论文部分内容阅读
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。
To establish a method for the preparation and identification of the cytoplasm in suspension culture cells and lay the foundation for cell remodeling. Human leukemia HL-60 cells were purified by density gradient centrifugation and low-speed centrifugation. The cells were centrifuged at 50 ℃ and 25 ℃ in density gradient centrifugation at 34 ℃ and 25 ℃ with cytochalasin B (CB) alone and combined with colchicine HL-60 cells were de-nucleated and then purified by centrifugation using 38% and 40% Percoll gradient centrifugation respectively. The Wright-Giem-sa staining and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) / carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to observe the morphological changes of the cytoplasm. Fluorescence cytometry (FCM) was used to detect the cytoplasmic phenotype and mitochondrial membrane potential (MMP) to evaluate its activity. The results showed that CB combined with colchicine-induced HL-60 cell de-nucleation rate was 91.98% ± 4.29%, significantly higher than the single CB group (74.95% ± 3.02%) (P <0.01) (P <0.01, P <0.05). The purity of cytoplasm in 38% Percoll density gradient centrifugation was higher than that in 40% Percoll group. The purified HL-60 cytoplasm No significant changes in body surface, within 12h of its activity up to 80%. The results showed that CB combined with colchicine-induced 50% Percoll density gradient centrifugation, 38% Percoll density gradient centrifugation, DAPI / CFSE double staining, and MMP detection were suitable methods for preparing and identifying the cytoplasm of suspension cultured cells .