重组人肿瘤坏死因子-α体内外对HL-60细胞作用的研究

Effects of Recombinant Human Tumor Necrosis Factor-alpha on HL-60 Cells In Vitro and In Vivo

为了研究重组人肿瘤坏死因子-α(recombinanthumantumornecrosisfactor-alpha,rhTNF-α)在体外及体内对人髓系白血病细胞株HL-60细胞的作用,应用MTT法和集落形成试验测定HL-60细胞的增殖抑制,采用AO/EB荧光染色法、流式细胞术和TdT酶介导的缺口末端标记(TUNEL)法检测凋亡细胞;利用HL-60细胞裸鼠异种移植瘤模型观察给药后瘤体重量、组织病理的改变,并采用透射电镜以及TUNEL法检测瘤组织细胞的凋亡。结果表明:rhTNF-α对HL-60细胞的增殖具有明显的抑制作用,呈时间和浓度依赖性;AO/EB染色后可见rhTNF-α处理组HL-60细胞核内染色质浓缩聚集或成碎片,呈现凋亡表象;流式细胞术显示随着rhTNF-α浓度的增加,细胞凋亡阳性率逐渐增高;TUNEL法检测显示,3200U/mlrhTNF-α作用于HL-60细胞48小时凋亡阳性率可达37.5%。在体内,rhTNF-α可抑制HL-60细胞裸鼠异种移植瘤生长,抑制率最高可达60.33%;病理检查发现,rhTNF-α处理组的瘤组织有程度不等的出血坏死区域;透射电镜及TUNEL法检测表明,处理组的瘤组织可见有较多凋亡细胞。结论:rhTNF-α在体内、外均可抑制HL-60细胞增殖,并诱导其凋亡。

To study the effects of recombinant human tumor necrosis factor-alpha （rhTNF-α） on HL-60 cells in vitro and in vivo, MTT and colony forming assay were used to examine the effects of rhTNF-α on proliferation of HL-60 cells; AO/EB （acridine orange-ethidium bromide） staining, Annexin-V flow cytometry analysis and TUNEL assay were used to detect apoptotic cells. The effect of rhTNF-α on xenograft growth of HL-60 cells was evaluated by tumor inhibition rate, histology, ultrastructure and TUNEL assay. The results showed that rhTNF-α inhibited the proliferation of HL-60 cells in a dose-dependent manner. Staining of cells with AO/EB revealed that rhTNF-α induced nuclear chromatin condensation and fragmentation . Positive Annexin V-FITC on cell membrane showed that rhTNF-α induced apoptosis of HL-60 cells in a dose-dependent manner. TUNEL assay showed that the apoptotic percentage of HL-60 cells reached 37. 5% when incubated with 3200 U/ml rhTNF-α for 48 hours. In vivo rhTNF-α inhibited xenograft growth of HL-60 cells with the highest inhibition rate of 60.33%. Pathologically it was found that there were necrotic areas in the tumors of groups treated with rhTNF-α. There were more apoptotic cells in treatment groups than in that control group by transmission electron microscopy （TEM） and TUNEL assay. It is concluded that rhTNF-α is able to inhibit the proliferation of HL-60 cells and to induce apoptosis of HL-60 cells in vitro and in vivo.