【作者】 刘芳遐；

【导师】 胡以平； 何志颖； 苏娟； 訾晓渊；

【作者基本信息】 第二军医大学 ， 基础医学， 2010， 硕士

【摘要】 肝移植是目前终末期肝脏疾病唯一有效的治疗手段,但供肝来源的缺乏使其临床应用受到很大的限制。近二十年来,作为原位肝移植的替代方案,临床上一直致力于开展肝细胞移植技术的研究,并取得了一定的疗效。然而,可用于分离肝细胞的高质量供体肝脏在数量上无法得到保证,严重制约着肝细胞移植的临床应用。干细胞生物学的研究已经表明,干细胞具有呈指数分裂的增殖能力,也具有重新形成干细胞巢,并分化产生特定功能细胞,以重新建立组织的基本生物学特性。特别令人鼓舞的是,在体外实验中已有可能使胚胎干细胞和来源于胎肝组织的肝干细胞分化为肝前体细胞、成熟的肝细胞及胆管细胞,并且在活体动物的移植实验中也观察到了外源的干细胞对损伤肝脏的再殖(repopulation),能够使损伤肝脏在结构上和功能上得到有效的恢复。这些进展的取得,充分显示了利用干细胞对人类肝脏疾病进行临床治疗的可能性。本实验室以4.5-6周的流产人胚胎作为分离肝干细胞的材料,通过克隆化培养建立了人胎肝来源的干细胞系(hFLSC)。hFLSC具有以下的特性：①具有强大的增殖能力；②表达多种类型的分子标记：不仅表达肝干细胞相关的分子标志(如AFP、ALB、CK8、CK18、CK19、GGT、DPPⅣ、c-Met、Thy-1和c-kit等),还表达间质细胞的部分分子标志(如CD44、CD29、SDF-1等),但不表达造血干细胞标志(如CD44、CD29、SDF-1等)；③具有多潜能性：在体外诱导实验中,能分化为肝细胞、胆管细胞、脂肪细胞、成骨细胞；④可以参与小鼠受损肝脏的再生：在四氯化碳急性损伤的重度联合免疫缺陷小鼠体内,检测到标记有绿色荧光蛋白基因的hFLSC细胞。然而,由于四氯化碳诱导的急性肝损伤模型本身的缺陷,不能对hFLSC移植后在小鼠体内的植入、归巢、再殖及其功能进行充分的评价。延胡索酰乙酰乙酸水解酶(fuarylacetoacetate hydrolase, FAH)基因剔除小鼠模型是评价外源性干细胞再殖损伤肝脏的理想动物模型,FAH基因剔除小鼠在细胞移植、再殖肝脏研究方面得到了广泛的应用。该小鼠存在广泛而持续的肝损伤,所造成的肝脏微环境特别适合于移植细胞的增殖。本课题在已建立的hFLSC细胞系基础上,选择FAH基因剔除小鼠模型作为评价hFLSC再殖损伤肝脏的基本动物模型,进一步探索hFLSC在小鼠体内的生物学特性以及其作为肝细胞移植细胞来源的可能性,为人胎肝来源的肝原始细胞的基础研究及临床应用建立基础。本课题分为两个部分：在第一部分实验中,通过对Fah-／-小鼠进行同品系野生型小鼠肝细胞的移植,观察Fah-／-小鼠肝再生环境促进移植肝细胞增殖的作用,为下一步进行hFLSC移植奠定基础。同时,虽然已知Fah-/-小鼠肝细胞自身的损伤是导致外源细胞植入和增殖的主要因素,然而对其病理学变化尤其采用电镜的方法分析肝细胞损伤的特点目前尚未见报道。本研究取Fah-／-小鼠停药后不同时间点的肝脏进行了HE染色和电镜检测,对Fah-/-小鼠肝脏损伤后的病理学变化进行了初步观察。在第二部分实验中,为了获得免疫缺陷的FAH基因剔除小鼠品系用于hFLSC移植,我们应用Fah-／-小鼠与Rag2-/-免疫缺陷小鼠进行杂交,建立了Fah-／-Rag2-／-小鼠品系。流式细胞仪检测表明Fah-/-Rag2-/-小鼠T细胞和B细胞功能缺失,可用于hFLSC的移植。将体外培养hFLSC细胞经脾脏注射移植Fah-／-Rag2-／-小鼠,取不同时间点的小鼠肝脏,通过免疫组织化学的方法检测hFLSC的植入、归巢及再殖效率,评价hFLSC肝向分化的能力。第一部分的研究表明,撤除NTBC的Fah-／-小鼠表现为毛刺、弓背、体重明显下降,撤药6-7周后小鼠死亡；而正常喂养NTBC的Fah-/-小鼠全部健康存活,体重稳定增长,并且具有正常的繁殖能力。我们成功的实现了野生型小鼠肝细胞在撤药的Fah-／-小鼠肝脏中的再殖。FAH免疫组织化学染色表明,肝细胞移植1周后即可在Fah-/-小鼠肝脏内检测到表达FAH蛋白的野生型肝细胞。随着时间的延长,移植肝细胞的再殖程度逐渐增加,3周时肝脏再殖22±6%,4周时肝脏再殖38±11%,6周时肝脏再殖67±8%,8周时肝脏再殖78±17%。8周时移植的肝细胞几乎完全重建了Fah-／-小鼠的肝脏,再殖的肝细胞进入Fah-/-小鼠肝脏结构,但并不破坏肝脏的小叶结构,嵌合体肝脏的肝板结构和肝小叶结构仍然正常,且不形成病理性的结节增生。HE染色和电镜检测进一步证明Fah-／-小鼠自体肝细胞发生进行性、不可逆转的损伤是促进移植肝细胞增殖的内在机制。第二部分的研究表明,首先,我们成功建立了Fah-／-Rag2-/-小鼠品系：以Fah-/-小鼠为参照,以CD3、CD19为标志物,流式细胞仪检测Fah-／-Rag2-/-小鼠骨髓细胞中T细胞(CD3+)、B细胞(CD19+)的数量变化,结果表明Fah-／-Rag2-／-小鼠T细胞和B细胞功能缺失。其次,hFLSC移植Fah-／-Rag2-／-小鼠后,在1、3、5、6、8周,应用Fah免疫组织化学染色都能检测到hFLSC存在,因为FAH酶的表达是检测再殖细胞(Fah+/+)的一个理想标志。我们进一步通过肝细胞功能蛋白——白蛋白免疫组织化学染色的方法,初步评价了hFLSC在Fah-/-Rag2-/-小鼠肝脏中的肝向分化能力。结果表明部分小鼠肝脏标本白蛋白染色为阳性。初步的实验结果提示：移植的hFLSC能够植入到Fah-／-Rag2-/-小鼠受损肝脏中,但其肝脏再殖的效率较低,小鼠的肝脏功能不能恢复正常。本研究的实验结果表明：Fah-／-小鼠是研究肝细胞移植的理想模型,Fah-／-小鼠自体肝细胞发生进行性、不可逆转的损伤是促进移植肝细胞增殖的内在机制；来源于4.5-6周的流产人胚胎的肝原始细胞可能是肝细胞移植的候选细胞之一,但其肝脏再殖效率可能与其成熟程度有密切的关系,临床应用前进行肝向诱导分化可能是必要的程序。更多还原

【Abstract】 Liver transplantation is currently the only effective treatment for end-stage liver disease. Clinical application of liver transplantation is limited by lack of donor livers. In the past two decades, as an alternative to liver transplantation, clinical hepatocyte transplantation has draw much attention and has achieved a certain effect. However, Resources for the isolation of cells for transplantation are limited and restrict the widespread application of liver cell therapies. The biological studies on stem cell have shown that stem cells divide exponentially, form a new nest, and differentiate into cells owning specific function and rebuild the organization. It is particularly encouraging that embryonic stem cells and hepatic stem cells derived from fetal liver may differentiate into liver precursor cells, mature hepatocytes and bile duct cells in vitro, and in vivo transplantation experiments also observed injury liver repopulation by