氨甲喋呤对映体对急性淋巴细胞白血病细胞株的体外诱导蛋白表达差异的研究

发布时间：2019-02-15 21:37

【摘要】：目的:建立耐甲氨蝶呤(MTX)对映体的人白血病细胞株CCRF-CEM和BALL-1细胞株,分析在加药前后蛋白表达的差异性。方法:采用低剂量诱导结合浓度递增的方法来建立MTX对映体诱导的两株亲本细胞的耐药细胞株,并且用倒置显微镜来观察在MTX对映体作用前后CCFR-CEM和BALL-1细胞株形态学变化和活性的差异。采用双向凝胶电泳技术来对MTX对映体作用后耐药细胞株与亲本细胞株的总蛋白进行分离,用凝胶扫描仪来进行对比找出差异蛋白点,结合质谱分析仪和数据库信息检索来鉴定差异蛋白点。结果:在蛋白定量上:CCRF-CEM,D-(-)MTX-CCRF-CEM,L-(+)-MTX-CCRF-CEM,BALL-1,D-(-)MTX-BALL-1,L-(+)-MTX-BALL-1的结果分别是36.17mg/ml,38.54mg/ml,41.09mg/ml,37.28mg/ml;作为亲本细胞的对照组CCRF-CEM和BALL-1检测到的平均蛋白点数分别为295±14和421±12个,平均蛋白点的匹配百分率为82.57%和85.13%。而加入药物的的实验组D-(-)MTX-CCRF-CEM,L-(+)-MTX-CCRF-CEM,D-(-)MTX-BALL-1,L-(+)-MTX-BALL-1检测到的平均蛋白点数分别为316±10,302±9,445±11,432±7个,平均蛋白点的匹配百分率为85.27%,84.72%,86.89%,89.35%。设定蛋白点体积差异倍数2倍以上为差异有统计学意义。与CCRF-CEM比较,D-(-)MTX-CCRF-CEM有15个差异明显的蛋白点,而L-(+)-MTX-CCRF-CEM中6个差异明显的蛋白点,有4个在实验组中表达,而另外两个是实验组不表达而对照组表达。与BALL-1相比较,D-(-)-MTX-BALL-1有17个差异明显的蛋白点在实验组中高表达,而L-(+)-MTX-CCRF-CEM则有12个差异明显的蛋白点,其中有10是实验组高表达,另外两个蛋白点是实验组不表达而对照组表达的蛋白点。结论:加入药物的CCRF-CEM和BALL-1的蛋白表达会多于对照组,其中加入D-(-)-MTX药物蛋白表达要明显多于L-(+)-MTX,而在加入L-(+)-MTX后的实验组会少表达某些正常细胞的蛋白。
[Abstract]:Aim: to establish CCRF-CEM and BALL-1 cell lines resistant to methotrexate (MTX) enantiomers and to analyze the difference of protein expression before and after administration of methotrexate. Methods: two parent cell lines induced by MTX enantiomer were established by low dose induction and increasing concentration. The morphological changes and activity of CCFR-CEM and BALL-1 cell lines before and after the enantiomeric action of MTX were observed by inverted microscope. Two-dimensional gel electrophoresis was used to isolate the total proteins of drug-resistant cell lines and parental cell lines treated with MTX enantiomers, and the differential protein spots were found by gel scanner. Differential protein spots were identified by mass spectrometer and database information retrieval. Results: in protein quantification: CCRF-CEM,D- (-) MTX-CCRF-CEM,L- () MTX-CCRF-CEM,BALL-1,D- (-) MTX-BALL-1,L- ()-MTX-BALL-1 results were 36.17 mg / ml, respectively. 38.54 mg / ml 41.09 mg / ml 37.28 mg / ml; The average protein points detected by CCRF-CEM and BALL-1 were 295 卤14 and 421 卤12, respectively, and the matching percentage of average protein points were 82.57% and 85.13% respectively. The average protein points detected by D- (-) MTX-CCRF-CEM,L- (-) MTX-CCRF-CEM,D- (-) MTX-BALL-1,L- ()-MTX-BALL-1 were 316 卤10302 卤9445 卤11432 卤7, respectively. The average matching percentage of protein points was 85.27% and 84.72%, 86.89% and 89.35%, respectively. It was statistically significant to set a multiple of 2 times the volume difference of the protein point. Compared with CCRF-CEM, D- (-) MTX-CCRF-CEM had 15 distinct protein spots, while in L- ()-MTX-CCRF-CEM, there were 6 distinct protein spots, 4 of which were expressed in experimental group. The other two were not expressed in the experimental group but expressed in the control group. Compared with BALL-1, D- (-)-MTX-BALL-1 had 17 distinct protein spots in high expression in experimental group, while L- ()-MTX-CCRF-CEM had 12 distinct protein spots, 10 of which were high expression in experimental group. The other two protein spots were not expressed in the experimental group and expressed in the control group. Conclusion: the protein expression of CCRF-CEM and BALL-1 was higher than that of control group, and the expression of D- (-)-MTX protein was significantly higher than that of L- ()-MTX,. After adding L-()-MTX, some normal cell proteins were less expressed in the experimental group.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.71

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