干细胞在PET上的三维扩增培养
发布时间：2019-01-07 11:33
【摘要】： 骨髓间充质干细胞(MSCs)具有多向分化潜能,可在体外大量扩增,并且具有调节细胞免疫反应的功能,其来源不存在伦理争议问题。因此是细胞治疗和组织工程中理想的种子细胞来源。胚胎干细胞(ESCs)可作为药物筛选的理想资源。而现今面临的主要问题是如何获得大量优质的种子干细胞来满足其在医学上的应用,现今的技术手段仍无法解决干细胞的大规模扩增问题,因此需要研究和发展干细胞的三维培养平台技术。针刺无纺聚对苯二甲酸乙二酯(Polyethylene Terephthalate,PET)纤维材料可作为三维的支架材料来支持MSCs和ESCs的增殖,它具备高孔隙率、比表面积大、渗透性好、机械强度高的优点,其独特的三维结构可提供给细胞一个仿生的环境。 研究目的:本实验采用PET纤维材料对hMSCs和mESCs进行三维扩增培养,探讨并优化hMSCs的培养条件及评定三维扩增的效果。为将来研发大规模生产干细胞的系统,进而建立一个相对成熟高效的培养系统平台做基础 实验方法:为了优化hMSCs在PET上的条件培养,CCK-8法检测了bFGF对hMSCs的增殖影响,分析了在PET纤维材料上的接种密度对细胞增殖的影响,检测了乳酸对细胞增殖的抑制作用;进一步探讨了hMSCs在PET上的增殖和代谢的特点;利用Cell tracker Green荧光染色,激光共聚焦显微镜检测细胞在材料上的活性;扫描电镜检测细胞在PET上的分布、形态及粘附情况;检测了低浓度的bFGF对PET上细胞的影响;流式细胞仪鉴定三维扩增后的hMSCs的表面标记物CD29、CD44、CD34和CD45。对hMSCs进行向成骨、成脂方向的诱导培养;最后通过倒置显微镜观察、接种量的优化和激光共聚焦显微镜、扫描电镜的观察检测PET对mESs的扩增培养情况。 结果:实验研究表明,培养基中5 ng/mL的bFGF,每孔接种1×104个hMSCs及保持培养基中的乳酸浓度少于0.9 g/L有利于促进PET上hMSCs的增殖。PET能够支持更长期的细胞的培养,而且PET上培养的hMSCs的对营养物的利用率更高。观察显示hMSCs及mESCs在PET纤维材料上成立体,多层生长,有较大的生长空间。三维扩增后的细胞保持了原有未分化状态和分化潜能;bFGF对三维材料上hMSCs的增殖有更明显的促进作用。1~2×104个细胞/孔为mESCs在PET纤维材料上接种的最适密度。
[Abstract]:Bone marrow mesenchymal stem cells (BMSCs) have the potential of multidirectional differentiation, can be expanded in vitro, and have the function of regulating cellular immune response. Therefore, it is an ideal seed cell source for cell therapy and tissue engineering. Embryonic stem cell (ESCs) can be used as an ideal resource for drug screening. But the main problem now is how to obtain a large number of high-quality seed stem cells to meet its medical application, but the current technology can not solve the problem of stem cell expansion on a large scale. Therefore, it is necessary to research and develop the three-dimensional culture platform technology of stem cells. The needle-free poly (ethylene terephthalate) (Polyethylene Terephthalate,PET) fiber can be used as a three-dimensional scaffold material to support the proliferation of MSCs and ESCs. It has the advantages of high porosity, large specific surface area, good permeability and high mechanical strength. Its unique three-dimensional structure provides a bionic environment for cells. Objective: to study and optimize the culture conditions of hMSCs and mESCs and evaluate the effect of three dimensional amplification by using PET fiber material. In order to develop a large-scale stem cell production system in the future, and then establish a relatively mature and efficient culture system platform for basic experimental methods: in order to optimize the condition culture of hMSCs on PET, The effect of bFGF on the proliferation of hMSCs, the effect of inoculation density on the proliferation of hMSCs and the inhibition of lactic acid on cell proliferation were analyzed by CCK-8 method. The characteristics of proliferation and metabolism of hMSCs on PET were further discussed. The activity of cells on the material was detected by Cell tracker Green fluorescence staining and confocal microscopy. The distribution, morphology and adhesion of cells on PET were detected by scanning electron microscope (SEM). The effect of low concentration of bFGF on the cells on PET was detected. The surface markers CD29,CD44,CD34 and CD45. of hMSCs were identified by flow cytometry HMSCs was cultured in the direction of osteogenesis and fat-forming. Finally, the amplification and culture of mESs by PET were detected by inverted microscope, optimization of inoculation amount and laser confocal microscope, scanning electron microscope (SEM). Results: the experimental study showed that 5 ng/mL bFGF, inoculated with 1 脳 104 hMSCs per well and kept lactic acid concentration less than 0.9 g / L in the culture medium could promote the proliferation of hMSCs on PET. PET could support the long-term cell culture. Moreover, the nutrient utilization of hMSCs cultured on PET was higher. The results showed that hMSCs and mESCs were three dimensional and multilayer growth on PET fiber material. The undifferentiated state and differentiation potential of the expanded cells were maintained, the proliferation of hMSCs on the three dimensional material was promoted more obviously by bFGF, and 1 2 脳 10 4 cells / well was the best density of mESCs inoculation on the PET fiber material. 2 脳 10 4 cells / well was the most suitable density of mESCs inoculation on PET fiber material.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2403609
