胎肝干细胞诱导分化为胰岛素分泌细胞的研究

发布时间：2018-01-22 11:19

本文关键词： 胎肝干细胞诱导分化胰岛素　出处：《第一军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 本文研究了胎肝干细胞的特性，并诱导其分化为胰岛素分泌细胞，着重进行了胎肝干细胞的体外扩增培养，探讨胎肝干细胞的生物学特性和性质，以及胚胎发育中胎肝前体细胞的类型；采用阶段性化学诱导、生物诱导以及二者相结合的方法，启动胰腺发育的相关基因，使胎肝干细胞分化为胰岛素分泌细胞。全文共分四个部分。第一部分，胎肝干细胞的分离、培养与鉴定。通过体外分离培养并扩增大鼠胎肝干细胞，研究其形态、生物学特性，采用免疫细胞化学分析胎肝干细胞表面标志物的表达情况，初步研究胎肝干细胞体外扩增培养的实验条件，并探讨肝脏的发生发育及肝干细胞的性质及增殖潜能。本实验选取胎龄12～16d SD大鼠，先用机械分离结合酶消化法分离培养胎肝细胞，继而用特异的干细胞培养基进行克隆筛选法获得了数量较多、较纯化的胎肝干细胞克隆。SABC法检测原代、传代后及细胞克隆中的肝干细胞特异表面标志物OV-6、CK-19及胰腺前体细胞特异标志蛋白Nestin的表达。以上研究表明，胎肝干细胞可通过克隆筛选法进行体外扩增，含有特殊细胞因子的干细胞培养基有助于肝干细胞的扩增，可获得较好的效果。培养3d左右开始出现小细胞团，1个月即形成肉眼可见的细胞集落，5～7d传代一次。原代、传代培养的胎肝细胞部分表达OV-6、CK-19及Nestin；细胞克隆几乎全部为干细胞标志阳性细胞，表明胎肝干细胞具有较强的分裂增殖能力。胎肝内存在Nestin阳性干细胞，我们称之为肝源性Nestin阳性祖细胞(nestin-positive hepatic-derived progenitor，NHP cells)，可能是一种更为原始的干细胞在胚胎发育中起重要作用。第二部分，胎肝组织细胞的鉴定及分类。本部分实验旨在研究胎肝干细胞性质的同时也为肝干细胞进行较系统的分类提供实验依据，探讨肝脏中干／祖细胞的类型并筛选出易于向胰腺方向分化的细胞类型，为下一步的诱导获取较佳的种子细胞。采用免疫荧光双标记法，针对OV-6、CK-19及Nestin三种抗体中二种抗体的不同组合，FITC和Cy3分别标记，DAPI染核，针对胎肝组织和原代、传代及克隆培养的细胞中不同的前体细胞类型进行初步的定位及分类。胎肝中包含了从原始到成熟不同发育程度的各种肝脏细胞：(1)多能前体细胞：OV-6~+nestin~+CK-19~-细胞，可能具有肝胰多向分化潜能；(2)双能前体细胞：OV-6~+CK-19~+nestin~-细胞，是肝脏的双能干细胞，可分化为肝细胞系和胆管细胞系；(3)定向前体细胞或过渡细胞：仅表达OV-6或仅表达CK-19，可能分别是仅有向肝脏方向分化能力的肝前体细胞和向胆管方向分化的细胞，或是正在分化为成熟细胞的过渡阶段；(4)较成熟细胞。体外获得的nestin~+细胞数量较少，大多是与OV-6一同表达，仅表达nestin的细胞数量很少。OV-6~+nestin~+细胞和nestin~+细胞可能具有向胰腺细胞分化的潜能，从胰岛发育的过程推测β细胞与肝细胞有共同的来源，它们共同的祖细胞是胰腺导管上皮内和肝内的nestin阳性细胞。因此我们选取此类肝源性nestin阳性祖细胞作为下一步诱导分化的种子细胞。第三部分，EGFP-PDX-1融合表达载体电穿孔转染大鼠胎肝干细胞的研究。本室构建了PDX-1绿色荧光蛋白融合表达载体，通过电穿孔转染入大鼠胎肝干细胞以得到较稳定的表达，期望借助于外源性生物因子启动胎肝干细胞中PDX-1基因的表达，从生物诱导方面研究胰腺内分泌发育的关键基因PDX-1在肝干细胞定向分化中的调控机制，为今后定向分化的深入研究提供物质平台。由PDX-1插入pEGFP-C1的多克隆位点中构建的重组质粒pEGFP-C1-PDX-1用电穿孔法转染体外培养的大鼠胎肝干细胞，分析转染前后细胞的生长曲线，荧光显微镜下观察GFP发光情况，RT-PCR鉴定PDX-1基因的表达情况。经电穿孔转染后GFP和PDX-1的融合蛋白能在胎肝干细胞中一起表达，并维持较长的时间，，对胎肝干细胞的生长增殖无显著影响(P＞0.05)。构建的PDX-1绿色荧光蛋白融合表达载体能在胎肝干细胞中较持续地表达，为研究PDX-1在干细胞定向分化为胰岛素分泌细胞中的调控作用提供了物质基础。第四部分，胎肝干细胞诱导分化为胰岛素分泌细胞的研究。采用阶段特异性的化学诱导、生物诱导以及二者相结合的方法，模拟胰腺发育的内外环境，使胎肝干细胞分化为胰岛素分泌细胞，并探讨肝干细胞诱导分化为胰岛素分泌细胞的内源性及外源性调控机制。将筛选出的nestin阳性胎肝细胞分组诱导，分别进行分阶段的以Nicotinamide为诱导剂的化学诱导、电穿孔转染PDX-1基因的生物诱导、生物诱导后化学诱导以及未诱导组。ELISA检测细胞上清液中胰岛素的含量，绘制标准曲线后换算出各组细胞释放的胰岛素量并比较；RT-PCR检测与胰腺发育相关的基因和肝细胞特异基因在各组的表达情况。 Nestin~+胎肝细胞经生物、化学及二者结合法诱导后分泌的胰岛素量分别为0.539、0.943和5.58ng／ml上清液(1×10~5细胞)，与阴性对照组相比均有显著性差异(P=0.000)，其中任意两组相比也有统计学意义(P=0.000)。各诱导组均表达胰岛素Ⅰ、GLUT-2及肝干细胞标志HNF-1、AFP和c-met，而不表达胰高血糖素、生长抑素和ALB。生物诱导组和生化诱导组表达PDX-1和胰岛素Ⅱ，化学诱导组弱表达这两个基因。生化诱导法通过模拟细胞内外的微环境能获得较高的胰岛素分泌水平，并表达一系列胰腺发育相关的基因，但是，nestin~+胎肝细胞诱导分化为胰岛素分泌细胞的机制还需更深入的研究，以实现胰岛素的生理性分泌调节。
