成人骨髓基质干细胞向肝细胞分化过程中细胞极化特征研究
发布时间：2019-05-11 13:46
【摘要】： 研究背景和目的： 细胞极化就是细胞依据其生物学功能的需要，而产生的方向性和不对称性，，极化细胞中细胞膜、细胞质内的细胞器及细胞骨架不对称地分布，分别面对不同的细胞外环境，并发挥不同的生物学功能。从低等生物如细菌到哺乳动物乃至人类，细胞极化对组织的发生，结构的形成及功能的发挥具有重要的作用。 肝细胞是体内高度极化的细胞。在成体肝脏中，肝板由极化的单细胞连接而成，将胆汁和血液截然分开。正常的细胞膜极化对于肝细胞发挥各种生理功能至关重要。但是，目前我们对肝细胞极化的发生和维持机制尚不清楚，对各功能膜区的结构和细胞质内转运途径等也只有初步认识。肝细胞极化紊乱与多种临床疾病密切相关，对肝细胞极化机制的深入研究不但是肝细胞生物学发展的需要，也是临床治疗各种肝病的理论基础。 骨髓干细胞(bone marrow stem cells，BMSCs)可以分化为肝前体细胞和肝细胞，并具有取材容易，增殖能力强，体外培养、传代及扩增容易的特点。本实验应用成人骨髓基质细胞体外诱导分化来源的肝细胞为模型，深入研究肝细胞极化产生的分子基础和形态、功能特征，探讨肝细胞极化形成所需的细胞外基质条件及主要影响因素，为临床治疗由于肝细胞极化紊乱引发的各种肝病提供理论基础。 实验方法： 1、提取成人骨髓基质细胞诱导其向肝细胞分化。通过形态学观察，ALB免疫荧光定位和RT-PCR验证肝细胞特征性分子HNF-4 alpha、ALB的mRNA的表达来证实成人骨髓基质干细胞在体外被诱导过程中，肝细胞特异性标志物产生。并且复苏冷冻保存的已分化肝细胞，观察复苏后细胞活率。 2、通过对成人骨髓基质干细胞来源肝细胞各个不同极化膜区标志性蛋白mRNA的RT-PCR研究和极化标志性蛋白在人骨髓基质干细胞分化来源的肝细胞中的免疫荧光染色，研究肝细胞极化发生的物质基础和骨髓基质干细胞来源肝细胞的分化程度。 3、通过小球样密集培养和从EHS中提取的细胞外基质的应用，用细胞免疫荧光染色方法观察SR-BI、MRP、DPPIV、LDLr等极化标志性蛋白在分化后的人骨髓基质干细胞来源的肝细胞不同膜区中的分布，用DiI AcLDL的摄取转运观察和actin的关系。并在UDCA诱导下，用FD观察细胞间胆管样结构形成特征。 研究结果： 1、成人骨髓基质干细胞经诱导分化后细胞具有成熟肝细胞的形态。应用ALB的特异性抗体进行的免疫荧光染色显示，骨髓基质干细胞在分化14d后，细胞质中充满了大量点状，或颗粒状的绿色的荧光染色。复苏后的肝细胞的最高存活率为92.4％±3.1％(n=16)，细胞形态完整。在成人骨髓基质干细胞向肝细胞分化的10d、14d、21d三个时间点，ALB和HNF-4 alpha mRNA表达逐渐增强，ALB mRNA表达明显高于非诱导培养(P＜0.05)。 2、肝细胞极化蛋白DPPIV，MRP，LDLr和SR-BI的mRNA在肝细胞分化过程中的表达有增加的趋势，部分极化分子显著性增加。在体内这些极化分子在组织内的分布是有严格的极性的。DPPIV和LDLr的双重免疫荧光染色可见二者在肝细胞膜上的分布是均匀的和弥散的，但二者几乎没有重合的定位。SR-BI定位在肝细胞表面的局部凹陷处，在肝细胞膜表面呈局部斑片状或呈簇状分布。而MRP呈散在均匀分布，和SR-BI的定位区域明显的不同。 3、在密集培养中，actin染色显示细胞骨架和细胞连接。在EHS提取的ECM中培养时，DPPIV、LDLr、SR-BI和MRP呈不对称分布，显示极化细胞形成。DiI AcLDL荧光颗粒沿actin骨架有方向性地分布。在UCDA作用下，可见到细胞间类似胆管的空腔内有FD浓集。 研究结论： 1、通过本部分实验研究，我们发现，体外成人骨髓基质干细胞在特定的诱导分化环境中可分化成具有一定功能的肝细胞。经冻存复苏后，分化成熟肝细胞存活率较高，且形态功能稳定，可作为体外实验研究、肝细胞移植及生物型人工肝较好的细胞来源。 2、本部分研究进一步验证了骨髓基质干细胞来源的肝细胞分化得较为成熟，并且已经在基因水平和蛋白质水平初步具备了极化发生和形成的物质基础。不同膜区的极化标志性蛋白在肝细胞分化特定环境下呈弥散的分布，或分布于特定区域，显示了细胞极化形成潜能。 3、通过本部分实验研究，我们发现成人骨髓基质干细胞来源的肝细胞在体外经特定的细胞外基质培养条件下，肝细胞极化初步形成，并且形成胆管样结构，开始有物质的摄取和转运，从而揭示了体外诱导分化的肝细胞在功能上进一步完善，在结构上趋于完整。可以用于研究肝细胞在分化过程中自然生理状态下极化的形成，物质和某些药物的转运和涉及细胞极化紊乱的一些疾病的发病机制。
[Abstract]:Background and purpose of the study: The cell polarization is the need of the cell according to its biological function, and the cell membrane in the polarized cell, the organelles in the cytoplasm and the cytoskeleton are not symmetrically distributed, facing different cells, The environment and the different biology Function. From low-grade organisms such as bacteria to mammals and even human, cell polarization plays an important role in the occurrence of tissue, the formation and function of the structure Effect. Hepatocytes are in-vivo height A polarized cell. In adult liver, the liver is formed by a polarized single cell, which connects the bile and the blood. The normal cell membrane polarization can exert various physiological functions on the liver cells. It is of great importance. However, at present, the mechanism of the occurrence and maintenance of the polarization of the liver cells is not clear, and the structure of each functional membrane region and the intra-cytoplasmic transport route and the like are also There is a preliminary understanding that the polarization disorder of the hepatocytes is closely related to a variety of clinical diseases, the in-depth study of the mechanism of the polarization of the hepatocytes is not only the need of the biological development of the liver cells, but also a clinical treatment of various liver diseases The bone marrow stem cells (BMSCs) can be differentiated into the liver precursor cells and the liver cells, and have the advantages of easy material selection, strong proliferation ability, in vitro culture and passage. And the characteristics of the molecular basis and the morphology and the function of the polarization of the hepatocytes are further studied, and the extracellular matrix strip required