外源性人多药耐药基因转染对兔骨髓单个核细胞生物学行为的影响

Effect of rAd-hMDR1 on the biologic behavior of bone marrow mononuclear cells of rabbits

目的：观察外源性人多药耐药基因（human multidrug resistance gene 1, hMDR1）对新西兰兔骨髓单个核细胞（mononuclear 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（multiplicity of 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72 h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 investigate the effects of human multidrug resistance gene 1 （hMDR1） on biologic behaviors of mono- nuclear cells （MNCs） from New Zealand rabbit bone marrow. Methods：Primary MNCs of rabbit bone marrow were transfected with a replication-deficient recombinant adenovirus expression vector carrrying hMDR1 and GFP （rAd-hMDR1-GFP） to obtain the hMDRlgene modified MNCs （MNCs-rAd-hMDR1-GFP）. The transfection efficiency of rAd-hMDR1-GFP was determined using fluorescence microscopy and flow cytometry （FCM）. The proliferative capacity was examined by [ 3-（4, 5 ）-dimethyhhiazol （-z-yl）3,5 diphenytetrazolium-romide] （MTI＇） assay. The cell cycle and the apoptosis of MNCs-rAd-hMDR1-GFP were determined by FCM. The mRNA expression of hMDR1 was measured by reverse transcription-polymerase chain reaction （RT-PCR） and the production of P-gp protein levels were determined by Western blotting. The functional expression of P-gp protein was detected by daunorubicin （DNR） efflux assay. Resuits：The transfection efficiency of rAd-hMDR1-GFP to MNCs of rabbit bone marrow was 30% to 35% when the multiplicity of infection （MOI） was 100. The transfection of rAd-hMDR1-GFP did not affect the cell proliferation capacity and had no effects on the apoptosis and cell cycle （all P 〉 0.05 ）. Both the mRNA transcription level of hMDR1 and the expression of P-gp were obviously up-regulated in MNCs after being modified with rAd-hMDR1-GFP （ P 〈 0.01 ）, The DNR efflux assay indicated that the exogenous hMDR1 exerted efflux function in MNCs （P 〈0.01 ）. Conclusion： Replication-deficient recombinant adenovirus expression vector efficiently introduces exogenous hMDR1 into the MNCs of rabbit bone marrow and the stably expressed hMDR1 gene does not affect the biological behaviors such as cell cycle, the apoptosis and the proliferative capacity of MNCs. The study has established a basis for the further research on myeloehemoprotective effects of hMDR1 gene on the bone marrow cells.