CAJ | 학술논문

目的：对Sut黑色素细胞和野生型黑色素细胞进行高分辨率差异蛋白质组学分析，探讨xCT缺陷引起细胞生长抑制的机制。方法分离Sut黑色素细胞和野生型黑色素细胞总蛋白，进行双向电泳和串联质谱分析，获得数值经Mascot软件处理后通过NCBI及Swiss-Prot蛋白质数据库检索，结合双向凝胶电泳相应点的表观等电点、相对分子质量、匹配肽段的多少和序列覆盖率等综合分析筛选差异蛋白质；逆转录-PCR分析Sut细胞差异蛋白mRNA表达水平，用蛋白免疫印迹法对xCT缺陷的Sut黑色素细胞进行自体吞噬分析。结果与结论得到20个明显上调或下调的蛋白，经MALDI-TOF质谱分析鉴定差异蛋白质，发现与自体吞噬和囊泡运输相关的调控因子（ Anxa3、Hist 1h2bk、NDRG1、CaM）以及与细胞转移相关的调控因子（S100A-4、S100A-6、波形蛋白）表达发生了改变；对自噬标志蛋白分析发现，在xCT缺陷的Sut细胞中发生了自体吞噬。结果表明，xCT缺陷可能通过与细胞外基质的黏附异常以及激活Sut黑色素细胞中自噬相关的信号通路，从而导致细胞的生长抑制和自噬性死亡。
목적：대Sut흑색소세포화야생형흑색소세포진행고분변솔차이단백질조학분석，탐토xCT결함인기세포생장억제적궤제。방법분리Sut흑색소세포화야생형흑색소세포총단백，진행쌍향전영화천련질보분석，획득수치경Mascot연건처리후통과NCBI급Swiss-Prot단백질수거고검색，결합쌍향응효전영상응점적표관등전점、상대분자질량、필배태단적다소화서렬복개솔등종합분석사선차이단백질；역전록-PCR분석Sut세포차이단백mRNA표체수평，용단백면역인적법대xCT결함적Sut흑색소세포진행자체탄서분석。결과여결론득도20개명현상조혹하조적단백，경MALDI-TOF질보분석감정차이단백질，발현여자체탄서화낭포운수상관적조공인자（ Anxa3、Hist 1h2bk、NDRG1、CaM）이급여세포전이상관적조공인자（S100A-4、S100A-6、파형단백）표체발생료개변；대자서표지단백분석발현，재xCT결함적Sut세포중발생료자체탄서。결과표명，xCT결함가능통과여세포외기질적점부이상이급격활Sut흑색소세포중자서상관적신호통로，종이도치세포적생장억제화자서성사망。
Objective To identify the mechanisms underlying xCT deficiency by high-resolution proteomic analysis of differential protein expression in Sut and wild melanocytes .Methods The proteins,extracted from Sut and wild melano-cytes,were analyzed by two dimensional electrophoresis and PDQuest software .Subsequent MALDI-TOF mass spectrometry analysis was carried out .The protein identification was based on peptide mass fingerprint combined with the pI and the rela -tive molecular mass ( Mr) .Sequence coverage was performed with the Peptldent software on the NCBnlm website .Also,the autophagy marker protein LC3-Ⅱ, and the autophagic cell death marker protein Beclin 1 were detected by Western blotting . Results and Conclusion Twenty apparently upregulated or downregulated proteins were identified .Strikingly, important modifications in regulators of trafficking and organization of vesicles and autophagy ( Anxa3; Hist 1h2bk, NDRG1, and CaM) and in regulators of invasion and metastasis of carcinoma (S100A-4;S100A-6 and vimentin)are likely to account for dysfunctions in cell viability and cell-extracellular adhesion .These results indicate that the proteins regulating autophagy , vesicles trafficking and MAP kinase related pathways are activated and play a role in xCT-deficiency .