【摘要】：目的:建立耐甲氨蝶呤(MTX)對映體的人白血病細胞株CCRF-CEM和BALL-1細胞株,分析在加藥前后蛋白表達的差異性。方法:采用低劑量誘導(dǎo)結(jié)合濃度遞增的方法來建立MTX對映體誘導(dǎo)的兩株親本細胞的耐藥細胞株,并且用倒置顯微鏡來觀察在MTX對映體作用前后CCFR-CEM和BALL-1細胞株形態(tài)學(xué)變化和活性的差異。采用雙向凝膠電泳技術(shù)來對MTX對映體作用后耐藥細胞株與親本細胞株的總蛋白進行分離,用凝膠掃描儀來進行對比找出差異蛋白點,結(jié)合質(zhì)譜分析儀和數(shù)據(jù)庫信息檢索來鑒定差異蛋白點。結(jié)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的結(jié)果分別是36.17mg/ml,38.54mg/ml,41.09mg/ml,37.28mg/ml;作為親本細胞的對照組CCRF-CEM和BALL-1檢測到的平均蛋白點數(shù)分別為295±14和421±12個,平均蛋白點的匹配百分率為82.57%和85.13%。而加入藥物的的實驗組D-(-)MTX-CCRF-CEM,L-(+)-MTX-CCRF-CEM,D-(-)MTX-BALL-1,L-(+)-MTX-BALL-1檢測到的平均蛋白點數(shù)分別為316±10,302±9,445±11,432±7個,平均蛋白點的匹配百分率為85.27%,84.72%,86.89%,89.35%。設(shè)定蛋白點體積差異倍數(shù)2倍以上為差異有統(tǒng)計學(xué)意義。與CCRF-CEM比較,D-(-)MTX-CCRF-CEM有15個差異明顯的蛋白點,而L-(+)-MTX-CCRF-CEM中6個差異明顯的蛋白點,有4個在實驗組中表達,而另外兩個是實驗組不表達而對照組表達。與BALL-1相比較,D-(-)-MTX-BALL-1有17個差異明顯的蛋白點在實驗組中高表達,而L-(+)-MTX-CCRF-CEM則有12個差異明顯的蛋白點,其中有10是實驗組高表達,另外兩個蛋白點是實驗組不表達而對照組表達的蛋白點。結(jié)論:加入藥物的CCRF-CEM和BALL-1的蛋白表達會多于對照組,其中加入D-(-)-MTX藥物蛋白表達要明顯多于L-(+)-MTX,而在加入L-(+)-MTX后的實驗組會少表達某些正常細胞的蛋白。
[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 鹵14 and 421 鹵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 鹵10302 鹵9445 鹵11432 鹵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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R733.71

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