for the polarization of the hepatocytes is discussed. The key factors and the main influencing factors, for clinical treatment of various diseases caused by the polarization disorder of the liver liver disease On the basis of experiment:1. Extraction of adult bone The expression of human bone marrow stromal stem cells in vitro was confirmed by morphological observation, ALB immunofluorescence localization and RT-PCR to verify the expression of the characteristic molecular HNF-4alpha, ALB in the liver cells. In the course, the hepatocyte-specific marker is generated and the recovered frozen-stored fraction After the recovery of the liver cells, the post-resuscitation cells were observed.2. The expression of the marker protein mRNA in the various polarized membrane regions of the source of the human bone marrow stromal stem cells was studied by RT-PCR and the polarized signature protein was dry and thin in human bone marrow Immunofluorescence staining in the liver cells of the source of cellular differentiation and the material basis for the study of the occurrence of the polarization of the hepatocytes Differentiation of bone marrow stromal stem cell-derived hepatocytes.3. The application of cell-like intensive culture and the extraction of extracellular matrix from EHS. The polarized symbolic proteins such as SR-BI, MRP, DPPIV and LDLr were observed in the differentiated human bone by the method of cell immunofluorescence staining. Distribution of the source of the marrow stromal stem cells in different membrane areas, with DiI A The relationship between the uptake and transport of cLDL and actin. Under the guide, use F D. The structure of the bile duct-like structure between cells was observed. The results were as follows:1. Human bone marrow stromal stem cells have the form of mature hepatocytes after induction of differentiation. Immunofluorescence staining using the specific antibodies of ALB shows that the bone marrow stromal stem cells are differentiated for 14 days. The cytoplasm is filled with a large number of dot-like, or granular, green fluorescent staining. The highest storage of the post-resuscitation hepatocytes The expression of ALB and HNF-4alpha in adult bone marrow stromal stem cells was gradually enhanced at three time points of 10 days,14 days and 21 days of the differentiation of human bone marrow stromal stem cells to hepatocytes. The expression of ALB mRNA was significantly higher than that of non-induction culture (P <0.05). In the process of the differentiation of the hepatocytes, there is a tendency to increase the expression of the partially polarized molecules. In vivo, the distribution of these polarized molecules in the tissues is strictly polar. The double immunofluorescent staining of DPPIV and LDLr can be seen in both. The distribution of the liver cell membrane is uniform and diffuse, but there is almost no coincidence of the two. The SR-BI is located in the liver The local depression of the surface of the cell, in the form of a local patch or a cluster in the surface of the cell membrane of the liver. and the positioning area of the SR-BI obviously does not In contrast to.3, in intensive culture, actin staining shows the cytoskeleton and cell connection. When cultured in the EHS-extracted ECM, DPP The asymmetric distribution of IV, LDLr, SR-BI and MRP shows polarization. Cell formation. DiI AcLDL fluorescent particles have directionality along the actin matrix To be distributed. Under the effect of UCDA, there was an FD-rich set in the cavity of a similar bile duct between the cells. In vitro adult bone marrow stromal cells can differentiate into hepatocytes with certain function in a specific induced differentiation environment. And can be used as a cell source for in-vitro