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背景:儿童急性白血病复发的主要根源是微小残留白血病细胞的存在,在试图清除微小残留病的众多治疗方法中,免疫生物治疗越来越受重视。以前的研究发现干扰素α-2b能有效抑制神经母细胞瘤和恶性淋巴瘤患儿体内肿瘤细胞的增长,其能否抑制白血病细胞的增长?目的:通过体外实验观察干扰素α-2b对白血病细胞的影响。设计:以人类早幼粒白血病细胞株HL-60为观察对象,对比观察实验。单位:青岛大学医学院附属医院儿科研究所细胞培养室、免疫实验室、细胞室。材料:HL-60细胞系由山东省医学科学院基础所提供。干扰素α-2b购自Megagene公司,FITC-兔抗鼠Ig工作液(CatEK001)和CD13抗人单克隆抗体工作液(Cat.DK013Y)购自协和干细胞基因工程有限公司。方法:体外建立HL-60细胞培养体系,调整细胞浓度为1×10~9 L~(-1),分为对照组和实验组,各实验组中加入终含量分别为5×10~5,1×10~6,2×10~6,5×10~6,1×10~7U/L的干扰素α-2b,对照组加入等量生理盐水,继续培养48 h。光镜下采用瑞氏染色观察HL60细胞的形态变化,采用吖啶橙/溴化乙锭(AO/EB)双染色观察细胞凋亡,通过CD13蛋白表达测定观察细胞膜抗原表达及成熟分化,通过MTT实验检测HL-60细胞增殖活性。主要观察指标:根据HL-60细胞核染色质是否均匀一致及着色情况,判断细胞是否发生凋亡;根据酶标仪检测的吸光度(A值)判断HL-60细胞增殖情况;根据CD13阳性细胞数量判断HL-60细胞成熟程度。结果:①HL-60细胞凋亡:培养48h,当干扰素α-2b为5×10~5U/L时,以早期凋亡细胞为主,当干扰素α-2b含量为1×10~7 U/L时,以晚期凋亡细胞为主,HL-60细胞凋亡率均高于对照组,差异有显著性意义(X~2= 23.79.29.57,P<0.01)。②HL-60细胞增殖情况:当干扰素α-2b含量为2×10~6 U/L和1×10~7 U/L时,A值均低于对照组,差异有显著性意义(t=5.65,11.31,P<0.01)。③HL-60细胞成熟程度:当干扰素α-2b含量为1×10~6U/L和1×10~7 U/L时,CD13~+细胞百分比均低于对照组,差异有显著性意义(x~2=6.89,14.73,P<0.01)。结论:干扰素α-2b有促进HL-60细胞凋亡、抑制HL-60细胞增殖和促进分化成熟的作用。
BACKGROUND: The primary source of childhood acute leukemia relapse is the presence of minimal residual leukemia cells. Immunobiological treatment is gaining more and more attention in numerous treatments that attempt to eradicate minimal residual disease. Previous studies found that interferon alpha-2b can effectively inhibit the growth of tumor cells in neuroblastoma and malignant lymphoma in vivo, and whether it can inhibit the growth of leukemia cells? Objective: To observe the effect of interferon alpha-2b on leukemia Effects of cells. Design: human promyelocytic leukemia cell line HL-60 as the observed object, the contrast observation experiment. Unit: Qingdao University Medical College Hospital Institute of Pediatrics cell culture room, immune laboratory, cell room. Materials: The HL-60 cell line was provided by Shandong Academy of Medical Sciences. Interferon alpha-2b was purchased from Megagene, Inc. FITC-rabbit anti-mouse Ig working solution (CatEK001) and CD13 anti-human monoclonal antibody working solution (Cat.DK013Y) were purchased from Pegasys Stem Cell Genetic Engineering Co., Methods: The HL-60 cell culture system was established in vitro and the cell concentration was adjusted to 1 × 10 ~ 9 L ~ (-1). The cells were divided into control group and experimental group. The final contents of each group were 5 × 10 ~ 5, 1 × 10 ~ 6, 2 × 10 ~ 6, 5 × 10 ~ 6, 1 × 10 ~ 7U / L interferon α-2b, the control group by adding an equal amount of saline, and cultured for 48 h. The morphological changes of HL60 cells were observed under Wright’s staining under light microscope. The apoptosis of HL60 cells was observed by acridine orange / ethidium bromide (EB) staining. The expression of CD13 protein and the differentiation of mature HL60 cells were observed by MTT The proliferation of HL-60 cells was tested by experiments. MAIN OUTCOME MEASURES: The apoptosis of HL-60 cells was judged according to whether the chromatin of HL-60 cells were uniform or not. The proliferation of HL-60 cells was judged by the absorbance (A value) detected by microplate reader. The number of CD13 positive cells HL-60 cell maturity. Results: ①HL-60cell apoptosis: 48h, when the interferon α-2b is 5 × 10 ~ 5U / L, mainly the early apoptotic cells, when the interferon α-2b content of 1 × 10 ~ 7U / L, the apoptotic rate of HL-60 cells was higher than that of the control group, the difference was significant (X ~ 2 = 23.79.29.57, P <0.01). ② The proliferation of HL-60 cells: when the content of interferon α-2b was 2 × 10 ~ 6 U / L and 1 × 10 ~ 7 U / L, the value of A was lower than the control group, the difference was significant (t = 5.65, 11.31, P <0.01). (3) The degree of maturation of HL-60 cells: The percentages of CD13 + cells were lower than that of control group when the content of interferon α-2b was 1 × 10-6 U / L and 1 × 10-7 U / L, the difference was significant x ~ 2 = 6.89,14.73, P <0.01). Conclusion: Interferon α-2b can promote HL-60 cell apoptosis, inhibit HL-60 cell proliferation and promote differentiation and maturation.