[Abstract]:This paper studied the characteristics of fetal liver stem cells, and induce their differentiation into insulin secreting cells, focus on the proliferation of fetal liver stem cells in vitro and explore the biological characteristics of fetal liver stem cells and the nature, and the type of embryo fetal liver progenitor cells; the stage of chemical induction, induction and biological method the combination of the two, start the related gene of pancreas. The fetal liver stem cells to differentiate into insulin secreting cells.
The full text is divided into four parts.
The first part, the isolation, culture and identification of fetal liver stem cells.
By in vitro culture and amplification of rat fetal liver stem cells and study its morphology, biological characteristics, analysis of cell surface marker expression of fetal liver stem cells by chemical amplification, experimental conditions of cultured cells in vitro study of fetal liver stem, and investigate the occurrence of liver development and liver stem cell proliferation properties and potential.
This experiment selects the gestational age of 12 ~ 16d SD rats by mechanical separation and enzyme digestion method cultured fetal liver cells, then use specific stem cell medium to obtain a large number of clone screening method, compared with the purified fetal liver stem cell clone.SABC was used to detect the primary cell specific surface marker OV-6. And cell clone in hepatic stem CK-19 and pancreatic precursor cells specific marker protein Nestin expression.
The above research results show that the cells were amplified in vitro by cloning and screening of fetal liver stem cells, containing special factors in stem cell medium contribute to liver stem cell expansion, good results can be obtained. 3D culture began to appear around the small cell groups, 1 months to form a visible cell colony, 5 ~ 7d passaged. Primary cultured fetal liver cells, the expression of OV-6 and CK-19 part, Nestin; cell clones for almost all stem cell marker positive cells showed that fetal liver stem cells with high proliferation ability. Nestin positive stem cells exist in fetal liver, we called hepatic Nestin positive progenitor cells (nestin-positive hepatic-derived progenitor, NHP cells), may be a more primitive stem cells play an important role in embryonic development.
The second part, the identification and classification of fetal liver tissue cells.
