled with green fluorescent protein were transplanted into the spleen of nude mice undergone 2/3 hepatectomized; Group B: treated with same cells as in A,but without hepatectomized; Group C: 0.2 saline was injected into the spleen of nude mice undergone 2/3 hepatectomized. The hAECs were detected with immunofluorescent microscope and their albumin expression were studied. Two and four weeks after the cell transplantation, Liver function and quantitative human albumin were measured respectively in all groups.Results The hAECs labeled green fluorescent protein were observed in liver and spleen tissue in group A and mostly expressed albumin.While in group B,C ,there is no such findings. The human albumin was detectable in serum of group A .The level of serum human albumin in group A in 4 weeks after transplantation was higher than that in 2 weeks after transplantation （3.9±1.34vs0.37±0.14, P<0.01）. Four weeks after transplantation, the liver functions in Group A were restored significantly compared with those of Group C,The liver functions in Group A in 4 weeks after transplantation were improved significantly compared with those in 2 weeks after transplantation while the level of ALT was also different between two groups（121.2±20.8 vs. 191.8±28.9, P＜0.05）.Conclusion The stimulus of liver damage might enhances the migration of hAECs to the liver ,in which a majority of the hAECs have the potential to possess hepatocyte-like function in nude mice . Transplantation of hAECs might amend the damaged liver function to a certain extent.更多还原