人毛囊源间充质干细胞体外立体与平面培养方法比较的实验研究
发布时间：2018-07-23 19:02
【摘要】：干细胞是体内一类具有自我更新、高度增殖和多向分化潜能的细胞群体，在一定条件下，可以分化成多种功能细胞。干细胞是再生医学的原基，可用于组织修复、器官再生、基因治疗等研究和临床治疗。目前研究比较深入的有胚胎干细胞、骨髓间充质干细胞、脐带血干细胞、毛囊间充质干细胞等。毛囊间充质干细胞，不仅自我更新，定向分化为脂肪、骨、软骨细胞，更重要的是毛囊间充质干细胞表达胚胎干细胞标志物（SOX2和Nanog）。毛囊来源丰富、获取方便、收获不受年龄的限制，几乎不会对机体造成任何损害，还可重复获取，并且免疫原性地下，是组织修复、器官重建和基因治疗重要的种子细胞来源。再生医学研究与治疗需要大量种子细胞，传统的平面培养方法很难在短时间获取大量种子细胞，而应用微载体、生物反应器技术立体培养就可以解决这一问题。微载体表面积/体积（S/V）大，单位体积培养液细胞产率高；立体培养兼悬浮和贴壁培养的优点，简化细胞生长环境因素的检测和控制，重现性好，成本低，劳动强度小。通过立体培养可以获得大量的毛囊间充质干细胞，然而其生长特性、生物学特性未知。 本实验研究目的是建立体外大量扩增人毛囊间充质干细胞（human hair folliclemesenchymal stem cells，hHF-MSCs）的生物反应器技术，，从而为再生医学研究与临床治疗提供未分化、大量的种子细胞。 拔取成人毛发，采用组织块培养法，成功获得了hHF-MSCs。我们将获取的hHF-MSCs成功的接种至载体上，经吖啶橙染色，共聚焦显微镜扫描，可以观察到hHF-MSCs贴附于载体表面并伸展呈长梭形。hHF-MSCs以1000cells/cm~2或500cells/cm~2密度接种至载体上，细胞在接种第3d时进入对数生长期，增殖至15d时细胞进入平台期；平面培养hHF-MSCs至第6天时进入对数生长期。间充质干细胞表面标志CD90、CD105免疫荧光染色，二种方法培养hHF-MSCs呈阳性表达；流式细胞术分析：立体培养hHF-MSCs CD90、CD105表达率分别为80.25%、45.36%，平面培养hHF-MSCsCD90、CD105表达率分别为85.65%、43.88%；这二种方法培养hHF-MSCs经成脂诱导后，油红O染色，脂滴呈红色；成骨诱导后，茜素红染色，可见钙盐沉积和钙结节被染成红色。 本研究成功获取hHF-MSCs，建立体外大量扩增hHF-MSCs生物反应器技术，应用该技术培养的hHF-MSCs仍能保持MSCs表面标志，并具有多向分化潜能，这为后期建立hHF-MSCs银行，构建组织工程化器官，移植修复病变的组织器官，作为基因治疗的靶细胞等相关实验研究及临床应用提供了丰富的种子细胞来源。
[Abstract]:Stem cells are a group of cells with self-renewal, high proliferative and multidirectional differentiation potential in vivo. Under certain conditions, stem cells can be differentiated into a variety of functional cells. Stem cells are the primordium of regenerative medicine and can be used in tissue repair, organ regeneration, gene therapy and clinical therapy. At present, embryonic stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells, hair follicle mesenchymal stem cells and so on. Hair follicle mesenchymal stem cells, not only self-renewal, directional differentiation into fat, bone, chondrocytes, and, more importantly, hair follicle mesenchymal stem cells express embryonic stem cell markers (SOX2 and Nanog).) Hair follicles are rich in source, easy to obtain, and not restricted by age. They can be obtained repeatedly and can be obtained repeatedly. They are important seed cells for tissue repair, organ reconstruction and gene therapy. The research and treatment of regenerative medicine require a large number of seed cells. The traditional plane culture method is difficult to obtain a large number of seed cells in a short time, which can be solved by using microcarriers and bioreactor technology. The microcarriers have large surface area / volume (S / V), high cell yield per unit volume, and the advantages of stereoscopic culture and suspension and adherent culture, which simplify the detection and control of the environmental factors of cell growth, with good reproducibility, low cost and low labor intensity. A large number of hair follicle mesenchymal stem cells can be obtained by stereoscopic culture. However, the growth and biological characteristics of hair follicle mesenchymal stem cells are unknown. The purpose of this study was to establish a bioreactor for the proliferation of human hair follicle mesenchymal stem cells (human hair folliclemesenchymal stem cells hHF-MSCs) in vitro, and to provide undifferentiated and large numbers of seed cells for the study of regenerative medicine and clinical treatment. The adult hair was extracted and the hHF-MSCs was successfully obtained by tissue mass culture. We successfully inoculated the obtained hHF-MSCs onto the vector, stained with acridine orange, and scanned by confocal microscope. We observed that the hHF-MSCs was attached to the surface of the carrier and stretched in the form of fusiform. 1000cells/cm~2 or 500cells/cm~2 density was used to inoculate the hHF-MSCs onto the carrier. The cells entered the logarithmic growth phase on the 3rd day of inoculation, the cell proliferation reached the plateau stage on the 15th day, and the logarithmic growth phase on the sixth day after hHF-MSCs was cultured on the plane. Flow cytometry analysis showed that the expression rates of CD90 CD105 were 80.25% and 45.36%, respectively, and the expression rates of CD105 were 85.65% in plane culture, 85.65% in hHF-MSCs and 43.88% in plane culture, respectively. After induced by lipogenesis, oil red O was stained with lipid droplets, and alizarin red was stained with alizarin red after osteogenic induction. Calcium salt deposition and calcium nodules were stained red after osteogenesis. In this study, hHF-MSCs was successfully obtained, and a large amount of hHF-MSCs bioreactor was established in vitro. The hHF-MSCs cultured with this technique can still maintain the surface marker of MSCs and have the potential of multi-differentiation. This is the late stage of establishing hHF-MSCs bank and constructing tissue engineering organs. Transplantation and repair of diseased tissues and organs, as target cells for gene therapy and other related experimental studies and clinical applications provide abundant seed cell sources.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2140358