[Abstract]:Bone marrow mesenchymal stem cells (BMSCs) have the potential of multidirectional differentiation, can be expanded in vitro, and have the function of regulating cellular immune response. Therefore, it is an ideal seed cell source for cell therapy and tissue engineering. Embryonic stem cell (ESCs) can be used as an ideal resource for drug screening. But the main problem now is how to obtain a large number of high-quality seed stem cells to meet its medical application, but the current technology can not solve the problem of stem cell expansion on a large scale. Therefore, it is necessary to research and develop the three-dimensional culture platform technology of stem cells. The needle-free poly (ethylene terephthalate) (Polyethylene Terephthalate,PET) fiber can be used as a three-dimensional scaffold material to support the proliferation of MSCs and ESCs. It has the advantages of high porosity, large specific surface area, good permeability and high mechanical strength. Its unique three-dimensional structure provides a bionic environment for cells. Objective: to study and optimize the culture conditions of hMSCs and mESCs and evaluate the effect of three dimensional amplification by using PET fiber material. In order to develop a large-scale stem cell production system in the future, and then establish a relatively mature and efficient culture system platform for basic experimental methods: in order to optimize the condition culture of hMSCs on PET, The effect of bFGF on the proliferation of hMSCs, the effect of inoculation density on the proliferation of hMSCs and the inhibition of lactic acid on cell proliferation were analyzed by CCK-8 method. The characteristics of proliferation and metabolism of hMSCs on PET were further discussed. The activity of cells on the material was detected by Cell tracker Green fluorescence staining and confocal microscopy. The distribution, morphology and adhesion of cells on PET were detected by scanning electron microscope (SEM). The effect of low concentration of bFGF on the cells on PET was detected. The surface markers CD29,CD44,CD34 and CD45. of hMSCs were identified by flow cytometry HMSCs was cultured in the direction of osteogenesis and fat-forming. Finally, the amplification and culture of mESs by PET were detected by inverted microscope, optimization of inoculation amount and laser confocal microscope, scanning electron microscope (SEM). Results: the experimental study showed that 5 ng/mL bFGF, inoculated with 1 脳 104 hMSCs per well and kept lactic acid concentration less than 0.9 g / L in the culture medium could promote the proliferation of hMSCs on PET. PET could support the long-term cell culture. Moreover, the nutrient utilization of hMSCs cultured on PET was higher. The results showed that hMSCs and mESCs were three dimensional and multilayer growth on PET fiber material. The undifferentiated state and differentiation potential of the expanded cells were maintained, the proliferation of hMSCs on the three dimensional material was promoted more obviously by bFGF, and 1 2 脳 10 4 cells / well was the best density of mESCs inoculation on the PET fiber material. 2 脳 10 4 cells / well was the most suitable density of mESCs inoculation on PET fiber material.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
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