【摘要】：目的:建立耐甲氨蝶呤(MTX)對(duì)映體的人白血病細(xì)胞株CCRF-CEM和BALL-1細(xì)胞株,分析在加藥前后蛋白表達(dá)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(lái)建立MTX對(duì)映體誘導(dǎo)的兩株親本細(xì)胞的耐藥細(xì)胞株,并且用倒置顯微鏡來(lái)觀察在MTX對(duì)映體作用前后CCFR-CEM和BALL-1細(xì)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(lái)對(duì)MTX對(duì)映體作用后耐藥細(xì)胞株與親本細(xì)胞株的總蛋白進(jìn)行分離,用凝膠掃描儀來(lái)進(jìn)行對(duì)比找出差異蛋白點(diǎn),結(jié)合質(zhì)譜分析儀和數(shù)據(jù)庫(kù)信息檢索來(lái)鑒定差異蛋白點(diǎn)。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(xì)胞的對(duì)照組CCRF-CEM和BALL-1檢測(cè)到的平均蛋白點(diǎn)數(shù)分別為295±14和421±12個(gè),平均蛋白點(diǎn)的匹配百分率為82.57%和85.13%。而加入藥物的的實(shí)驗(yàn)組D-(-)MTX-CCRF-CEM,L-(+)-MTX-CCRF-CEM,D-(-)MTX-BALL-1,L-(+)-MTX-BALL-1檢測(cè)到的平均蛋白點(diǎn)數(shù)分別為316±10,302±9,445±11,432±7個(gè),平均蛋白點(diǎn)的匹配百分率為85.27%,84.72%,86.89%,89.35%。設(shè)定蛋白點(diǎn)體積差異倍數(shù)2倍以上為差異有統(tǒng)計(jì)學(xué)意義。與CCRF-CEM比較,D-(-)MTX-CCRF-CEM有15個(gè)差異明顯的蛋白點(diǎn),而L-(+)-MTX-CCRF-CEM中6個(gè)差異明顯的蛋白點(diǎn),有4個(gè)在實(shí)驗(yàn)組中表達(dá),而另外兩個(gè)是實(shí)驗(yàn)組不表達(dá)而對(duì)照組表達(dá)。與BALL-1相比較,D-(-)-MTX-BALL-1有17個(gè)差異明顯的蛋白點(diǎn)在實(shí)驗(yàn)組中高表達(dá),而L-(+)-MTX-CCRF-CEM則有12個(gè)差異明顯的蛋白點(diǎn),其中有10是實(shí)驗(yàn)組高表達(dá),另外兩個(gè)蛋白點(diǎn)是實(shí)驗(yàn)組不表達(dá)而對(duì)照組表達(dá)的蛋白點(diǎn)。結(jié)論:加入藥物的CCRF-CEM和BALL-1的蛋白表達(dá)會(huì)多于對(duì)照組,其中加入D-(-)-MTX藥物蛋白表達(dá)要明顯多于L-(+)-MTX,而在加入L-(+)-MTX后的實(shí)驗(yàn)組會(huì)少表達(dá)某些正常細(xì)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(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.71

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 許維東;何曉東;孫利;李道靜;張永娟;張白銀;沈佐君;;Luminex液相芯片技術(shù)測(cè)定氨甲喋呤對(duì)映體耐藥A549細(xì)胞株中磷酸化酪氨酸激酶受體[J];臨床檢驗(yàn)雜志;2011年08期

2 楊沐;鐘文英;侯雯;;手性藥物分析方法研究進(jìn)展[J];藥學(xué)進(jìn)展;2014年03期

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