[Abstract]:Stem cells are a group of cells with self-renewal, high proliferative and multidirectional differentiation potential in vivo. Under certain conditions, stem cells can be differentiated into a variety of functional cells. Stem cells are the primordium of regenerative medicine and can be used in tissue repair, organ regeneration, gene therapy and clinical therapy. At present, embryonic stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells, hair follicle mesenchymal stem cells and so on. Hair follicle mesenchymal stem cells, not only self-renewal, directional differentiation into fat, bone, chondrocytes, and, more importantly, hair follicle mesenchymal stem cells express embryonic stem cell markers (SOX2 and Nanog).) Hair follicles are rich in source, easy to obtain, and not restricted by age. They can be obtained repeatedly and can be obtained repeatedly. They are important seed cells for tissue repair, organ reconstruction and gene therapy. The research and treatment of regenerative medicine require a large number of seed cells. The traditional plane culture method is difficult to obtain a large number of seed cells in a short time, which can be solved by using microcarriers and bioreactor technology. The microcarriers have large surface area / volume (S / V), high cell yield per unit volume, and the advantages of stereoscopic culture and suspension and adherent culture, which simplify the detection and control of the environmental factors of cell growth, with good reproducibility, low cost and low labor intensity. A large number of hair follicle mesenchymal stem cells can be obtained by stereoscopic culture. However, the growth and biological characteristics of hair follicle mesenchymal stem cells are unknown. The purpose of this study was to establish a bioreactor for the proliferation of human hair follicle mesenchymal stem cells (human hair folliclemesenchymal stem cells hHF-MSCs) in vitro, and to provide undifferentiated and large numbers of seed cells for the study of regenerative medicine and clinical treatment. The adult hair was extracted and the hHF-MSCs was successfully obtained by tissue mass culture. We successfully inoculated the obtained hHF-MSCs onto the vector, stained with acridine orange, and scanned by confocal microscope. We observed that the hHF-MSCs was attached to the surface of the carrier and stretched in the form of fusiform. 1000cells/cm~2 or 500cells/cm~2 density was used to inoculate the hHF-MSCs onto the carrier. The cells entered the logarithmic growth phase on the 3rd day of inoculation, the cell proliferation reached the plateau stage on the 15th day, and the logarithmic growth phase on the sixth day after hHF-MSCs was cultured on the plane. Flow cytometry analysis showed that the expression rates of CD90 CD105 were 80.25% and 45.36%, respectively, and the expression rates of CD105 were 85.65% in plane culture, 85.65% in hHF-MSCs and 43.88% in plane culture, respectively. After induced by lipogenesis, oil red O was stained with lipid droplets, and alizarin red was stained with alizarin red after osteogenic induction. Calcium salt deposition and calcium nodules were stained red after osteogenesis. In this study, hHF-MSCs was successfully obtained, and a large amount of hHF-MSCs bioreactor was established in vitro. The hHF-MSCs cultured with this technique can still maintain the surface marker of MSCs and have the potential of multi-differentiation. This is the late stage of establishing hHF-MSCs bank and constructing tissue engineering organs. Transplantation and repair of diseased tissues and organs, as target cells for gene therapy and other related experimental studies and clinical applications provide abundant seed cell sources.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 郭黎黎;毛囊干细胞修复皮肤损伤的实验研究[D];吉林大学;2013年
本文编号：2140358
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