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目的本实验室前期研究在锰染毒体内实验模型的大鼠脑脉络丛组织中鉴定到32个锰毒性相关的差异蛋白。本试验挑选出其中7个差异表达蛋白,包括抑制素蛋白(PHB1)电压阴离子通道(VDAC)、肌动蛋白β亚型(β-Actin)、应激性磷蛋白1(STI1)、热休克蛋白70(HSP70)、转甲状腺素蛋白(TTR)和中间丝波形蛋白(Vimentin),在大鼠永生化脉络丛上皮Z310细胞体外染锰模型中,对其蛋白和mRNA水平的变化趋势进行验证。方法氯化锰(0、50、100和200μmol/l)染毒Z310细胞24 h,采用蛋白印迹法(Western Blot)测定7个锰毒性相关蛋白的蛋白表达水平;同样剂量的氯化锰染毒Z310细胞12 h,以实时定量逆转录聚合酶链式反应法(Real time-RT PCR)测定7个蛋白在mRNA水平的表达情况。结果随着剂量的增加PHB1、β-Actin和STIP1在蛋白表达与mRNA水平均表现为上调的效应趋势,VDAC、HSP70、TTR和Vimentin在蛋白表达与mRNA水平均表现为下调的效应趋势;PHB1、β-actin、STIP1和TTR在Z310细胞中的表达效应趋势与体内动物模型脑脉络丛中锰毒性差异蛋白的变化趋势相符,而VDAC、HSP70和Vimentin在Z310细胞中的表达效应趋势则与之相反。结论氯化锰对PHB1、β-actin、STIP1和TTR的毒性效应在体内和体外是一致的,染锰后其蛋白表达和mRNA水平的变化趋势是可靠准确的。这为开展锰致脑脉络丛组织及其上皮细胞的毒性效应及其分子机制研究提供了有价值的线索。
PURPOSE: Our previous study identified 32 differential proteins associated with manganese toxicity in rat brain choroid plexus tissue in a manganese-exposed in vivo model. In this study, seven differentially expressed proteins were selected, including VDA, β-Actin, STI1, Hsp 70 HSP70, TTR and Vimentin were used to detect the changes of protein and mRNA levels in the immortalized choroid plexus Z310 cells in vitro. Methods Z310 cells were treated with manganese chloride (0, 50, 100 and 200 μmol / l) for 24 h. Western blotting was used to determine the protein expression levels of seven manganese-related proteins. The same dose of manganese chloride Z310 cells for 12 h, the expression levels of seven proteins at the mRNA level were determined by Real-time reverse transcription-polymerase chain reaction (Real time-RT PCR). Results With the increase of dose, the expression of PHB1, β-Actin and STIP1 showed up-regulation trend both in protein expression and mRNA level. VDAC, HSP70, TTR and Vimentin showed down-regulation trend both in protein expression and mRNA level. The trend of the expression of β-actin, STIP1 and TTR in Z310 cells was consistent with that of the manganese chondrocytes in vivo, while the expression of VDAC, HSP70 and Vimentin in Z310 cells was the opposite . Conclusion The toxic effects of manganese chloride on PHB1, β-actin, STIP1 and TTR are consistent in vivo and in vitro. The trends of the protein expression and mRNA level after manganese dying are reliable and accurate. This provides valuable clues for studying the toxic effect of manganese-induced choroid plexus and its epithelial cells and their molecular mechanisms.