本文選題：CFSE　切入點：PI　出處：《吉林大學》2007年博士論文　論文類型：學位論文

【摘要】： 羧基熒光素二乙酸鹽琥珀酰亞胺酯(CFDA-SE)是一種膜通透性的熒光素染料, CFDA-SE標記細胞后轉變成CFSE形式,隨細胞分裂被平均分配到兩個子代細胞中,其熒光強度是親代細胞的一半。這樣,在一個增殖的細胞群中,各連續(xù)幾代細胞的熒光強度以2倍遞減為特征,利用流式細胞術在FL1檢測通道進行分析。 本實驗以CFDA-SE作為細胞標記物,結合流式細胞術分析小鼠淋巴結、胸腺、脾淋巴細胞,在不同多克隆刺激劑作用下的增殖分裂;分析PHA刺激的人PBMC、誘導的CIK細胞的增殖情況,從而對CFDA-SE檢測淋巴細胞增殖的意義進行評價。結果證明:用流式細胞術通過分析CFSE熒光強度可以在單細胞水平上判斷細胞的分裂次數,比較外周淋巴細胞、胸腺細胞、CIK細胞以及T、B細胞在不同刺激劑作用下的分裂增殖程度,并結合熒光標記特異抗體可以進一步分析淋巴細胞亞群的增殖。 以不同濃度的CFDA-SE標記不同腫瘤細胞系,研究熒光強度變化的時間動力學;研究CFDA-SE對細胞的毒性;選擇CFSE標記細胞的最佳濃度、最佳標記時間。通過實驗證明CFDA-SE標記細胞4h后熒光強度趨于穩(wěn)定;CFDA-SE標記細胞能力強,不同細胞CFDA-SE最佳標記濃度不同。CFDA-SE無毒性,不影響細胞活性。CFDA-SE標記細胞8分鐘即可。這為其更好的應用于檢測細胞毒實驗提供依據。 本實驗用CFDA-SE標記靶細胞,以區(qū)分效應細胞,再用DNA染料碘化丙啶PI標記膜受損的靶細胞,用流式細胞術計數CFSE/PI雙陽性細胞,在單細胞水平上精確量化靶細胞死亡的百分率,從而判定人PBMC、CIK細胞、小鼠脾細胞三種效應細胞的細胞毒活性;檢測CIK細胞殺傷不同腫瘤細胞及凋亡途徑;并與51Cr釋放法進行比較,證實本方法適用于低效靶比和共孵育時間較短的細胞毒檢測,其敏感性高于51Cr釋放法。結合熒光標記特異抗體還可以多參數定性分析靶細胞。優(yōu)化了實驗參數,用少量的效應細胞,可以提供連續(xù)可比較的結果。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392.12

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