骨髓基质干细胞向肝细胞的定向诱导分化及其机理的实验研究

发布时间：2018-02-14 12:08

  本文关键词： 骨髓基质干细胞 诱导分化 肝细胞 细胞移植 鼠　出处：《青岛大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：目前认为治疗肝功能衰竭的有效方法是肝器官移植，但由于供肝缺乏和免疫排斥反应，制约了肝移植的应用。骨髓基质干细胞(BMSCs)具有易获得、自我复制能力强、在特定的条件下可向各胚层来源的细胞分化的特点。将BMSCs定向诱导分化成为肝细胞，进行细胞移植，以替代损伤的肝细胞，对肝病治疗有非常重要的临床价值。本实验探索和筛选诱导BMSCs向肝细胞定向分化为的最佳条件、探索Ca~(2+)在BMSCs向肝细胞定向分化过程中的作用、以及体内移植对肝损伤的治疗作用，为临床上应用BMSCs进行细胞移植治疗肝损伤提供实验依据。 方法：用全骨髓法从大鼠骨髓中分离BMSCs，纯化和扩增BMSCs。MTT比色法筛选BMSCs的生长曲线，选择最佳生长状态的BMSCs，培养体系中分别加入肝细胞提取液(G，200μg／ml、500μg/ml)、大鼠和人来源的β-神经生长因子—β-NGF from rat(β-NGF-r，20ng/ml、50ng/ml)、β-NGF from human(β-NGF-h 20ng/ml、50ng/ml)和肝细胞生长因子HGF(50μg/ml)诱导BMSCs向肝细胞分化，通过镜下观察分化细胞的形态、RT-PCR法和免疫细胞化学染色法检测肝细胞的特异性标志物白蛋白(ALB)和a-抗胰蛋白酶(AAT)、吲哚靛青绿(ICG)摄取实验鉴定肝样分化细胞。应用流式细胞技术分别检测BMSCs和G诱导分化细胞内游离[Ca~(2+)]i。将BrdU标记后的BMSCs异体移植入慢性肝损伤和对照组的小鼠体内，免疫组织化学法检测BMSCs在肝内的迁移和分化，血清学指标观察BMSCs移植后对慢性肝损伤的治疗作用。 结果：(1)全骨髓分离培养法是纯化、培养和扩增BMSCs的简便有效方法；BMSCs标准生长曲线表明体外培养扩增第3-5代的细胞具有较高的增殖活力。(2)添加G(200μg/ml，500μg/ml)、β-NGF from rat(20ng/ml，50ng/ml)、β-NGF from human(20ng/ml，50ng/ml)和HGF(50μg/ml)诱导后，，分化细胞逐渐趋短变圆呈圆形和卵圆形，圆形细胞可以摄取ICG，呈现绿色。免疫细胞化学染色显示分化细胞呈AAT阳性表达；各诱导组分化细胞的AAT表达显著性高于对照组(P＜0.05)，其中G、β-NGF组AAT表达强于HGF组(P＜0.05)，G、β-NGF组间无显著性差异(P＞0.05)，两种不同来源的β-NGF诱导效果无差异显著性(P＞0.05)。(3)RT-PCR结果显示四种诱导剂诱导21d后，分化细胞均表达ALB mRNA。(4)G诱导形成的肝样分化细胞中[Ca~(2+)i较对照组细胞显著性升高(P＜0.05)，加入尼莫地平可以阻断BMSCs向肝细胞的诱导分化，并影响细胞的增殖活性。(5)BMSCs移植后7d、14d、28d损伤组受体肝脏内均可见BrdU标记的移植细胞，移植细胞大部分呈现由血管逐渐向肝实质内迁移的过程，BMSCs移植后20d、40d的慢性肝损伤小鼠血清学指标与对照组有明显的改善(P＜0.05)。 结论：(1)体外培养BMSCs第3-5代的细胞具有较高的活力。(2)G、β-NGF from rat、β-NGF from human和HGF均可诱导BMSCs向肝细胞分化。(3)Ca~(2+)在BMSCs的增殖和向肝细胞定向分化过程中发挥重要作用。(4)静脉移植的BMSCs可迁移至异种动物损伤的肝脏，可以改善受损肝脏的功能。
[Abstract]:Objective: the effective method for the treatment of liver failure is liver transplantation, but because of the lack of donor liver and immune rejection, which restricts the application of liver transplantation. Bone marrow stromal stem cells (BMSCs) are easy to obtain, self replication ability, characteristics under certain conditions to the source of the germ cell differentiation the BMSCs directional induced differentiation into liver cells, cell transplantation, to replace the damaged liver cells, has very important clinical value for the treatment of liver diseases. The optimal experimental conditions to explore and screening to induce BMSCs to differentiate into liver cells, to explore the Ca~ (2+) differentiation into hepatocyte function in the process of BMSCs and, in vivo therapeutic effect on liver injury and provide experimental basis for clinical application of BMSCs damage on cell transplantation in the treatment of liver.
Methods: BMSCs were isolated from rat bone marrow by whole bone marrow method, growth curve of purification and amplification of BMSCs.MTT assay BMSCs, select the best growth status of BMSCs culture system were added to the hepatocyte extracts (G, 200 g / ml, 500 g/ml), rat and human derived beta nerve growth factor beta -NGF from rat (beta -NGF-r, 20ng/ml, 50ng/ml), -NGF from human (beta beta -NGF-h, 20ng/ml, 50ng/ml) and hepatocyte growth factor HGF (50 g/ml) to induce BMSCs differentiation into liver cells, observe the differentiation of cell morphology by microscope, RT-PCR assay and immune cell chemical staining method for the detection of liver cell specific markers albumin (ALB) and a- antitrypsin (AAT), indocyanine green (ICG) uptake experiments to identify liver like cells differentiation. BMSCs and G were used to detect the differentiation of intracellular [Ca~ by flow cytometry (2+)]i. BrdU labeled BMSCs after allogeneic implantation In vivo, the migration and differentiation of BMSCs in liver were detected by immunohistochemical method in mice with chronic liver injury and control group. Serological indexes were used to observe the therapeutic effect of BMSCs after transplantation on chronic liver injury.
缁撴灉锛

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