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作者采用原位杂交、间接免疫荧光和免疫印染等技术,从细胞水平检测TNF、RNA和TNF蛋白,以研究TNF的基因在急性髓性白血病细胞中的表达。首先对早幼粒白血病细胞系HL-60进行了研究。用生物素标记的TNF、cDNA探针与HL-60细胞作原位杂交,然后再用碱性磷酸酶标法检测。发现只有在TPA诱导下向单核细胞方向分化的HL-60细胞,才能测出TNF的基因表达,阳性细胞可高达99%。用核糖核酸酶处理过的细胞及不能整合到染色体上的,用生物素标记的pBR322质粒处理的细胞作为阴性对照。用同样方法对9例AML病人进行了分析。结果是;9例病人中有8例的白血病细胞可测出TNF RNA,阳性细胞达89-98%。这些TNFRNA阳性的细胞用间接免疫荧光法检测其TNF蛋白也是阳性,说明AML病
The authors used in situ hybridization, indirect immunofluorescence, and immunoblotting techniques to detect TNF, RNA, and TNF proteins at the cellular level to study the expression of TNF genes in acute myeloid leukemia cells. First, the promyelocytic leukemia cell line HL-60 was studied. Biotin-labeled TNF, cDNA probes were used for in situ hybridization with HL-60 cells and then detected by alkaline phosphatase standard method. It was found that only HL-60 cells differentiated into mononuclear cells by TPA could detect TNF gene expression, and positive cells could be as high as 99%. Cells treated with ribonuclease and unable to integrate on chromosomes were treated with biotin-labeled pBR322 plasmid as a negative control. Nine cases of AML patients were analyzed by the same method. As a result, TNF RNA was detected in 8 out of 9 patients and the positive cells were 89-98%. These TNFRNA-positive cells were positive for TNF protein detected by indirect immunofluorescence, indicating AML disease.