Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility
Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility
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DOI：
10.1016/s0016-5085(03)00664-4
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发表时间：
2003-07-01
期刊：
影响因子：
29.4
通讯作者：
Corinaldesi, R
中科院分区：
文献类型：
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作者：
De Giorgio, R;Bovara, M;Corinaldesi, R
背景与目的：自身免疫在副肿瘤性肠道动力障碍中的作用尚未确定。由于抗-Hu抗体可能损害肠神经元功能，我们测试了来自副肿瘤性肠动力障碍患者的抗-HuD阳性血清或商业抗-HuD抗体是否激活神经母细胞瘤细胞系和培养的肌间神经元中的凋亡级联。方法：采用免疫荧光和免疫印迹法对严重副肿瘤性肠动力障碍患者的抗HuD抗体进行表征。将SH-Sy 5 Y成神经细胞和培养的肌间神经元暴露于含有抗HuD抗体或2种市售抗HuD抗体的血清。采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记（TUNEL）技术检测细胞凋亡。免疫荧光法用于鉴定活化的caspase-3和apaf-1，沿着微管相关蛋白2。结果如下：在SH-Sy 5 Y细胞中，暴露于抗HuD阳性血清（32% +/-7%）或抗HuD抗体（23% +/-2%）后观察到的TUNEL阳性细胞核的百分比显著大于对照血清或胎牛血清（P < 0.001）。时程分析显示，与对照相比，在24、48和72小时，由2种商业抗HuD抗体诱发的凋亡神经母细胞瘤细胞的数量显著更大。暴露于抗HuD抗体的TUNEL阳性肌间神经元的数量（60% +/-14%）显著大于胎牛血清（7% +/-2%; P < 0.001）。Apaf-1和caspase-3免疫标记显示，与对照组相比，暴露于抗HuD阳性血清或商业抗HuD抗体的细胞比例显著更大，细胞质染色强烈。结论：抗HuD抗体诱发神经元凋亡，可能导致肠神经系统损害，潜在的副肿瘤性肠道动力障碍。Apaf-1的激活表明参与了一个依赖于细胞凋亡途径。
Background & Aims: The role of autoimmunity underlying paraneoplastic gut dysmotility remains unsettled. Because anti-Hu antibodies may impair enteric neuronal function, we tested whether anti-HuD-positive sera from patients with paraneoplastic gut dysmotility or commercial anti-HuD antibodies activated the apoptotic cascade in a neuroblastoma cell line and cultured myenteric neurons. Methods: Anti-HuD antibodies from patients with severe paraneoplastic gut dysmotility were characterized by immunofluorescence and immunoblot. SH-Sy5Y neuroblasts and cultured myenteric neurons were exposed to sera containing anti-HuD antibodies or 2 commercial anti-HuD antibodies. Cells were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique to evaluate apoptosis. Immunofluorescence was used to identify activated caspase-3 and apaf-1, along with microtubule-associated protein 2. Results: In SH-Sy5Y cells, the percentage of TUNEL-positive nuclei observed after exposure to anti-HuD-positive sera (32% +/- 7%) or anti-HuD antibodies (23% +/- 2%) was significantly greater than that of control sera or fetal calf serum (P < 0.001). The time-course analysis showed a significantly greater number of apoptotic neuroblastoma cells evoked by the 2 commercial anti-HuD antibodies at 24, 48, and 72 hours versus controls. The number of TUNEL-positive myenteric neurons exposed to anti-HuD antibodies (60% +/- 14%) was significantly greater than that of fetal calf serum (7% +/- 2%; P < 0.001). Apaf-1 and caspase-3 immunolabeling showed intense cytoplasmic staining in a significantly greater proportion of cells exposed to anti-HuD-positive sera or to commercial anti-HuD antibodies compared with controls. Conclusions: Anti-HuD antibodies evoked neuronal apoptosis that may contribute to enteric nervous system impairment underlying paraneoplastic gut dysmotility. Apaf-1 activation suggests participation of a mitochondria-dependent apoptotic pathway.