人真皮干细胞标记示踪方法的比较研究

发布时间：2018-06-24 07:50

本文选题：标记 + 示踪　；参考：《第三军医大学》2012年硕士论文

【摘要】：研究背景：干细胞具有自我更新和多向分化潜能的特性。利用干细胞移植进行疾病治疗已成为干细胞研究的热点。干细胞的标记和示踪能够为阐明干细胞在移植受体内的定植、分布、迁移和分化等生物学行为提供重要依据，对干细胞移植研究具有重要意义。本课题拟探讨利用荧光染料PKH26、核酸标记物BrdU以及人源性细胞特异性识别抗体等三种方法用于干细胞体内示踪，，分析三种方法的优缺点，为研究干细胞在体移植后的生物学评价提供依据。研究方法：第一部分，以人真皮干细胞为研究对象，观察荧光染料PKH26、核酸标记物BrdU以及三种人源性细胞特异性识别抗体来体外标记和检测干细胞的效果。第二部分，以不同剂量全身辐射C57BL/6J小鼠动物损伤模型作为受试体，以2×106个细胞/只尾静脉移植经不同方法标记的人真皮干细胞，于不同时间点取材，应用普通光学显微镜或荧光显微镜观察各种方法检测细胞在组织器官中的分布情况。研究结果：本研究主要结果如下： 1.经BrdU体外标记的人真皮干细胞经静脉移植受试小鼠后，可通过抗BrdU抗体检测到人真皮干细胞在受试小鼠肺内的定植； 2.经PKH26体外标记的人真皮干细胞经静脉移植受试小鼠后，未能检测到人真皮干细胞在受试小鼠各个脏器的定植情况； 3.本研究中所尝试的三种商品化的人源性细胞特异性识别抗体，其中小鼠抗人β-2-Microglobulin抗体(Santa Cruz, sc-80668)和小鼠抗人endoglin抗体(Dako, M3527)在体外均不能特异性识别人源性细胞。小鼠抗人nuclei抗体(Millipore, MAB1281)能够在体外特异性识别人源性细胞，但人真皮干细胞经静脉移植受试小鼠后，通过小鼠抗人nuclei抗体未能检测到人真皮干细胞在受试小鼠各个脏器的定植情况。结论：本课题研究中，我们通过利用荧光染料PKH26、核酸标记物BrdU以及三种人源性细胞特异性识别抗体来体外标记干细胞并进行体内示踪，比较了三种标记方法的优缺点。本课题取得的主要研究结论包括： 1.核酸标记物BrdU具有较高的体外标记效率和较为稳定的体内示踪特性，可作为干细胞体外标记及体内示踪方法应用于干细胞移植研究； 2.荧光染料PKH26具有较高的体外标记效率，但用于干细胞体内示踪时，其分辨率和信号强度等问题不够理想； 3.基于人源性细胞特异性识别抗体的检测方法在研究干细胞体内示踪的过程中很大程度上受限于抗体质量和稳定性的影响，本研究所采用的小鼠抗人nuclei抗体（Millipore公司）、小鼠抗人β-2-Microglobulin抗体（Santa Cruz公司）、小鼠抗人endoglin抗体（Dako公司）均未获得理想的结果。
[Abstract]:Background: stem cells have self-renewal and multi-differentiation potential. Stem cell transplantation for disease treatment has become a hot spot in stem cell research. The markers and tracers of stem cells can provide important basis for elucidating the biological behaviors of stem cells in vivo, such as the colonization, distribution, migration and differentiation of stem cells, and are of great significance to the research of stem cell transplantation. The purpose of this study was to explore the use of fluorescent dye PKH26, nucleic acid marker BrdU and human derived cell-specific recognition antibody to trace stem cells in vivo, and to analyze the advantages and disadvantages of the three methods. To provide the basis for studying the biological evaluation of stem cell transplantation in vivo. Methods: in the first part, the effect of fluorescent dye PKH26, nucleic acid labeled BrdU and three human derived cell-specific recognition antibodies on the labeling and detection of human dermal stem cells in vitro was observed. In the second part, the animal injury model of C57BL / 6J mice irradiated with different doses of whole body radiation was used as the experimental body. Human dermal stem cells labeled by different methods were transplanted with 2 脳 106 cells / tail vein, and the samples were obtained at different time points. The distribution of cells in tissues and organs was observed by ordinary optical microscope or fluorescence microscope. Results: the main results of this study are as follows: 1. Human dermal stem cells labeled with BrdU in vitro can be detected by anti-BrdU antibody in the lung of the tested mice after intravenous transplantation of human dermal stem cells; 2. Human dermal stem cells labeled by PKH26 in vitro could not be detected after intravenous transplantation of human dermal stem cells in each organ of the tested mice. 3. In this study, three commercial human cell-specific recognition antibodies, sc-80668 and Dako, M3527, could not specifically recognize human derived cells in vitro, among them, mouse anti-human 尾 -2-microglobulin antibody (sc-80668) and mouse anti-human endoglin antibody (Dako, M3527) could not specifically recognize human derived cells in vitro. Mouse anti-human nuclei antibody (MAB1281) was able to specifically recognize human derived cells in vitro, but human dermal stem cells were transplanted via vein to mice. The colonization of human dermal stem cells in various organs of the tested mice was not detected by mouse anti-human nuclei antibody. Conclusion: in this study, we used fluorescent dye PKH26, nucleic acid marker BrdU and three human cell specific antibodies to label stem cells in vitro and trace them in vivo. The advantages and disadvantages of three marking methods were compared. The main conclusions of this paper are as follows: 1. The nucleic acid marker BrdU has higher in vitro labeling efficiency and more stable in vivo tracer properties, and can be used as in vitro labeling and in vivo tracer of stem cell transplantation. 2. Fluorescent dye PKH26 has high labeling efficiency in vitro, but its resolution and signal intensity are not satisfactory when it is used for in vivo tracer of stem cells. 3. The detection of antibodies based on the specific recognition of human cells is largely limited to the quality and stability of antibodies in the process of studying the tracing of stem cells in vivo. No ideal results were obtained for mouse anti-human nuclei antibody (Millipore), mouse anti-human 尾 -2-microglobulin antibody (Santa Cruz) and mouse anti-human endoglin antibody (Dako).
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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