三种不同来源间充质干细胞比较研究

发布时间：2018-05-06 15:22

本文选题：间充质干细胞 + 脐带　；参考：《北京协和医学院》2009年硕士论文

【摘要】：研究背景：间充质干细胞(mesenchymal stem cells, MSCs)是一种具有自我更新能力和多向分化潜能的成体干细胞,它来源广泛、取材方便,同时具有造血支持及免疫调节功能,这使其成为有前途的临床使用种子细胞。成体骨髓来源的间充质干细胞是研究较早、较深入的间充质干细胞,但是其取材侵袭性强,对供者伤害较大：细胞的数量、增殖和分化能力随供者年龄增长而显著下降；容易被病毒感染,这些因素限制了骨髓来源间充质干细胞的应用。大量研究已证实,间充质干细胞还存在于多种组织中,本实验采用了脐带及脂肪来源的间充质干细胞与成人骨髓来源的间充质干细胞进行生物学比较,期望获得更佳的种子细胞来源。目的：分离培养成人骨髓、脂肪和脐带来源的间充质干细胞,比较三种不同来源间充质干细胞的形态特征、增殖能力、表面标志及诱导分化能力,为间充质干细胞临床应用选择细胞来源提供实验依据和理论依据。方法：在本研究中我们利用直接贴壁培养法分离培养成人骨髓间充质干细胞,利用酶消化法分离培养脂肪和脐带来源的间充质干细胞,并通过贴壁培养的方法进一步纯化获得三种间充质干细胞。使用显微镜技术比较观察三种来源间充质干细胞的形态特征。用MTT法检测P3代的三种间充质干细胞的增殖能力。通过体外连续传代试验研究其体外传代能力。用流式细胞学方法检测三种P3/P4代间充质干细胞的表面标记。体外诱导三种间充质干细胞向脂肪和成骨细胞分化,通过油红0和von-Kossa染色鉴定比较分化结果。结果：三种间充质干细胞体外培养时呈长梭型、旋涡状、贴壁生长,与脐带来源的间充质干细胞相比,脂肪和骨髓来源的间充质干细胞体积较大。通过生长曲线分析,计算倍增时间发现三种间充质干细胞的增殖能力由强至弱依次为脐带来源间充质干细胞、脂肪来源间充质干细胞、骨髓来源间充质干细胞。通过体外长期传代实验,我们发现脐带来源间充质干细胞的传代能力最强,而脂肪来源的间充质干细胞次之,传代能力最差的是骨髓来源的间充质干细胞。三种间充质干细胞均表达CD90,CD105 (SH2), CD73 (SH3/4), CD166, CD29;不表达CD31、CD133、CD19、CDllb、CD34、CD45和HLA-DR (HLA-Ⅱ)。此外,三种间充质干细胞经油红0染色证实均可向成脂细胞分化；von Kossa染色证实均可向成骨细胞分化。结论：我们成功从成人骨髓、脂肪和脐带组织中分离并培养出间充质干细胞,这三种不同来源的间充质干细胞的形态、增殖传代及诱导分化等生物学特征存在一定差异,而表面标记基本相同。研究背景：端粒是位于真核细胞染色体末端的特殊结构,由端粒DNA和端粒蛋白质组成,其对维持染色体的稳定和完全有重要作用。一般情况下动物细胞染色体的端粒随着细胞分裂而缩短,当缩短到某种程度时细胞将停止增殖并调亡。端粒酶在一般细胞中不表达,只在生殖细胞、干细胞和肿瘤细胞中表达,而在间充质干细胞中端粒酶的表达情况研究结果不统一。脂肪源间充质干细胞是基础实验研究和临床应用较多的间充质干细胞之一,本研究测定脂肪源间充质干细胞的端粒酶活性,为其今后的实验研究及临床应用提供更详尽的实验依据。目的：分离鉴定成人脂肪来源的间充质干细胞,测定成人脂肪来源的间充质干细胞是否表达端粒酶活性。方法：按国际细胞治疗协会(the International Society for Cellular Therapy, ISCT)提出的标准鉴定从脂肪中分离、培养得到间充质干细胞。体外诱导成人脂肪来源间充质干细胞向成脂和成骨细胞分化,通过油红0和von-Kossa染色鉴定。采用逆转录PCR和端粒酶重复序列扩增法-酶链免疫吸附法(TRAP-ELISA法),检测不同供者来源和不同传代次数的脂肪源间充质干细胞是否表达端粒酶活性。结果：研究发现用我们的方法分离得到的脂肪源间充质干细胞符合国际细胞治疗协会提出的标准。不同供者来源和不同传代次数的脂肪源间充质干细胞均不表达端粒酶mRNA,也无端粒酶活性。结论：成人脂肪源间充质干细胞不表达端粒酶活性。
[Abstract]:Background: mesenchymal stem cells (MSCs) is a kind of adult stem cells with self-renewal ability and multidirectional differentiation potential. It has a wide range of sources, convenient to take materials, and also has hematopoiesis support and immunomodulatory function. This makes it a promising seedbed using seed cells. The mesenchymal stem cells from adult bone marrow stem cells are dry and fine. Cell is an early and deeper mesenchymal stem cell, but it has strong invasiveness and great damage to the donor: the number of cells, proliferation and differentiation ability decrease significantly with the age of the donor; it is easy to be infected by the virus. These factors restrict the application of mesenchymal stem cells derived from bone marrow. A large number of studies have confirmed that the mesenchymal stem is dry and fine. The cells also exist in a variety of tissues. This experiment uses mesenchymal stem cells derived from umbilical and fat sources to compare with adult mesenchymal stem cells derived from adult bone marrow, hoping to obtain better seed cell sources.
Objective: to isolate and culture mesenchymal stem cells from adult bone marrow, fat and umbilical cord, and compare the morphological characteristics, proliferation ability, surface markers and differentiation ability of three different sources of mesenchymal stem cells, and provide experimental basis and theoretical basis for the selection of cell sources in the clinical application of mesenchymal stem cells.
Methods: in this study, adult bone marrow mesenchymal stem cells were isolated and cultured by direct adherence culture, and mesenchymal stem cells derived from fat and umbilical cord were isolated and cultured by enzyme digestion, and three kinds of mesenchymal stem cells were further purified by adherent culture. The three sources were compared with the microscope technique. The morphological characteristics of the mesenchymal stem cells. The proliferation ability of three mesenchymal stem cells of P3 generation was detected by MTT method. The external transmission capacity was studied by continuous passages in vitro. Flow cytometry was used to detect the surface markers of three P3/P4 generations of mesenchymal stem cells. In vitro, three mesenchymal stem cells were induced to differentiate into adipose and osteoblasts. The differentiation results were identified by oil red 0 and von-Kossa staining.
