小鼠脾淋巴细胞对人HepG2细胞杀伤活性检测方法的建立
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摘要
目的在建立既能保留人肿瘤生物学特性的,且免疫功能相对正常的人肿瘤异种移植动物模型的过程中,为保证小鼠脾淋巴细胞对人肿瘤细胞杀伤作用的考察效率,摸索建立基于流式细胞术的评价小鼠脾淋巴细胞对人HepG2细胞杀伤活性的方法。方法用羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记人HepG2细胞,利用无水乙醇模拟杀伤人HepG2细胞,用碘化丙啶(PI)染色,流式细胞仪检测,确定CFSE和PI的浓度及作用时间。以小鼠脾细胞为效应细胞,人HepG2细胞为靶细胞,从效靶作用时间和效靶比方面进行优化,确定试验方法的最佳条件。结果采用CFSE/PI双标记将细胞分为CFSE~+PI-、CFSE~+PI~+、CFSE-PI~+和CFSE-PI-4个组群,可有效区分活细胞和被杀伤细胞。CFSE标记靶细胞的浓度采用2.5μmol/mL,效靶作用时间为12 h,效靶比10∶1。结论建立了基于流式细胞术的评价小鼠脾淋巴细胞对人HepG2细胞杀伤活性的方法,该方法的建立可为评价动物脾淋巴细胞对异种细胞的杀伤作用提供参考。
Objective To develop a method for evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells based on flow cytometry(FCM), in addition to ensure the efficiency of evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells in the process of establishing an animal model of human tumor xenotransplantation, which can preserve the biological characteristics of human tumors, meanwhile, has relatively normal immune function.Methods Human HepG2 cells were labeled with CFSE(carboxyfluorescein diacetate, succinimidyl ester) and treated with anhydrous ethanol to mimic the cytotoxic effect of cytotoxic killer cells, then determined by FCM after propidium iodide(PI) staining, based on which the concentrations of CFSE and PI and time for treatment were optimized. The lymphocytes were isolated from spleen of mice as effector cells and human HepG2 cells were target cells. A procedure was developed based on the optimization of conditions including the action time and ratio between effector and target cells. Results The cells were divided into CFSE~+PI-, CFSE~+PI~+, CFSE-PI~+ and CFSE-PI-groups by CFSE/PI staining, and the survival and killed cells were well distinguished. The concentration of CFSE was 2.5μmol/mL, the optimal action time between effector and target cells was 12 h, and the effector to target cell ratios of 10∶1 was adopted. Conclusions A method for evaluation of the killing activity of mice splenic lymphocytes to human HepG2 cells based on FCM was successfully developed, which provided a reference for evaluating the killing effect of animal splenic lymphocytes on xenogeneic cells.
Objective To develop a method for evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells based on flow cytometry(FCM), in addition to ensure the efficiency of evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells in the process of establishing an animal model of human tumor xenotransplantation, which can preserve the biological characteristics of human tumors, meanwhile, has relatively normal immune function.Methods Human HepG2 cells were labeled with CFSE(carboxyfluorescein diacetate, succinimidyl ester) and treated with anhydrous ethanol to mimic the cytotoxic effect of cytotoxic killer cells, then determined by FCM after propidium iodide(PI) staining, based on which the concentrations of CFSE and PI and time for treatment were optimized. The lymphocytes were isolated from spleen of mice as effector cells and human HepG2 cells were target cells. A procedure was developed based on the optimization of conditions including the action time and ratio between effector and target cells. Results The cells were divided into CFSE~+PI-, CFSE~+PI~+, CFSE-PI~+ and CFSE-PI-groups by CFSE/PI staining, and the survival and killed cells were well distinguished. The concentration of CFSE was 2.5μmol/mL, the optimal action time between effector and target cells was 12 h, and the effector to target cell ratios of 10∶1 was adopted. Conclusions A method for evaluation of the killing activity of mice splenic lymphocytes to human HepG2 cells based on FCM was successfully developed, which provided a reference for evaluating the killing effect of animal splenic lymphocytes on xenogeneic cells.
引文
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[10]李姗姗,舒昀,陈来,等.妊娠小鼠子宫内胎鼠腹腔注射人肿瘤HepG2细胞对胎鼠生长发育的影响[J].中国比较医学杂志,2018,28(7):33-42.
[2]Desmond-Hellman S,Rosen ST.China plans large center for PDX models[J].Cancer Discov,2014,4(2):136-137.
[3]Ada GL,Nossal G.The clonal-selection theory[J].Sci Am,1987,257(2):62-69.
[4]王甫厚,陈绍先,郭彩云,等.LDH释放法测NK细胞毒的方法学研究[J].中国免疫学杂志,1990(2):115-117.
[5]童骁,沈弢,梁华,等.利用流式细胞术测定CTL活性方法在EIAV免疫学中的应用[J].中国病毒学,2005.20(6):642-646.
[6]卫江波,徐然然,付志浩,等.疫苗诱导的特异性细胞杀伤活性检测方法的建立[J].中国生物制品学杂志,2012,25(12):1680-1683.
[7]包晶晶,林海霞,马璟.CFSE标记技术的研究进展[J].免疫学杂志,2010,5:461-464.
[8]Bocharov G,Luzyanina T,Cupovic J,et al.Asymmetry of cell division in CFSE-Based lymphocyte proliferation analysis[J].Front Immunol,2013,4:264.
[9]刘英杰,李景华.低浓度乙醇对人肝癌HepG2细胞的杀伤作用[J].解剖学报,2016,47(2):228-233.
[10]李姗姗,舒昀,陈来,等.妊娠小鼠子宫内胎鼠腹腔注射人肿瘤HepG2细胞对胎鼠生长发育的影响[J].中国比较医学杂志,2018,28(7):33-42.