利用SELDI技术检测大鼠肝纤维化组织的实验研究

目的初步建立表面增强激光解吸离子化飞行时间质谱(SELDI-TOF/MS)技术检测大鼠肝纤维化组织样本的方法。方法采用SELDI技术及弱阳离子交换表面蛋白芯片(CM10)对组织样本进行分析,从组织样本制备、组织裂解液选择、上样浓度控制、重复性验证等方面进行实验条件的优化,并对大鼠肝纤维化组和对照组肝组织各4例进行差异蛋白分析。结果液氮研磨法结合含两性电解液和蛋白酶抑制剂的裂解液处理样本,以及组织蛋白样本浓度为5μg/μl时,芯片检测得到的蛋白峰强度和蛋白数量最优;对两组样本重复检测3次,蛋白丰度的平均变异系数(CV值)分别为0.105和0.155;两组样本共检测到292个蛋白峰,其中差异表达蛋白54个,在肝纤维化组中表达上调16个,表达下调38个。结论初步建立了SELDI技术检测组织样本的方法,并初步筛选出大鼠肝纤维化差异表达蛋白。

Application of SELDI in Researching Hepatic Fibrosis Tissue of Rat

Objective To initially establish the process for detecting the rat hepatic fibrosis tissue by surface enhanced laser desorption/ionization time of flight mass spectrometry（SELDI-TOF-MS） technique.Methods SELDI-TOF-MS（PBSⅡ） and CM10 Protein Chip were used to detect the tissue samples,which including 4 cases in each of the hepatic fibrosis group and control group.In the article,we introduced and discussed the following aspects of the issue,containning the collection and preparation of samples,the selection and conservation of reagents,the concentration of the loading samples,the calibration and attendance of laboratory apparatus,moreove,the reproduction and analysis of the experimental result.Results LN-grinding and lysate Ⅲ were proved to be the most effective factors for schizolysis.Tissue samples with the concentration of 5 μg/μl had the optimal detecting result.While reproduct the experiment triple times with the optimized conditions,the corresponding CV values of the experimental and controlling group were 0.105 and 0.155 respectively,which suggested the optimized progect possessed favourable reproducibility.Meanwhile,there were 292 protein peaks detected by SELDI-TOF-MS,in 18.5% of which were differential expression ones,including 16 up-regulating and 38 down regulating protein peaks.Conclusion The optimized progect for the tissue applied in SELDI-TOF-MS was initially established,trough which the preliminary screening of the rat hepatic fibrosis related biomarkers was completed.