exogenous stem cells, so that the liver damage on the structure and function is effectively recovered. The success achieved, which fully demonstrates the use of stem cells to human liver disease and the possibility of clinical treatment.The laboratory used human fetal liver of 4.5-6 weeks as the material to isolate stem cells by the method of cloning, then established a cell line named human fetal liver-derived stem cell lines (hFLSC). hFLSC has the following characteristics:①powerful proliferation;②expression of multiple types of molecular markers:not only the liver stem cell-related markers (such as AFP, ALB, CK8, CK18, CK19, GGT, DPPIV, c-Met, Thy-1 and c-kit, etc.), but also the interstitial cells of some molecular markers (such as CD44, CD29, SDF-1, etc.), but it did not express stem cell markers (such as CD44, CD29, SDF-1, etc.);③with multiple potential resistance:it can differentiate into hepatocyte, bile duct cells, fat cells, osteoblasts in vitro;④it can participate in the regeneration of liver damage in mice:using the mice model of acute liver damage induced by carbon tetrachloride(CCL4) with severe combined immunodeficiency, we detected hFLSC cells with green fluorescent protein marker gene. However, due to the defects of acute liver injury model induced by carbon tetrachloride, we are not able to fully evaluate the ability of implantation, homing, repopulation of hFLSC as well as its function. The model of Fumaric acid acetoacetate hydrolase (fuarylacetoacetate hydrolase, FAH) knockout mouse is an ideal animal model of liver injury to evaluate the repopulation of exogenous stem cells, FAH knockout mice has been widely used in the cell transplantation researches, the mice existent extensive and continuous mouse liver injury that caused liver micro-environment which is particularly suitable for the proliferation of transplanted cells. Our researches establishes on the basis of hFLSC, and choose FAH knockout mouse model as the basic animal models to evaluate the repopulation of hFLSC in liver injury, and further explore the biological characteristics of in mice and the possibilities of hFLSC as a cell source for liver cell transplantation, and establish a basis for the basic research and clinical application of hepatic progenitor cells derived from fetal liver.The research is divided into two parts:in the first part of the experiment, by transplantation of hepatocyte isolated from wild-type strains mice into Fah-/-mice, we observe the role of liver regeneration environment of Fah-/- mice in stimulating the proliferation of transplanted hepatocytes, and preparing for the next study that is transplantation of hFLSC. Furthermore, though we knew that the injury of liver cell of Fah-/- mouse is the major factor which cause the proliferation and repopulation of exogenous cells, however, its pathological changes, particularly analyzing the characteristics of liver cell injury by electron microscopy has not been reported. In our research, we obtained the liver of Fah-/- mouse at different time after the withdrawal of drug, then HE staining and electron microscopy were performed on liver section, in order to observe preliminarily the pathological changes of Fah-/- mouse liver after the liver injury. In the second part of the experiment, in order to obtain immunodeficiency FAH knockout mouse strains which is prepared for the latter hFLSC transplantation, the Fah-/- mice was hybridized with Rag2-/- immunodeficient mice, Fah-/- Rag2-/- mouse strain was