建立表达增强型绿荧光蛋白的小鼠多药耐药白血病模型

Establishment of murine multi-drug resistant leukemia model expressing enhanced green fluorescent protein

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 gene marking of murine multi- drug resistant leukemia model with enhanced green fluorescent protein. Methods MSCV- eGFP plasmid was transferred into ΦNX- E paking cell by Ca3(PO4)2 precipitation using retrovirus vector. EGFP virus producer cells were generated with liposome mediated transfection. The murine P388/VCR cell line was transduced with EGFP retrovirus. The biological characteristics of P388/VCR- G were compared with P388/VCR, in respect of cell growth curve, cell growth cycle by flow cytometer(FCM), and the expression of mdr1a gene by RT- PCR. Its change of drug resistant was compared in murine. Result The EGFP virus was obtained, when pLNG- EGFP plasmid was transferred into ΦNX- E packed cell, and its titer was (4.5±3.4)×106 infectious particles/mL. The gene transferred P388/VCR cell could express bright green fluorescence steadily. There were no significant difference of cell growth curve and cell cycle analysis between P388/VCR- G and P388/VCR cell lines. The expression of mdr1a gene in P388/VCR was negative through the detection of RT- PCR, but it was positive in P388/VCR- G. All of the mice inoculated P388/VCR- G and P388/VCR cells died of leukemia with ascites. Conclusions It was established that model expressing high level green fluorescence .This model may provide an useful tool for the study of multi- drug resistant.