發(fā)布時間：2018-03-03 18:43

  本文選題：CFSE　切入點：PI　出處：《吉林大學(xué)》2007年博士論文　論文類型：學(xué)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(biāo)記細(xì)胞后轉(zhuǎn)變成CFSE形式,隨細(xì)胞分裂被平均分配到兩個子代細(xì)胞中,其熒光強(qiáng)度是親代細(xì)胞的一半。這樣,在一個增殖的細(xì)胞群中,各連續(xù)幾代細(xì)胞的熒光強(qiáng)度以2倍遞減為特征,利用流式細(xì)胞術(shù)在FL1檢測通道進(jìn)行分析。 本實驗以CFDA-SE作為細(xì)胞標(biāo)記物,結(jié)合流式細(xì)胞術(shù)分析小鼠淋巴結(jié)、胸腺、脾淋巴細(xì)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(dǎo)的CIK細(xì)胞的增殖情況,從而對CFDA-SE檢測淋巴細(xì)胞增殖的意義進(jìn)行評價。結(jié)果證明:用流式細(xì)胞術(shù)通過分析CFSE熒光強(qiáng)度可以在單細(xì)胞水平上判斷細(xì)胞的分裂次數(shù),比較外周淋巴細(xì)胞、胸腺細(xì)胞、CIK細(xì)胞以及T、B細(xì)胞在不同刺激劑作用下的分裂增殖程度,并結(jié)合熒光標(biāo)記特異抗體可以進(jìn)一步分析淋巴細(xì)胞亞群的增殖。 以不同濃度的CFDA-SE標(biāo)記不同腫瘤細(xì)胞系,研究熒光強(qiáng)度變化的時間動力學(xué);研究CFDA-SE對細(xì)胞的毒性;選擇CFSE標(biāo)記細(xì)胞的最佳濃度、最佳標(biāo)記時間。通過實驗證明CFDA-SE標(biāo)記細(xì)胞4h后熒光強(qiáng)度趨于穩(wěn)定;CFDA-SE標(biāo)記細(xì)胞能力強(qiáng),不同細(xì)胞CFDA-SE最佳標(biāo)記濃度不同。CFDA-SE無毒性,不影響細(xì)胞活性。CFDA-SE標(biāo)記細(xì)胞8分鐘即可。這為其更好的應(yīng)用于檢測細(xì)胞毒實驗提供依據(jù)。 本實驗用CFDA-SE標(biāo)記靶細(xì)胞,以區(qū)分效應(yīng)細(xì)胞,再用DNA染料碘化丙啶PI標(biāo)記膜受損的靶細(xì)胞,用流式細(xì)胞術(shù)計數(shù)CFSE/PI雙陽性細(xì)胞,在單細(xì)胞水平上精確量化靶細(xì)胞死亡的百分率,從而判定人PBMC、CIK細(xì)胞、小鼠脾細(xì)胞三種效應(yīng)細(xì)胞的細(xì)胞毒活性;檢測CIK細(xì)胞殺傷不同腫瘤細(xì)胞及凋亡途徑;并與51Cr釋放法進(jìn)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(xì)胞毒檢測,其敏感性高于51Cr釋放法。結(jié)合熒光標(biāo)記特異抗體還可以多參數(shù)定性分析靶細(xì)胞。優(yōu)化了實驗參數(shù),用少量的效應(yīng)細(xì)胞,可以提供連續(xù)可比較的結(jié)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R392.12

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 牛青霞,趙承彥,靖志安;酶反應(yīng)比色法檢測細(xì)胞介導(dǎo)的細(xì)胞毒作用的原理與問題[J];癌變.畸變.突變;2000年04期

2 段秀梅;譚巖;宋燕;王曉祺;劉力華;方艷秋;許淑芬;;流式細(xì)胞術(shù)檢測CIK的免疫表型和體外殺傷活性[J];吉林大學(xué)學(xué)報(醫(yī)學(xué)版);2006年04期

3 劉蘋;李平;熊仁平;周元國;;MTT法與BrdU ELISA法檢測成纖維細(xì)胞增殖的可靠性比較[J];第三軍醫(yī)大學(xué)學(xué)報;2006年11期

4 李經(jīng)倫;~3H-TdR應(yīng)用研究現(xiàn)狀(文獻(xiàn)綜述)[J];放射免疫學(xué)雜志;2004年01期

5 陳必成,昌盛,杜敦峰,唐莉,張鑫,袁勁,周潔,陳忠華;羧基熒光素乙酰乙酸在移植免疫研究中的應(yīng)用[J];醫(yī)學(xué)研究生學(xué)報;2005年02期

6 肇靜嫻,曾耀英,何賢輝,王南,狄靜芳,曾山;活體染料CFDA-SE在淋巴細(xì)胞增殖研究中的應(yīng)用[J];細(xì)胞與分子免疫學(xué)雜志;2003年02期

7 韓作寧，劉白，，馬樹俊，陶家平，張志欣，志宏，龍振洲;PMA對人外周血T細(xì)胞和CD~（4＋）細(xì)胞表型影響的研究[J];中華微生物學(xué)和免疫學(xué)雜志;1994年03期

8 段秀梅;譚巖;方艷秋;楊廣民;王曉祺;劉曉琳;劉力華;許淑芬;;羧基熒光素琥珀酰亞胺酯標(biāo)記腫瘤細(xì)胞及其在細(xì)胞毒檢測中的價值[J];中華檢驗醫(yī)學(xué)雜志;2006年10期

9 王曉祺,譚巖,段秀梅,劉玲麗,方艷秋,姜艷芳,許淑芬,劉力華;流式細(xì)胞術(shù)檢測細(xì)胞毒方法的建立[J];中國免疫學(xué)雜志;2004年10期

本文編號：1562233