【摘要】： 研究背景和目的： 細(xì)胞極化就是細(xì)胞依據(jù)其生物學(xué)功能的需要，而產(chǎn)生的方向性和不對(duì)稱性，，極化細(xì)胞中細(xì)胞膜、細(xì)胞質(zhì)內(nèi)的細(xì)胞器及細(xì)胞骨架不對(duì)稱地分布，分別面對(duì)不同的細(xì)胞外環(huán)境，并發(fā)揮不同的生物學(xué)功能。從低等生物如細(xì)菌到哺乳動(dòng)物乃至人類，細(xì)胞極化對(duì)組織的發(fā)生，結(jié)構(gòu)的形成及功能的發(fā)揮具有重要的作用。 肝細(xì)胞是體內(nèi)高度極化的細(xì)胞。在成體肝臟中，肝板由極化的單細(xì)胞連接而成，將膽汁和血液截然分開。正常的細(xì)胞膜極化對(duì)于肝細(xì)胞發(fā)揮各種生理功能至關(guān)重要。但是，目前我們對(duì)肝細(xì)胞極化的發(fā)生和維持機(jī)制尚不清楚，對(duì)各功能膜區(qū)的結(jié)構(gòu)和細(xì)胞質(zhì)內(nèi)轉(zhuǎn)運(yùn)途徑等也只有初步認(rèn)識(shí)。肝細(xì)胞極化紊亂與多種臨床疾病密切相關(guān)，對(duì)肝細(xì)胞極化機(jī)制的深入研究不但是肝細(xì)胞生物學(xué)發(fā)展的需要，也是臨床治療各種肝病的理論基礎(chǔ)。 骨髓干細(xì)胞(bone marrow stem cells，BMSCs)可以分化為肝前體細(xì)胞和肝細(xì)胞，并具有取材容易，增殖能力強(qiáng)，體外培養(yǎng)、傳代及擴(kuò)增容易的特點(diǎn)。本實(shí)驗(yàn)應(yīng)用成人骨髓基質(zhì)細(xì)胞體外誘導(dǎo)分化來源的肝細(xì)胞為模型，深入研究肝細(xì)胞極化產(chǎn)生的分子基礎(chǔ)和形態(tài)、功能特征，探討肝細(xì)胞極化形成所需的細(xì)胞外基質(zhì)條件及主要影響因素，為臨床治療由于肝細(xì)胞極化紊亂引發(fā)的各種肝病提供理論基礎(chǔ)。 實(shí)驗(yàn)方法： 1、提取成人骨髓基質(zhì)細(xì)胞誘導(dǎo)其向肝細(xì)胞分化。通過形態(tài)學(xué)觀察，ALB免疫熒光定位和RT-PCR驗(yàn)證肝細(xì)胞特征性分子HNF-4 alpha、ALB的mRNA的表達(dá)來證實(shí)成人骨髓基質(zhì)干細(xì)胞在體外被誘導(dǎo)過程中，肝細(xì)胞特異性標(biāo)志物產(chǎn)生。并且復(fù)蘇冷凍保存的已分化肝細(xì)胞，觀察復(fù)蘇后細(xì)胞活率。 2、通過對(duì)成人骨髓基質(zhì)干細(xì)胞來源肝細(xì)胞各個(gè)不同極化膜區(qū)標(biāo)志性蛋白mRNA的RT-PCR研究和極化標(biāo)志性蛋白在人骨髓基質(zhì)干細(xì)胞分化來源的肝細(xì)胞中的免疫熒光染色，研究肝細(xì)胞極化發(fā)生的物質(zhì)基礎(chǔ)和骨髓基質(zhì)干細(xì)胞來源肝細(xì)胞的分化程度。 3、通過小球樣密集培養(yǎng)和從EHS中提取的細(xì)胞外基質(zhì)的應(yīng)用，用細(xì)胞免疫熒光染色方法觀察SR-BI、MRP、DPPIV、LDLr等極化標(biāo)志性蛋白在分化后的人骨髓基質(zhì)干細(xì)胞來源的肝細(xì)胞不同膜區(qū)中的分布，用DiI AcLDL的攝取轉(zhuǎn)運(yùn)觀察和actin的關(guān)系。并在UDCA誘導(dǎo)下，用FD觀察細(xì)胞間膽管樣結(jié)構(gòu)形成特征。 研究結(jié)果： 1、成人骨髓基質(zhì)干細(xì)胞經(jīng)誘導(dǎo)分化后細(xì)胞具有成熟肝細(xì)胞的形態(tài)。