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  本文選題：標記 + 示蹤　； 參考：《第三軍醫(yī)大學》2012年碩士論文

【摘要】：研究背景： 干細胞具有自我更新和多向分化潛能的特性。利用干細胞移植進行疾病治療已成為干細胞研究的熱點。干細胞的標記和示蹤能夠為闡明干細胞在移植受體內(nèi)的定植、分布、遷移和分化等生物學行為提供重要依據(jù)，對干細胞移植研究具有重要意義。本課題擬探討利用熒光染料PKH26、核酸標記物BrdU以及人源性細胞特異性識別抗體等三種方法用于干細胞體內(nèi)示蹤，，分析三種方法的優(yōu)缺點，為研究干細胞在體移植后的生物學評價提供依據(jù)。 研究方法： 第一部分，以人真皮干細胞為研究對象，觀察熒光染料PKH26、核酸標記物BrdU以及三種人源性細胞特異性識別抗體來體外標記和檢測干細胞的效果。 第二部分，以不同劑量全身輻射C57BL/6J小鼠動物損傷模型作為受試體，以2×106個細胞/只尾靜脈移植經(jīng)不同方法標記的人真皮干細胞，于不同時間點取材，應用普通光學顯微鏡或熒光顯微鏡觀察各種方法檢測細胞在組織器官中的分布情況。 研究結(jié)果： 本研究主要結(jié)果如下： 1.經(jīng)BrdU體外標記的人真皮干細胞經(jīng)靜脈移植受試小鼠后，可通過抗BrdU抗體檢測到人真皮干細胞在受試小鼠肺內(nèi)的定植； 2.經(jīng)PKH26體外標記的人真皮干細胞經(jīng)靜脈移植受試小鼠后，未能檢測到人真皮干細胞在受試小鼠各個臟器的定植情況； 3.本研究中所嘗試的三種商品化的人源性細胞特異性識別抗體，其中小鼠抗人β-2-Microglobulin抗體(Santa Cruz, sc-80668)和小鼠抗人endoglin抗體(Dako, M3527)在體外均不能特異性識別人源性細胞。小鼠抗人nuclei抗體(Millipore, MAB1281)能夠在體外特異性識別人源性細胞，但人真皮干細胞經(jīng)靜脈移植受試小鼠后，通過小鼠抗人nuclei抗體未能檢測到人真皮干細胞在受試小鼠各個臟器的定植情況。 結(jié)論： 本課題研究中，我們通過利用熒光染料PKH26、核酸標記物BrdU以及三種人源性細胞特異性識別抗體來體外標記干細胞并進行體內(nèi)示蹤，比較了三種標記方法的優(yōu)缺點。本課題取得的主要研究結(jié)論包括： 1.核酸標記物BrdU具有較高的體外標記效率和較為穩(wěn)定的體內(nèi)示蹤特性，可作為干細胞體外標記及體內(nèi)示蹤方法應用于干細胞移植研究； 2.熒光染料PKH26具有較高的體外標記效率，但用于干細胞體內(nèi)示蹤時，其分辨率和信號強度等問題不夠理想； 3.基于人源性細胞特異性識別抗體的檢測方法在研究干細胞體內(nèi)示蹤的過程中很大程度上受限于抗體質(zhì)量和穩(wěn)定性的影響，本研究所采用的小鼠抗人nuclei抗體（Millipore公司）、小鼠抗人β-2-Microglobulin抗體（Santa Cruz公司）、小鼠抗人endoglin抗體（Dako公司）均未獲得理想的結(jié)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).
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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本文編號：2060687