荧光染料CFDA-SE标记在检测淋巴细胞增殖及细胞介导的细胞毒活性中的应用研究

发布时间：2018-03-03 18:43

本文选题：CFSE　切入点：PI　出处：《吉林大学》2007年博士论文　论文类型：学位论文

【摘要】： 羧基荧光素二乙酸盐琥珀酰亚胺酯(CFDA-SE)是一种膜通透性的荧光素染料, CFDA-SE标记细胞后转变成CFSE形式,随细胞分裂被平均分配到两个子代细胞中,其荧光强度是亲代细胞的一半。这样,在一个增殖的细胞群中,各连续几代细胞的荧光强度以2倍递减为特征,利用流式细胞术在FL1检测通道进行分析。本实验以CFDA-SE作为细胞标记物,结合流式细胞术分析小鼠淋巴结、胸腺、脾淋巴细胞,在不同多克隆刺激剂作用下的增殖分裂;分析PHA刺激的人PBMC、诱导的CIK细胞的增殖情况,从而对CFDA-SE检测淋巴细胞增殖的意义进行评价。结果证明:用流式细胞术通过分析CFSE荧光强度可以在单细胞水平上判断细胞的分裂次数,比较外周淋巴细胞、胸腺细胞、CIK细胞以及T、B细胞在不同刺激剂作用下的分裂增殖程度,并结合荧光标记特异抗体可以进一步分析淋巴细胞亚群的增殖。以不同浓度的CFDA-SE标记不同肿瘤细胞系,研究荧光强度变化的时间动力学;研究CFDA-SE对细胞的毒性;选择CFSE标记细胞的最佳浓度、最佳标记时间。通过实验证明CFDA-SE标记细胞4h后荧光强度趋于稳定;CFDA-SE标记细胞能力强,不同细胞CFDA-SE最佳标记浓度不同。CFDA-SE无毒性,不影响细胞活性。CFDA-SE标记细胞8分钟即可。这为其更好的应用于检测细胞毒实验提供依据。本实验用CFDA-SE标记靶细胞,以区分效应细胞,再用DNA染料碘化丙啶PI标记膜受损的靶细胞,用流式细胞术计数CFSE/PI双阳性细胞,在单细胞水平上精确量化靶细胞死亡的百分率,从而判定人PBMC、CIK细胞、小鼠脾细胞三种效应细胞的细胞毒活性;检测CIK细胞杀伤不同肿瘤细胞及凋亡途径;并与51Cr释放法进行比较,证实本方法适用于低效靶比和共孵育时间较短的细胞毒检测,其敏感性高于51Cr释放法。结合荧光标记特异抗体还可以多参数定性分析靶细胞。优化了实验参数,用少量的效应细胞,可以提供连续可比较的结果。
[Abstract]:Carboxyl fluorescein diacetate succinimide (CFDA-SE) is a membrane permeable fluorescein dye, which is labeled with CFDA-SE and transformed into CFSE form, which is evenly distributed to two progenies with cell division. The fluorescence intensity of the cells was half that of the parental cells. In a proliferating cell group, the fluorescence intensity of each successive generation of cells was reduced by 2 times, and the flow cytometry was used to analyze the FL1 detection channel. In this study, CFDA-SE was used as a cell marker and flow cytometry was used to analyze the proliferation and division of mouse lymph nodes, thymus and spleen lymphocytes under the action of different polyclonal stimulators, and to analyze the proliferation of CIK cells induced by PHA. The results showed that flow cytometry could be used to determine the number of cell division at the single cell level by analyzing the fluorescence intensity of CFSE, and the peripheral lymphocytes could be compared by flow cytometry. The mitotic and proliferative degree of thymocytes CIK cells and Thunb cells under different stimulators, combined with fluorescent labeled specific antibodies, can be used to further analyze the proliferation of lymphocyte subsets. Different tumor cell lines were labeled with different concentrations of CFDA-SE to study the time-dynamics of fluorescence intensity changes, to study the cytotoxicity of CFDA-SE, and to select the best concentration of CFSE labeled cells. The best labeling time. The results showed that the fluorescence intensity of CFDA-SE labeled cells tended to be stable after 4 hours. The best concentration of CFDA-SE in different cells was not toxic, but the ability of CFDA-SE labeled cells was stronger than that of CFDA-SE cells. CFDA-SE labeled cells did not affect cell activity for 8 minutes, which provided a basis for its better application in the detection of cytotoxicity. In this study, CFDA-SE was used to label target cells to distinguish effector cells, then DNA dye was used to label target cells with Pi membrane damage. Flow cytometry was used to count CFSE/PI double positive cells, and to accurately quantify the percentage of target cell death at single cell level. The cytotoxic activity of three effector cells of human PBMC-CIK cells and mouse spleen cells were determined, the cytotoxicity of CIK cells to different tumor cells and apoptotic pathways were detected, and the results were compared with 51Cr release method. It is proved that this method is suitable for the detection of cytotoxicity with low target ratio and short incubation time, and its sensitivity is higher than that of 51Cr release method. Combined with fluorescent labeled specific antibody, the target cells can be qualitatively analyzed with multiple parameters. The experimental parameters are optimized. With a small number of effector cells, continuous comparable results can be provided.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392.12

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