experimental study, hepatocyte transplantation and biological artificial liver. The cellular differentiation is mature and has a material base for the occurrence and formation of the polarization at the level of the gene and the protein level. The distribution of the sex protein in the specific environment of the differentiation of the hepatocytes, or the distribution in a specific region, shows the potential of the polarization of the cells.3. In this part of the experiment, we find that the liver cells of the source of the adult bone marrow stromal stem cells are transfused in vitro. Under the conditions of extracellular matrix culture, the polarization of the hepatocytes was primarily formed and the bile duct-like structure was formed. It can be used to study the differentiation of the liver cells.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
本文编号：2474536
[Abstract]:Background and purpose of the study: The cell polarization is the need of the cell according to its biological function, and the cell membrane in the polarized cell, the organelles in the cytoplasm and the cytoskeleton are not symmetrically distributed, facing different cells, The environment and the different biology Function. From low-grade organisms such as bacteria to mammals and even human, cell polarization plays an important role in the occurrence of tissue, the formation and function of the structure Effect. Hepatocytes are in-vivo height A polarized cell. In adult liver, the liver is formed by a polarized single cell, which connects the bile and the blood. The normal cell membrane polarization can exert various physiological functions on the liver cells. It is of great importance. However, at present, the mechanism of the occurrence and maintenance of the polarization of the liver cells is not clear, and the structure of each functional membrane region and the intra-cytoplasmic transport route and the like are also There is a preliminary understanding that the polarization disorder of the hepatocytes is closely related to a variety of clinical diseases, the in-depth study of the mechanism of the polarization of the hepatocytes is not only the need of the biological development of the liver cells, but also a clinical treatment of various liver diseases The bone marrow stem cells (BMSCs) can be differentiated into the liver precursor cells and the liver cells, and have the advantages of easy material selection, strong proliferation ability, in vitro culture and passage. And the characteristics of the molecular basis and the morphology and the function of the polarization of the hepatocytes are further studied, and the extracellular matrix strip required for the polarization of the hepatocytes is discussed. The key factors and the main influencing factors, for clinical treatment of various diseases caused by the polarization disorder of the liver liver disease On the basis of experiment:1. Extraction of adult bone The expression of human bone marrow stromal stem cells in vitro was confirmed by morphological observation, ALB immunofluorescence localization and RT-PCR to verify the expression of the characteristic molecular HNF-4alpha, ALB in the liver cells. In the course, the hepatocyte-specific marker is generated and the recovered frozen-stored fraction After the recovery of the liver cells, the post-resuscitation cells were observed.2. The expression of the marker protein mRNA in the various polarized membrane regions of the source of the human bone marrow stromal stem cells was studied by RT-PCR and the polarized signature protein was dry and thin in human bone marrow Immunofluorescence staining in the liver cells of the source of cellular differentiation and the material basis for the study of the occurrence of the polarization of the