應(yīng)用ALB的特異性抗體進(jìn)行的免疫熒光染色顯示，骨髓基質(zhì)干細(xì)胞在分化14d后，細(xì)胞質(zhì)中充滿了大量點(diǎn)狀，或顆粒狀的綠色的熒光染色。復(fù)蘇后的肝細(xì)胞的最高存活率為92.4％±3.1％(n=16)，細(xì)胞形態(tài)完整。在成人骨髓基質(zhì)干細(xì)胞向肝細(xì)胞分化的10d、14d、21d三個(gè)時(shí)間點(diǎn)，ALB和HNF-4 alpha mRNA表達(dá)逐漸增強(qiáng)，ALB mRNA表達(dá)明顯高于非誘導(dǎo)培養(yǎng)(P＜0.05)。 2、肝細(xì)胞極化蛋白DPPIV，MRP，LDLr和SR-BI的mRNA在肝細(xì)胞分化過程中的表達(dá)有增加的趨勢(shì)，部分極化分子顯著性增加。在體內(nèi)這些極化分子在組織內(nèi)的分布是有嚴(yán)格的極性的。DPPIV和LDLr的雙重免疫熒光染色可見二者在肝細(xì)胞膜上的分布是均勻的和彌散的，但二者幾乎沒有重合的定位。SR-BI定位在肝細(xì)胞表面的局部凹陷處，在肝細(xì)胞膜表面呈局部斑片狀或呈簇狀分布。而MRP呈散在均勻分布，和SR-BI的定位區(qū)域明顯的不同。 3、在密集培養(yǎng)中，actin染色顯示細(xì)胞骨架和細(xì)胞連接。在EHS提取的ECM中培養(yǎng)時(shí)，DPPIV、LDLr、SR-BI和MRP呈不對(duì)稱分布，顯示極化細(xì)胞形成。DiI AcLDL熒光顆粒沿actin骨架有方向性地分布。在UCDA作用下，可見到細(xì)胞間類似膽管的空腔內(nèi)有FD濃集。 研究結(jié)論： 1、通過本部分實(shí)驗(yàn)研究，我們發(fā)現(xiàn)，體外成人骨髓基質(zhì)干細(xì)胞在特定的誘導(dǎo)分化環(huán)境中可分化成具有一定功能的肝細(xì)胞。經(jīng)凍存復(fù)蘇后，分化成熟肝細(xì)胞存活率較高，且形態(tài)功能穩(wěn)定，可作為體外實(shí)驗(yàn)研究、肝細(xì)胞移植及生物型人工肝較好的細(xì)胞來源。 2、本部分研究進(jìn)一步驗(yàn)證了骨髓基質(zhì)干細(xì)胞來源的肝細(xì)胞分化得較為成熟，并且已經(jīng)在基因水平和蛋白質(zhì)水平初步具備了極化發(fā)生和形成的物質(zhì)基礎(chǔ)。不同膜區(qū)的極化標(biāo)志性蛋白在肝細(xì)胞分化特定環(huán)境下呈彌散的分布，或分布于特定區(qū)域，顯示了細(xì)胞極化形成潛能。 3、通過本部分實(shí)驗(yàn)研究，我們發(fā)現(xiàn)成人骨髓基質(zhì)干細(xì)胞來源的肝細(xì)胞在體外經(jīng)特定的細(xì)胞外基質(zhì)培養(yǎng)條件下，肝細(xì)胞極化初步形成，并且形成膽管樣結(jié)構(gòu)，開始有物質(zhì)的攝取和轉(zhuǎn)運(yùn)，從而揭示了體外誘導(dǎo)分化的肝細(xì)胞在功能上進(jìn)一步完善，在結(jié)構(gòu)上趨于完整。可以用于研究肝細(xì)胞在分化過程中自然生理狀態(tài)下極化的形成，物質(zhì)和某些藥物的轉(zhuǎn)運(yùn)和涉及細(xì)胞極化紊亂的一些疾病的發(fā)病機(jī)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.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

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相關(guān)期刊論文 前4條

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