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目的 :建立高效、稳定的大鼠 CRBH- 7919多药耐药细胞系。 方法 :利用逆转录病毒转染法将带有 mdr1c DNA全序列的逆转录病毒载体 p Ha MDR转入到大鼠 CRBH- 7919细胞中 ,MTT法检测细胞系在不同化疗药物作用下的存活率 ;免疫组化检测细胞的 P-糖蛋白 (P- GP)表达 ,RT- PCR检测细胞内 mdr1m RNA的表达量 ,PCR检测 mdr1基因转移到细胞内的基因片段。 结果 :转基因的细胞系对阿霉素、丝裂霉素的耐药性分别提高 9和 7.9倍 ,免疫组化见转基因细胞系 P- GP表达增加 ,RT- PCR示细胞内 mdr1m RNA的表达量增加 ,PCR表明转基因细胞内扩增出 m dr1片段。结论 :利用逆转录病毒转染法成功建立了大鼠 CRBH - 7919多药耐药细胞系 ,该细胞系具有耐药强度高、耐药性稳定等特点
Objective: To establish an efficient and stable rat CRBH- 7919 multidrug-resistant cell line. Methods: The retroviral vector p Ha MDR with mdr1c DNA sequence was transfected into rat CRBH- 7919 cells by retroviral transfection method. The survival rate of the cell lines under different chemotherapeutic drugs was determined by MTT assay. The expression of P-glycoprotein (P-GP) was detected by immunohistochemistry, the expression of mdr1m RNA was detected by RT-PCR and the gene fragment transfected by mdr1 gene into cells was detected by PCR. Results: The resistance to doxorubicin and mitomycin increased by 9 and 7.9 times respectively in the transgenic cell lines. The expression of P-GP in the transgenic cell line was increased by immunohistochemistry. The expression of mdr1m RNA in the transfected cells was detected by RT-PCR Increasing, PCR showed that m dr1 fragment was amplified in transgenic cells. Conclusion: The CRBH - 7919 multidrug resistant cell line was established successfully by retroviral transfection. The cell line is characterized by high drug resistance and stable drug resistance