hepatocytes Differentiation of bone marrow stromal stem cell-derived hepatocytes.3. The application of cell-like intensive culture and the extraction of extracellular matrix from EHS. The polarized symbolic proteins such as SR-BI, MRP, DPPIV and LDLr were observed in the differentiated human bone by the method of cell immunofluorescence staining. Distribution of the source of the marrow stromal stem cells in different membrane areas, with DiI A The relationship between the uptake and transport of cLDL and actin. Under the guide, use F D. The structure of the bile duct-like structure between cells was observed. The results were as follows:1. Human bone marrow stromal stem cells have the form of mature hepatocytes after induction of differentiation. Immunofluorescence staining using the specific antibodies of ALB shows that the bone marrow stromal stem cells are differentiated for 14 days. The cytoplasm is filled with a large number of dot-like, or granular, green fluorescent staining. The highest storage of the post-resuscitation hepatocytes The expression of ALB and HNF-4alpha in adult bone marrow stromal stem cells was gradually enhanced at three time points of 10 days,14 days and 21 days of the differentiation of human bone marrow stromal stem cells to hepatocytes. The expression of ALB mRNA was significantly higher than that of non-induction culture (P <0.05). In the process of the differentiation of the hepatocytes, there is a tendency to increase the expression of the partially polarized molecules. In vivo, the distribution of these polarized molecules in the tissues is strictly polar. The double immunofluorescent staining of DPPIV and LDLr can be seen in both. The distribution of the liver cell membrane is uniform and diffuse, but there is almost no coincidence of the two. The SR-BI is located in the liver The local depression of the surface of the cell, in the form of a local patch or a cluster in the surface of the cell membrane of the liver. and the positioning area of the SR-BI obviously does not In contrast to.3, in intensive culture, actin staining shows the cytoskeleton and cell connection. When cultured in the EHS-extracted ECM, DPP The asymmetric distribution of IV, LDLr, SR-BI and MRP shows polarization. Cell formation. DiI AcLDL fluorescent particles have directionality along the actin matrix To be distributed. Under the effect of UCDA, there was an FD-rich set in the cavity of a similar bile duct between the cells. In vitro adult bone marrow stromal cells can differentiate into hepatocytes with certain function in a specific induced differentiation environment. And can be used as a cell source for in-vitro experimental study, hepatocyte transplantation and biological artificial liver. The cellular differentiation is mature and has a material base for the occurrence and formation of the polarization at the level of the gene and the protein level. The distribution of the sex protein in the specific environment of the differentiation of the hepatocytes, or the distribution in a specific region, shows the potential of the polarization of the cells.3. In this part of the experiment, we find that the liver cells of the source of the adult bone marrow stromal stem cells are transfused in vitro. Under the conditions of extracellular matrix culture, the polarization of the hepatocytes was primarily formed and the bile duct-like structure was formed. It can be used to study the differentiation of the liver cells.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
【参考文献】
相关期刊论文 前4条
1 闫旭;曲强;周立;;肝细胞极化的基础与临床研究现状[J];细胞与分子免疫学杂志;2006年06期
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3 李秉璐,曲强,赵玉沛,何小东,王磊,陈翠珠,刘振元;人骨髓基质干细胞向肝细胞分化过程中白蛋白的表达研究[J];中华外科杂志;2005年11期
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