蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞的毒性

本研究旨在探讨一定理化条件下形成的蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞（SH-SY5Y）的毒性作用。采用高温（57±0.1）℃、pH 2.0甘氨酸溶液、振荡条件下孵育100 μmol·L^-1的蛋清溶菌酶蛋白，使其聚集成为蛋清溶菌酶淀粉样纤维；透射电子显微镜观察蛋清溶菌酶淀粉样纤维的超微结构，ANS荧光法测定纤维的疏水性，圆二色谱法测定并计算纤维的二级结构含量；将此纤维作用于培养的SH-SY5Y神经细胞，MTT法检测细胞活力，核染色法检测细胞凋亡状况。透射电镜结果显示，蛋清溶菌酶在孵育4 d后形成了呈短杆状超微结构的淀粉样纤维；与天然蛋清溶菌酶蛋白相比，纤维的疏水性和二级结构β折叠含量均显著提高；且1~3 μmol·L^-1纤维作用于SH-SY5Y细胞12、24和48 h均产生了显著的毒性作用（P<0.01），1、2和3 μmol·L^-1纤维作用细胞12 h时的细胞活力分别为75.16%±15.51%、67.54%±12.13%和67.89%±10.26%，作用24 h的细胞活力分别为75.78±13.01%、58.41%±5.55%和61.90%±8.94%，作用48 h的细胞活力分别为71.59%±14.75%、55.65%±5.78%和46.45%±6.23%，细胞活力降低呈现明显的浓度依赖性和时间依赖性，且细胞核染色呈现典型的核凋亡变化。结果提示，蛋清溶菌酶在一定理化条件下可形成具有神经细胞毒性的淀粉样纤维，为解释朊蛋白或其他蛋白质形成淀粉样纤维的机制提供参考。

The Cytotoxicity of Amyloid Fibrils Formed by Hen Egg-White Lysozyme on SH-SY5Y Cells

The aim of this study was to investigate the cytotoxicity effect of amyloid fibrils of hen egg-white lysozyme (HEWL) on SH-SY5Y cells. The 100 μmol·L^-1 HEWL protein was incubated with agitation at 57℃ in glycine solution of pH 2.0. The ultrastructure of HEWL amyloid fibrils was observed under transmission electron microscopy, the hydrophobicity was detected by ANS fluorescence, the secondary structural contents were measured and analyzed by circular dichroism. After SH-SY5Y cells were treated by the formed HEWL fibrils, the cell viability was determined by MTT assay and the apoptosis features were detected by nuclear staining. The transmission electron microscopy results showed that HEWL protein formed short and rod-shaped amyloid fibrils after 4 days of incubation. Meanwhile, compared with the native HEWL protein, the surface hydrophobicity and β-sheet structure of aggregates were both greatly increased. Furthermore, 1-3 μmol·L^-1 HEWL fibrils all produced significant cytotoxicity in SH-SY5Y cells after 12, 24 and 48 h treatments(P<0.01). For different concentrations of 1, 2 and 3 μmol·L^-1 HEWL fibrils, the cell viability were 75.16%±15.51%, 67.54%±12.13% and 67.89%±10.26% for 12 h treatment,respectively; 75.78±13.01%, 58.41%±5.55% and 61.90%±8.94% for 24 h treatment,respectively; 71.59%±14.75%, 55.65%±5.78% and 46.45%±6.23% for 48 h treatment,and the decreased cell viability displayed time-dependent and concentration-dependent manner. Nuclear staining results further confirmed the apoptosis features. These results suggest that HEWL protein could form into toxic amyloid fibrils under certain physicochemical conditions, which could provide basis for understanding the mechanisms of amyloid fibrils forming of prion protein and other amyloid proteins.