established, the result of flow cytometry showed that Fah-/- Rag2-/- mouse lose the function of T cells and B cell, it can be used in hFLSC transplantation. HFLSC cells were cultured in vitro and transplanted into Fah-/-Rag2-/-mice through spleen, mouse livers were obtained at different time points of, the implantation, homing and the efficiency of repopulation of hFLSC were detected by the method of immunohistochemistry, the capability of hepatic differentiation is evaluated.The first part of the study show that after the withdrawl of NTBC, Fah-/- mice showed spikes, bowing, body weight decreased significantly mice died after 6-7 weeks after drug withdrawal; and Fah-/- mice with normal feeding NTBC all survive healthily, their weight growth stably, and have normal reproductive capacity. We successfully achieved the liver repopulation of wild-type mouse’s hepatocyte in Fah-/-mouse after the withdrawal of the drug. The result of immnuohistochemistry of Fah protein showed that wild-type hepatocyte with the expression of Fah protein was detected in the Fah-/- mice liver after 1 week of liver cell transplantation. As time passed by, the degree of liver repopulation of transplanted hepatocyte gradually increased, the yield level is 22±6%in 3rd week, In the 4th weeks, the liver repopulate about 38±11%, in the 6th week, the liver is repopulated 67±8%, in the 8th week, the liver repopulate about 78±17%. In the 8th week, the Fah-/- mice liver is almost rebuilt completely by transplanted hepatocytes. The transplanted hepatocytes engraft into the Fah-/- mouse liver structure, but does not damage the liver lobule structure, chimeric liver’s board structure and the hepatic lobules was normal, without the formation of pathological nodular hyperplasia. HE staining and electron microscopy further demonstrated that the progressive, irreversible damage of Fah-/- mouse hepatocyte is the internal mechanism of promoting the proliferation of transplanted hepatocytes.In the second part of the study, first of all, we have successfully established a Fah-/- Rag2-/- mouse strains:using CD3, CD 19 as markers, flow cytometry detects the changes in number of T cells (CD3+), B cells (CD 19+) in the bone marrow cells of Fah-/-Rag2-/-, compared with Fah-/- mice. The results show that the function of T cells and B cells are lost in Fah-/-Rag2-/-mouse. Secondly, hFLSC was transplanted into Fah-/-Rag2-/-mice, in 1,3,5,6,8 weeks, through the immnuohistochemistry of Fah enzyme, hFLSC can be detected, because the expression of Fah enzyme is a good sign for the detection of transplanted cells (Fah-/-). We further apply the immnuohistochemistry of liver cell functional protein-albumin, we preliminarily evaluate the ability of hepatocyte differentiation of hFLSC in the liver of Fah-/-Rag2-/- mice. The preliminary resulst shows that hFLSC could engrafte into the liver of Fa h-/-Rag2-/- mice, but its repopulation of the liver is less efficient, mice cannot restore the normal liver function.The results of this study show that:Fah-/- mice is an ideal model for the study of liver cell transplantation. The progressive, irreversible damage of Fah-/- mouse hepatocyte is the internal mechanism of promoting the proliferation of transplanted hepatocytes. Hepatocyte progenitor cells from human fetal liver (4.5-6 weeks) may be one of the candidate cells, but its efficiency may be related to the cell’s mature degree. Hepatocyte differentiation induced in vitro may be necessary before its clinical application.更多还原