EXPRESSION AND FUNCTION OF CATHEPSIN E IN ACTIVATED MICROGLIA FOLLOWING FACIAL NERVE INJURY

组织蛋白E(Cthepsin E，CE)是一种细胞内的天冬氨酸氨基转移酶，在小胶质细胞激活后，主要以成熟酶的形式存在于内吞体样结构中，但其在激活的小胶质细胞中的功能目前尚不清楚。在本研究中，我们用天冬氨酸氨基转移酶抑制剂胃抑素A来研究CE在小胶质细胞中的可能作用。当原代培养的大鼠小胶质细胞被胃抑素A处理时，小胶质细胞的数量以时间和剂量依赖的方式增加。同时观察到变形体和双极形的小胶质细胞转变为杆状细胞。胃抑素A也以剂量依赖的方式增加对溴脱氧尿苷的摄取。在第二系列实验中，我们检测了激活的小胶质细胞对神经元存活的影响，因为激活的小胶质细胞与r-…有关。对神经细胞死亡的更多监管。用干扰素-γ/脂多糖处理的小胶质细胞与神经元型PC12细胞共同培养时，神经元型PC12细胞发生凋亡，并伴有caspase-3样蛋白酶活性和DNA片段化。用Caspas-3样蛋白酶抑制剂N-乙酰-Asp-Glu-Val-Asp醛预处理后，虽然细胞内的Caspase-3样蛋白酶活性被完全抑制，但并不能逆转这种细胞死亡。这表明，抑制caspase-3样蛋白酶活性不足以抑制激活的小胶质细胞诱导的神经元死亡。虽然在神经细胞中过表达Bcl2可明显抑制caspase-3样蛋白酶活性和DNA片段化，但不能抑制激活的小胶质细胞诱导的神经元死亡。在电子显微镜下，高表达Bcl2的变性神经元PCl2细胞的特点是染色质轻微凝集形成不规则形状的团块，核周严重解体，空泡化明显。这些结果表明，激活的小胶质细胞在过度表达BCI-2的神经细胞中诱导非凋亡性细胞死亡。总之，我们的研究为激活的小胶质细胞通过阻断caspase-3蛋白酶级联而导致神经元死亡提供了另一种死亡途径，可能导致坏死性死亡。较少

Although cathepsin E (CE), an intracellular aspartic proteinase, is existed predominantly as the mature enzyme in the endosome-like structures following cellular activation of microglia, functions of CE in activated microglia are currently unknown. In the present study, we have initiated to investigate possible functions of CE in microglia by using the aspartic proteinases inhibitor pepstatin A.When primaiy cultured rat microglia were treated with pepstatin A, the number of microglia was increased in both time- and dose-dependent manners. At the same time, it was noted that microglia with ameboid and bipolar-shape was changed into rod-like cells. The uptake of bromodeoxyuridine was also increased by pepstatin A in a dose-dependent manner. These effects of pepstatin A on microglia were significantly suppressed by staurosporine.In the second series of experiments, we have examined effects of activated microglia on neuronal survival, since activated microglia have been implicated in the r … More egulation of neuronal cell death. When neuronal PC12 cells were cocultured with microglia that were treated with interferon-gamma /lipopolysaccharide, neuronal PC12 cells caused apoptosis accompanied by caspase-3-like protease activation and DNA fragmentation. Pretreatment with the caspas-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde did not reverse this cell death, although it resulted in complete inhibition of the caspase-3-like protease activity in the cells. This suggests that the inhibition of caspase-3-like protease activity is not sufficient to inhibit the activated microglia-induced neuronal death. Although Bcl-2 overexpression in the neuronal cells caused the apparent inhibition of caspase-3-like protease activity and DNA fragmentation, it could not suppress the activated microglia-induced neuronal death. At electronmicroscopic level, the degenerating neuronal PCl2 cells with high levels of Bcl-2 were characterized by slightly condensed chromatins forming irregular shaped masses, severely disintegrated perikarya, and marked vacuolation. These results suggest that activated microglia induce non- apoptotic cell death in the neuronal cells overexpressing BcI-2. Altogether, our study provides an alternative death pathway for the activated microglia-induced neuronal death by blockage of the caspase-3 protease cascade, probably leading to necrotic death. Less