Results: three kinds of mesenchymal stem cells were cultured in vitro, with a long shuttle type, vortexed and adherent growth. Compared with mesenchymal stem cells derived from umbilical cord, the volume of mesenchymal stem cells from adipose and bone marrow derived mesenchymal stem cells was larger. Through the analysis of growth curve, the multiplication time of three mesenchymal stem cells was found to be the umbilical cord from strong to weak. Derived mesenchymal stem cells derived from mesenchymal stem cells, adipose derived mesenchymal stem cells and bone marrow mesenchymal stem cells. Through long term passages in vitro, we found that mesenchymal stem cells derived from umbilical cord derived mesenchymal stem cells have the strongest passages, but the adipose derived mesenchymal stem cells are the next, and the most poor passages of mesenchymal stem cells are mesenchymal stem cells derived from bone marrow. Three kinds of mesenchymal stem cells are used. The stem cells all express CD90, CD105 (SH2), CD73 (SH3/4), CD166, CD29, and do not express CD31, CD133, CD19, CDllb, CD34, and CDllb (CD34). In addition, three mesenchymal stem cells can differentiate into adipocytes confirmed by oil and red 0 staining.
Conclusion: we successfully isolated and cultured mesenchymal stem cells from adult bone marrow, fat and umbilical cord tissue. There are some differences in the biological characteristics of the three different sources of mesenchymal stem cells, such as the morphology, proliferation and differentiation of mesenchymal stem cells, and the surface markers are basically the same.
Background: telomere is a special structure at the terminal of eukaryotic cells. It is composed of telomere DNA and telomere protein. It plays an important role in maintaining the stability and integrity of the chromosomes. In general, the telomere of the chromosomes of an animal cell is shortened with cell division. When a certain degree is shortened, the cell will stop proliferating and terminating. Granulase is not expressed in general cells and is expressed only in germ cells, stem cells and tumor cells. However, the results of telomerase expression in mesenchymal stem cells are not unified. Adipose derived mesenchymal stem cells are one of the mesenchymal stem cells which are widely used in basic experimental research and clinical application. This study was to determine the stem cells of adipose derived mesenchymal stem cells. The telomerase activity provides a more detailed experimental basis for its future research and clinical application.
Objective: to isolate and identify adult adipose derived mesenchymal stem cells, and to determine whether adult adipose derived mesenchymal stem cells express telomerase activity.
Methods: mesenchymal stem cells were isolated from fat and cultured in accordance with the standard of the International Society for Cellular Therapy, ISCT. In vitro, adult adipose derived mesenchymal stem cells were induced to differentiate into fat and osteoblast cells, identified by oil red 0 and von-Kossa staining. Reverse transcriptase PCR was used. Telomerase repeated sequence amplification, enzyme chain immunoadsorption (TRAP-ELISA), was used to detect the expression of telomerase activity in adipose derived mesenchymal stem cells with different donor sources and different passages.
Results: the study found that the adipose derived mesenchymal stem cells derived from our method were in conformity with the standards proposed by the international cell therapy association. The adipose derived mesenchymal stem cells of different donor sources and different passages did not express telomerase mRNA or telomerase activity.
Conclusion: adult adipose derived mesenchymal stem cells do not express telomerase activity.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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