【摘要】 建立悬浮培养细胞胞质体制备及鉴定方法,为细胞重构奠定基础。采用密度梯度离心法和低速离心法纯化人白血病HL-60细胞,在单独细胞松弛素B(CB)和联合秋水酰碱介导下,分别于34℃和25℃,50%Percoll等密度梯度离心对HL-60细胞进行脱核,然后分别用38%和40%Percoll密度梯度离心纯化胞质体;采用Wright-Giem-sa染色和4,6-联脒-2-苯基吲哚二盐酸盐(DAPI)/羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)荧光染料双染色观察胞质体形态学变化;采用流式细胞术(FCM)检测胞质体表型和线粒体膜电位(MMP)以评价其活性。结果显示,CB联合秋水酰碱介导HL-60细胞脱核率为91.98%±4.29%,明显高于单独CB组(74.95%±3.02%)(P<0.01);34℃组的脱核率和直径≥5μm胞质体比例均明显高于25℃组(P<0.01,P<0.05);38%Percoll密度梯度离心纯化胞质体纯度高于40%Percoll组;纯化的HL-60胞质体表型无明显变化,12h内其活性达80%以上。结果表明,CB联合秋水酰碱介导下50%Percoll密度梯度离心脱核、38%Percoll密度梯度离心纯化、DAPI/CFSE双染鉴定、MMP检测是制备和鉴定悬浮培养细胞胞质体的适宜方案。更多还原

【Abstract】 This experimental research was aimed to establish an optimum system of enucleation,purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination.Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min,respectively.The purified HL-60 cells were treated with cytochalasin B(CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25℃ and 34℃,respectively.Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%,38% and 40% Percoll,respectively.The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride(DAPI)/5,6-carboxyflu-orescein diacetate succinimidyl ester(CFSE) double-staining.The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry.The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91.98%±4.29%,which was significantly higher than that in CB alone group(74.95%±3.02%)(P<0.01).The rates of enucleation and cytoplast with diameter over 5μm in 34℃ group were higher than those in 25℃ group(all P<0.01).The cytoplast purities were(95.43±0.59)% in 38% Percoll groups,which were higher than those of 40% Percoll(P<0.05).Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling.The results further showed that the phenotype of HL-60 cytoplasts had no significant change,and the activity of the cytoplasts was above 80% within 12h.It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine,38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.更多还原