羊水来源多潜能干细胞的培养鉴定及其定向分化能力的研究

发布时间：2018-04-25 10:12

本文选题：羊水干细胞 + 细胞培养　；参考：《第四军医大学》2011年硕士论文

【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。
[Abstract]:With the rapid development of regenerative medicine, stem cells play a more and more important role in the basic research and clinical application of modern medicine. The research and clinical application of embryonic stem cells have been greatly restricted due to ethical and moral problems. The ability of differentiation and proliferation of adult stem cells is limited, and only some cells and tissues can be differentiated. The discovery of amniotic fluid stem cells opens up a new field for stem cell research. Amniotic fluid derived stem cells are expected to be used as a new seed cell in tissue engineering research and cell therapy. Although the culture methods of amniotic fluid stem cells have been improved continuously, the stem cells obtained by using the existing techniques are not suitable for the research of human organ regeneration medicine. Although the purity of stem cells obtained by immunomagnetic bead method was high, but the cells were easily contaminated by animal antibodies, the continuous passage culture method could avoid the contamination of stem cells by other components, but the culture cycle in vitro was long. We established a high-speed and effective method to obtain amniotic fluid stem cells. Objective: 1. A high speed and effective culture system of human amniotic fluid derived pluripotent stem cells was established and its biological characteristics were discussed. 2. The cultured amniotic fluid stem cells were identified. 3. Amniotic fluid stem cells were induced to differentiate into osteoblasts, adipocytes and nerve cells under suitable culture conditions. Methods: 1. Human amniotic fluid specimens were collected for primary culture. Long fusiform fibroid cells were selected after adherent to the wall of human amniotic fluid. The cells were called initial cells and stem cell colonies were produced by the initial cells. 2. Flow cytometry (FCM) and RT-PCR technique were used to detect the expression of the partial marker gene Oct-4SEA-4 of embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), and to identify the isolated and cultured amniotic fluid stem cells (amniotic fluid stem cells). 3. Under suitable conditions, amniotic fluid stem cells were induced to differentiate into osteoblasts, adipocytes and nerve cells. The differentiated cells were identified by alizarin red S staining, oil red O staining and immunocytochemistry. Results: 1. Using the improved culture method, the colony uniformity of amniotic fluid stem cells was good. The multiplication time was only 24 h, which was faster than the conventional culture method. 2. Flow cytometry was used to detect the expression of embryonic stem cell and mesenchymal stem cell marker genes such as Oct-4SSEA-4 + CD49 + CD105- CD117. The results of RT-PCR showed that Oct-4 gene mRNA and Nanog gene mRNA were significantly expressed in the cells without expression of hematopoietic stem cell surface marker gene CD45 + CD133. 3. 3 weeks after osteogenesis induction, alizarin red S staining showed the formation of calcium nodules in the cells, oil red O staining showed a large number of lipid droplets in the cells after 3 weeks of adipogenic induction. The positive expression of nestin was detected by immunocytochemistry after induction of nerve cells. Conclusion: 1. By combining the improved culture method with the conventional method, the cell culture cycle was shortened obviously, and the colony homogeneity and purity of amniotic fluid stem cells were obtained. The stem cells obtained by this method can be used as an ideal seed cell for regenerative medicine. 2. AFS expressed some marker genes of embryonic stem cells and mesenchymal stem cells. 3. Under suitable culture conditions, amniotic fluid stem cells can differentiate into osteoblasts, adipocytes and nerve cells.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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