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目的:观察外源性人多药耐药基因(human multidrug resistance gene1,hMDR1)对新西兰兔骨髓单个核细胞(mono-nuclear cells,MNCs)生物学行为的影响。方法:用携带目的基因hMDR1和绿色荧光蛋白(green fluorescence protein,GFP)报告基因的复制缺陷型重组腺病毒(rAd-hMDR1-GFP)转染原代培养的MNCs以获取hMDR1基因修饰的MNCs(MNCs-rAd-hMDR1-GFP),荧光显微镜和FCM法检测转染效率;MTT法绘制MNCs-rAd-hMDR1-GFP细胞生长曲线;FCM检测MNCs-rAd-hMDR1-GFP细胞周期及细胞凋亡情况;RT-PCR及Western印迹法从基因和蛋白水平检测MNCs-rAd-hMDR1-GFP细胞内hMDR1的表达;柔红霉素(daunorubicin,DNR)泵出实验检测导入hMDR1基因的外排泵生理功能。结果:感染复数(multiplicityof infection,MOI)为100时,rAd-hMDR1-GFP对MNCs的转染效率为30%~35%,且不影响MNCs增殖生长;FCM结果提示,rAd-hMDR1-GFP对MNCs细胞周期及凋亡无影响(P>0.05);rAd-hMDR1-GFP转染72h后hMDR1转录水平和P-糖蛋白(P-glycoprotein,P-gp)表达水平均明显升高(P<0.01),且外源性hMDR1能发挥外排泵功能(P<0.01)。结论:rAd-hMDR1-GFP能将外源性hMDR1基因高效导入兔骨髓MNCs并稳定功能性表达,不影响MNCs细胞周期、细胞凋亡及细胞增殖生长等生物学特性,为进一步研究hMDR1基因的骨髓保护作用提供实验依据。
OBJECTIVE: To observe the effect of exogenous human multidrug resistance gene1 (hMDR1) on the biological behavior of New Zealand rabbit bone marrow mononuclear cells (MNCs). Methods: Primary cultured MNCs were transfected with replication-defective recombinant adenovirus (rAd-hMDR1-GFP) carrying target gene hMDR1 and green fluorescence protein (GFP) reporter gene to obtain hMDR1 gene-modified MNCs (MNCs The expression of MNCs-rAd-hMDR1-GFP was detected by fluorescence microscopy and FCM. The cell growth curve of MNCs-rAd-hMDR1-GFP was plotted by MTT assay. The cell cycle and apoptosis of MNCs-rAd- The expression of hMDR1 in MNCs-rAd-hMDR1-GFP cells was detected by PCR and Western blotting. The daunorubicin (DNR) pumpout assay was used to detect the physiological function of efflux pump transduced with hMDR1 gene. Results: The transfection efficiency of rAd-hMDR1-GFP to MNCs was 30% -35% at a multiplicity of infection (MOI) of 100, and did not affect the proliferation and proliferation of MNCs. FCM showed that rAd-hMDR1- (P> 0.05). The expression of hMDR1 and the expression of P-glycoprotein (P-glycoprotein, P-gp) were significantly increased at 72 h after transfection with rAd-hMDR1-GFP , And exogenous hMDR1 can play a role of efflux pump (P <0.01). Conclusions: rAd-hMDR1-GFP can efficiently transfer exogenous hMDR1 gene into MNCs of rabbit bone marrow and stable functional expression without affecting the biological characteristics of cell cycle, apoptosis and proliferation and proliferation of MNCs. To further investigate the biological characteristics of hMDR1 gene in bone marrow Protection provides experimental evidence.