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目的:采用蛋白质组学技术检测LIM矿化蛋白-1(LIM mineralization protein-1,LMP-1)转染人胎盘源间充质干细胞(Human placenta-derived mesenchymal stem cells,hPDMSCs)前后蛋白质组学改变。方法:提取hPDMSCs LMP-1和hPDMSCsvector细胞总蛋白,通过二维凝胶电泳(two-dimensional gel electrophoresis,2-DE)制备两组细胞的二维电泳图谱,并用PDQuest软件进行图像分析,寻找差异表达的蛋白质点,并用基质辅助激光解吸粒子-飞行时间电离质谱(matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry,MALDI-TOF-MS)分析技术对这些差异表达的蛋白质点进行鉴定,并选取6个与成骨相关的蛋白(PCNA,vimentin,annexin A1,annexin A2,eEF2和peroxiredoxin-1)作为验证对象,蛋白质印迹法检测各蛋白在两组细胞中的表达水平。结果:采用2-DE技术建立了hPDMSCsLMP-1和hPDMSCsvector两组细胞的二维电泳图谱;PDQuest软件分析图像发现两组细胞有22个差异蛋白质点,采用MALDI-TOF-MS质谱初步鉴定出15个蛋白质点,其中表达上调9个,下调6个。Western blot检测结果与蛋白质组学一致。结论:LMP-1基因转染hPDMSCs后共筛选获得15个差异表达的蛋白,它们是成骨分化潜在的重要参与者,为进一步阐明LMP-1对hPDMSCs细胞功能和成骨分化影响的分子机制奠定基础。
OBJECTIVE: To study the proteomic changes of human placenta-derived mesenchymal stem cells (hPDMSCs) transfected with LIM mineralization protein-1 (LMP-1) using proteomic techniques . Methods: The total proteins of hMPD and hPDMSCsvector cells were extracted and two-dimensional gel electrophoresis (2D-gel electrophoresis, 2-DE) was used to prepare two-dimensional gel electrophoresis profiles. The PDQuest software was used for image analysis to find differential expression The protein spots were identified and the differentially expressed protein spots were identified using matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) Six osteoblast-associated proteins (PCNA, vimentin, annexin A1, annexin A2, eEF2 and peroxiredoxin-1) were selected as the validation subjects. The expression of each protein in the two groups of cells was detected by Western blotting. Results: Two-dimensional electrophoresis patterns of hPDMSCs, LMP-1 and hPDMSCsvector cells were established by 2-DE technique. PDQuest software showed that there were 22 differential protein spots in two groups of cells, and MALDI-TOF- Protein spots, of which 9 were up-regulated and 6 down-regulated. Western blot results are consistent with proteomics. CONCLUSION: Fifteen differentially expressed proteins were screened after hPDMSCs were transfected with LMP-1 gene, which are potentially important players in osteogenic differentiation. The molecular mechanism of LMP-1 on the function and osteogenic differentiation of hPDMSCs was established basis.