This experiment aims to study the properties of fetal liver stem cells and provide experimental basis for liver stem cells are classified systematically, discusses the types of liver stem / progenitor cells were selected to differentiate into pancreatic cell type direction, to obtain better seed cells for the induction of the next step.
Double immunofluorescence labeling for OV-6, different combinations of two kinds of CK-19 antibody and Nestin three antibodies, FITC and Cy3 were labeled, DAPI staining, the fetal liver tissue and cultured in different cell types were cloned and precursor cells cultured in the localization and classification of.
Fetal liver contains from original to mature in different developmental stages of hepatic cells: (1) multipotent progenitor cells: OV-6~+nestin~+CK-19~- cells, liver and pancreas may have multiple differentiation potential; (2) two precursor cells: OV-6~+CK-19~+nestin~- cells, the liver is the double stem cells, can differentiate into hepatic cells and bile duct cell line; (3) directional precursor cells or transitional cells expressing only OV-6 or only CK-19 expression may be cells and somatic cells to differentiate into hepatic bile duct only differentiation ability of the liver, or is the transitional stage differentiation into mature cells; (4) mature cells in vitro. The number of nestin~+ cells are mostly expressed with the OV-6, only the expression of the number of nestin cells rarely.OV-6~+nestin~+ cells and nestin~+ cells may have potential to differentiate into pancreatic cells, from pancreatic island development push Beta cells and hepatocytes share a common source. Their common progenitor cells are nestin positive cells in the pancreatic ductal epithelium and liver. Therefore, we select this hepatocyte derived nestin positive progenitor cells as the next seed cells to induce differentiation.
The third part, the transfection of rat fetal liver stem cells by electroporation with EGFP-PDX-1 fusion expression vector.
The room was constructed PDX-1 fusion expression vector of green fluorescent protein by electroporation into rat fetal liver stem cells to obtain stable expression, with expectations to exogenous biological factors start fetal liver stem cells PDX-1 gene expression and induction of key gene PDX-1 from biological research in the development of pancreatic secretion in liver stem regulation the mechanism of cell differentiation, to provide material platform for future in-depth study of directed differentiation.
The recombinant plasmid pEGFP-C1-PDX-1 was constructed by inserting pEGFP-C1 multiple cloning sites in PDX-1 by electroporation transfection of cultured rat fetal liver stem cells, growth curve analysis before and after transfection, fluorescence microscope GFP light, the expression of RT-PCR PDX-1 gene was identified.
By GFP after electroporation and PDX-1 fusion protein in fetal liver stem cells expressed together, and maintain a longer period of time, no significant effect on the proliferation of fetal liver stem cells (P > 0.05). The expression vector is stably expressed in fetal liver stem cell fusion PDX-1 green fluorescent protein construct. For the study of PDX-1 in the differentiation of stem cells into insulin secreting cells in the regulation provides a material basis.
The fourth part is the study of the induced differentiation of fetal liver stem cells into insulin secreting cells.
Stage specific induced by chemical, biological induction method and the combination of the two, the simulation of pancreatic development internal and external environment, the fetal liver stem cells to differentiate into insulin secreting cells, and to explore the induction and differentiation of hepatic stem cells into insulin secreting cells of endogenous and exogenous control mechanism.
Nestin positive fetal liver cell group screened were induced in phases induced by Nicotinamide chemical inducers, electroporation of PDX-1 gene induced by biological, biological and chemical induced induced content did not induce insulin group.ELISA cell supernatants were measured in the standard curve after the calculated amount of insulin cells were released and compared; RT-PCR detection and development of pancreas and liver cell specific gene related gene expression in each group.
Nestin~+ fetal liver cells by biological, chemical and the combination of the two amount of insulin induced secretion were 0.539,0.943 and 5.58ng / ml supernatant (1 x 10~5 cells), compared with the negative control group were significantly different (P=0.000), which compared to any two groups had statistical significance (P=0.000). The induction group both the expression of insulin I, HNF-1 and hepatic stem cell marker GLUT-2, AFP and c-Met, but the expression of glucagon, somatostatin and ALB. biological induction group and biochemical induction group PDX-1 and insulin expression induced by chemical group II, weak expression of these two genes. The biochemical induced secretion of insulin can be obtained through the simulation of micro environment the higher the inside and outside of the cells, and the expression of related genes, a series of pancreatic development but the mechanism of nestin~+ induced differentiation of fetal liver cells into insulin secreting cells also need more in-depth research, to achieve physiological insulin secretion Adjust.

【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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