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目的建立增强型绿荧光蛋白(EGFP)基因标记的小鼠多药耐药白血病模型。方法以磷酸钙沉淀法将pLNG蛳EGFP质粒导入ΦNX蛳E包装细胞,用脂质体转染的方法获得高滴度的EGFP病毒,并转导入小鼠多药耐药白血病细胞;采用悬浮细胞培养观察该细胞的生长规律;流式细胞术分析细胞周期分布;RT蛳PCR方法检测mdr1a基因表达;体内接种观察白血病发病情况并比较其耐药性的变化。结果将pLNG蛳EGFP质粒导入ΦNX蛳E包装细胞后,收集细胞培养上清,病毒滴度为(4.5±3.4)×106CFU/mL。病毒转染后的小鼠多药耐药白血病细胞P388/VCR蛳G能稳定地表达绿色荧光。与P388/VCR细胞相比,P388/VCR蛳G细胞倍增时间、周期分析无明显差异;RT蛳PCR检测mdr1a基因的结果为:P388/S蛳G细胞阴性,P388/VCR蛳G细胞阳性。P388/VCR蛳G细胞在DBA小鼠体内也具有耐药性。所有接种P388/VCR蛳G细胞的DBA小鼠均死于腹水型白血病。结论建立了在体内稳定表达EGFP的小鼠多药耐药白血病模型,为研究多药耐药提供一种新的工具。
Objective To establish a mouse multidrug resistance leukemia model with enhanced green fluorescent protein (EGFP) gene. Methods The pLNG 蛳 EGFP plasmid was introduced into ΦNX 蛳 E packaging cells by calcium phosphate precipitation method. High titer of EGFP virus was obtained by lipofection and transfected into mouse multidrug resistant leukemia cells. The growth of the cells was observed. The cell cycle distribution was analyzed by flow cytometry. The mdr1a gene expression was detected by RT 蛳 PCR. The inoculation was observed in vivo and the changes of drug resistance were compared. Results The pLNG 蛳 EGFP plasmid was introduced into ΦNX 蛳 E packaging cells and the cell culture supernatant was collected. The virus titer was (4.5 ± 3.4) × 106CFU / mL. Viral transfected mouse multidrug-resistant leukemia cells P388 / VCR 蛳 G can stably express green fluorescence. Compared with P388 / VCR cells, the doubling time and cycle analysis of P388 / VCR 蛳 G cells showed no significant difference. RT-PCR showed that the mdr1a gene was negative in P388 / S 蛳 G cells and positive in P388 / VCR 蛳 G cells. P388 / VCR 蛳 G cells are also resistant in DBA mice. All DBA mice vaccinated with P388 / VCR G cells died of ascitic leukemia. Conclusion The mouse model of multidrug-resistant leukemia that stably expresses EGFP in vivo was established, which provided a new tool for the study of multidrug resistance.