【摘要】 正常成熟外周血粒细胞提取物含有多种抑制因子(granulocyte-derived inhi-bitor,简称G-INH)。G-INH抑制骨髓CFU-GM及白血病细胞系(HL-60、K562、CEM、Raji-Li和Molt4)体外集落生长。形态学及DNA电泳技术证实:G-INH亦可诱导正常骨髓细胞、外周血粒细胞、白血病细胞系HL-60、K562和CEM细胞进入程序性死亡,但Molt4(一种T细胞白血病细胞系)则例外。集落抑制活性存在于分子量<3kD、3-100kD以及>100kD各组分中;而细胞程序性死亡诱导活性则存在于>3kD组分,主要存在于分子量>100kD组分。生物活性及ELISA检测提示:G-INH不含有TNF-α、IFN-α、GM-CSF、IL-3、乳铁蛋白以及前列腺素活性;热处理60℃,30min不影响活性。癌基因表达水平检测表明:G-INH显著降低HL-60细胞内bcl-2表达而p53无显著改变;同样实验条件下,CEM细胞并未显示bcl-2表达差异而表现为细胞内p53表达明显提高。提示这些癌基因在细胞程序性死亡之机理中具有重要调控作用,推测这些细胞死亡与基因调控有关。正常骨髓和外周血细胞并无bcl-2及p53阳性表达。G-INH各成分尚在分离纯化中。更多还原

【Abstract】 Growth inhibitors produced by mature cells are important negative regulators in maintaining hematopoietic homeostasis. An granulocytes-derived inhibitor(G-INH) obtained from normal people contains substance that suppressed in vitro colony growth of normal human marrow progenitors and leukemia cell lines. The effects of G-INH on human marrow and leukemia (K562, HL-60, CEM, Molt4, and Raji-Li) were tested in semi-solid culture and dose-dependent inhibition was indicated. Molt4 was exquisitely sensitive to G-INH while K562, CEM and Raji-Li cells showed less sensitive. HL-60 cells were intermediate in sensitivity. Effect of G-INH on cultured HL-60 cells and ficoll-separated bone marrow cells indicated that colony-suppression activity present in <3kD, >3kD-100kD and > 100kD fractions but the activity triggering the cells undergoing apoptosis was only in the > 100kD fraction. Apoptosis in leukemia cell line HL-60, K562, and CEM caused by G-INH was identified by both low MW DNA fragments on agarose gel and the appearance of typical morphological changes. 21.3%, 16.4% and 14.5% cells in apoptosis was noted in HL-60, K562 and CEM cells, respectively, while 5.6-6.2% in controls within 3 hours of exposure to G-INH but not in Molt4. Normal marrow cells failed to show apoptosis at equivalent concentration and exposure period until extending of exposure time to 24 hours. G-INH heated at 60℃ for 30 min did not influence the apoptosis inducing activity. ELISA assays of G-INH were undetectable for TNF-α, IL-3 and GM-CSF. Blocking antibodies to INF-α did not decrease inhibition produced by G-INH. The > 100kD fraction was a potent inducer of apoptosis. Expression of bcl-2 and p53 were detected by APAAP immunological assay. After exposed 30 minutes with G-INH, down regulated bcl-2’expression was seen in HL-60; Under the same condition, p53 overexpression was found in CEM cells. It suggests heterogeneous oncogenes response interfering into apoptosis course in different cell lines. G-INH promotes these leukemia cells undergoing apoptosis and inhibits their growth. However, normal marrow cells are not as sensitive as the leukemia cells. These results indicate an effect of G-INH which possibly confers on the leukemia cells a selective growth disruption but not the normal marrow cells.更多还原