伪狂犬病病毒Ea株诱导牛肾细胞凋亡及核基质蛋白的变化

目的研究伪狂犬病病毒(PrV)是否具有诱导组织培养细胞发生细胞凋亡的功能及探讨凋亡细胞核基质蛋白表达的变化。方法利用锥虫蓝(台盼蓝)染色绘制PrV在不同细胞上的增殖曲线；荧光染色观察凋亡细胞核的形态特征；琼脂糖凝胶电泳分析细胞染色体DNA的片段化；高分辨率双向电泳分析细胞凋亡前后核基质蛋白的表达差异。结果膜通透性的DNA特异性结合染料Hoechst 33342染色显示，PrV感染后36h，牛肾(MDBK)细胞核染色质开始固缩、凝聚，细胞核碎裂；DNA提取及琼脂糖凝胶电泳分析表明，细胞染色体DNA发生片段化，形成“DNA梯状”条带(DNA ladder)；尽管PrV可以诱导MDBK细胞发生细胞凋亡，但不能诱导IBRS-2，BHK-21及PK-15细胞发生凋亡。MDBK细胞发生凋亡前后核基质蛋白的表达发生改变，存在表达上调、下调、诱导表达及表达遏制等情况。结论PrV诱导细胞发生细胞凋亡是其致细胞死亡的形式之一，间接反映了PrV与宿主细胞间复杂的相互作用。细胞凋亡前后核基质蛋白表达存在的差异说明PrV致MDBK细胞发生凋亡是自身基因产物或诱导或抑制宿主细胞基因表达的结果。

INDUCTION OF APOPTOSIS BY PSEUDORABIES VIRUS Ea STRAIN IN BOVINE KIDNEY(MDBK) CELLS AND THE ALTERATION OF NUCLEAR MATRIX PROTEIN

Objective In order to observe the phenomena of apoptosis induced by PrV-Ea strain in tissue cultured cells and the altered expression of nuclear matrix proteins when cells took on the apoptotic character. Methods PrV progration in different cells was determined by typan blue staining. Apoptotic nuclear morphological change was observed with fluorescent microscopy by fluorescent staining Hoechst 33342 and oligonucleosomal ladders were detected in a 1.5% agarose gel. The alteration of nuclear matrix proteins was analyzed in MDBK cells induced apoptosis by PrV with 2-dimensional gel electrophoresis. Results PrV could induce MDBK cells apoptosis 36?h post-infection while it couldn't induce other several cells, PK-15, BHK-21 and IBRS-2, apoptosis. The expression of nuclear matrix proteins was changed when MDBK cells were induced apoptosis compared with the mock-infected cells. Some nuclear matrix proteins (NMPs) were significantly up-regulated and/or down-regulated. Moreover, some proteins were appeared only when cells took on apoptosis even though some of NMPs were inhibited. Conclusion Apoptosis was only one form of PrV to make cells to cytopathic effect (CPE), and these phenomena indicated that the interaction between PrV and host cells was very complex. The altered expression of NMPs suggested that apoptosis resulted from the regulation of host cell's and/or PrV' gene and nuclear matrix played an important role on the interaction of PrV infection and host ce1ls.