【作者】 顾潇；

【导师】 陈必良； 张建芳；

【作者基本信息】 第四军医大学 ， 妇产科学， 2011， 硕士

【摘要】 再生医学的飞速发展使干细胞在现代医学的基础研究与临床应用中发挥越来越重要的作用。因伦理道德问题,胚胎干细胞的研究和临床应用受到了极大的制约;成体干细胞自身分化潜能和增殖能力均有限,只能分化出部分细胞和组织。羊水干细胞的发现为干细胞的研究开辟了新的领域。羊水来源干细胞有望作为一种新的种子细胞,应用于组织工程领域的研究与细胞治疗。虽然羊水干细胞的培养方法不断改进,但利用现有技术获得的干细胞并不适合应用于人体器官再生医学的研究。通过免疫磁珠法分选获得的干细胞虽然纯度高,但细胞易受动物抗体的污染;连续传代培养法虽可避免干细胞被其他成分污染,但体外培养周期长。我们在实验中通过改良培养方法,建立了一种高速有效的获得羊水干细胞的新方法。目的:1.通过改良培养方法,建立一种高速有效的人羊水来源多潜能干细胞的体外培养体系,并对其生物学特性进行探讨。2.对培养的羊水干细胞进行鉴定。3.在适宜的培养条件下诱导羊水干细胞向成骨细胞、脂肪细胞、神经细胞进行分化,检测其体外分化能力。方法:1.取人孕中期羊水标本进行原代培养,细胞贴壁后挑选出长梭形、呈纤维样细胞,将其称之为起始细胞,利用起始细胞产生干细胞集落。2.流式细胞仪和RT-PCR技术检测胚胎干细胞和间充质干细胞部分标志基因Oct-4、SSEA-4、Nanog、CD29、CD44、CD105和CD117的表达情况,鉴定分离培养的羊水干细胞。3.使用特定培养基,在适宜条件下诱导羊水干细胞向成骨细胞、脂肪细胞、神经细胞进行分化。分别用茜素红S染色、油红O染色以及免疫细胞化学方法对分化出的细胞进行鉴定。结果:1.应用改良后的培养方法,获得的羊水干细胞集落均一性好。体外增殖能力强,倍增时间仅为24h,明显快于常规使用的培养方法。2.流式细胞仪检测细胞表达Oct-4、SSEA-4、CD29、CD44、CD105、CD117等胚胎干细胞和间充质干细胞标志基因,不表达造血干细胞表面标志基因CD45、CD133。RT-PCR结果显示细胞中Oct-4基因mRNA和Nanog基因mRNA均明显表达。3.成骨诱导3周后茜素红S染色见细胞内有钙结节的形成;成脂诱导3周后油红O染色见细胞内有大量脂滴形成;向神经细胞诱导后免疫细胞化学方法检测细胞巢蛋白(Nestin)的表达呈阳性。结论:1.将改良的培养方法与常规方法良好的结合,使细胞的培养周期明显缩短,获得的羊水干细胞集落均一性好,纯度高。此方法培养得到的干细胞可以作为一种理想的再生医学的种子细胞。2. AFS表达胚胎干细胞和间充质干细胞部分标志基因,体外增值能力强,符合多潜能干细胞的特点。3.在适宜的培养条件下,羊水干细胞可以向成骨细胞、脂肪细胞、神经细胞分化。更多还原

【Abstract】 With the rapid growth of regenerative medicine, stem cells have played more and more important role in the basic research and clinical application of modern medicine. Because of ethical problem,the research of embryonic stem cells and clinical application has greatly restricted; adule stem cells have limited ability to differentiate into some cells and tissues. When the human amniotic fluid-derived stem cells have been discovered , they pioneer a new field for study the stem cells. Human amniotic fluid-derived stem cells (hAFS) may be a potential source of cells for tissue engineering and cellular therapy. Although several hAFS cells derivation techniques have now been developed, the existing techniques are unsuitable for hAFS cells production for medical purposes because these methods often result in contamination with other cell types or contamination with antibodies raised from animals. Additionally, these techniques require a long period of time for stem cell production. Hence, a better method which allows utilization of these cells for cell-based therapy needs to be developed. In the current study, we present an improved method as an efficient technique that is suitable to derive hAFS cells for therapeutic purposes.Objective1. To establish an improved device of culture amniotic fluid-derived multipotential stem cells in vitro and study their biological characteristics.2. The phenotypes of hAFS cells were tested by flow cytometry.3. Inducing hAFS cells into neurogenic, adipogenic and osteogenic by definite culture medium to detection their differentiation potentiality.Methods1. The technique starts by selecting adherent stem cells from second-trimester human amniotic fluid primary culture. A selective individual hAFS cell is called a "starter cell". The starter cell is used as a beginner cell for generating a clonal hAFS cell line.2. hAFS cell markers were characterized using flow cytometry analysis and RT-PCR technique, the tested antibody marker of some embryonic stem cells and mesenchymal stem cells such as: Oct-4、SSEA-4、Nanog、CD29、CD44、CD105 and CD117.3. Inducing amniotic fluid-derived stem cells into neurogenic, adipogenic and osteogenic by definite culture medium, The osteogenesis was assessed by determinating cell mineralization,using alizarin red S staining. The Oil Red O staining was used for detection of intracellular lipid droplet formation for evaluating adipogenesis. For evaluation of neural differentiation, the neuron specific marker nestin was used.Results1. The hAFS cells derived by our technique proliferated rapidly and have a high purity, with a population doubling time of 24h,it is significantly accelerated compared with the previous research.2. The hAFS cells showed high positive signals for embryonic stem cells and mesenchymal stem cells markers :Oct-4、SSEA-4、CD29、CD44、CD105and CD117. These cells show a negative signal for hematopoietic stem cell markers CD45 and CD133 by flow cytometry. RT-PCR show that the mRNA of Oct-4 and Nanog were expressed.3. Osteogenic differentiation for 3 weeks , calcium mineralization were appeared in cytoplasm of hAFS-derived osteogenic cells, they verified by a photochemical reaction using alizarin red S .For adipogenic differentiation, the appearance of endogenous lipid droplets was observed by staining with Oil Red O after 3 weeks of culture, the lipid droplets appeared in cytoplasm and bright red. For neurogenic differentiation, the cells showed positive staining for neuron-specific marker nestin. Above all,hAFS cells can differentiation into neurogenic,adipogenic and osteogenic in vitro.Conclusions1. The starter cell method as a simple approach to provide good quality hAFS cells that fit the requirements for use in therapeutic purposes. This technique should allow for future cell-based therapy.2. hAFS cells have a great potential for proliferation, which express both embryonic and mesenchymal stem cells markers. Their characteristics are in accord with multipotential stem cells.3. AFS cells have the ability to differentiate into neurogenic, adipogenic and osteogenic in suitable condition in vitro.更多还原