杨向东+王兴丽+杨文华+赵磊+颜田赅

[摘要] 该实验采用BALB/c小鼠多药耐药（MDR）白血病模型，观察蝎毒多肽（PESV）对小鼠瘤体MDR白血病干细胞（LSC）P-gp（P-gp）蛋白上游信号的影响，研究PESV逆转白血病干细胞（LSC）多药耐药的效果及机制；同时通过流式细胞术，RT-PCR，Western blot，Elisa法分别检测 P-gp，MDR1 mRNA，PI3-K，NF-κB表达，并通过组织病理学方法检测小鼠肝脏、脾脏等。结果显示，正常对照组小鼠无明显变化，其余6组小鼠均出现弓背、消瘦等表现，肝脏肿大，肝内均出现炎症坏死；PESV下调小鼠瘤块中LSC细胞膜P-gp，PI3K蛋白表达下调；胞浆中MDR1 mRNA表达在PESV低剂量组显著下降，在中、高剂量组出现不同程度的升高；PESV显著降低LSC细胞核内NF-κB因子水平。PESV具有明显下调P-gp上游信号PI3K/NF-κB/MDR1的作用，在一定程度上增强了LSC对化疗药物阿霉素（ADM）的敏感度，从而闡述了PESV逆转LSC多药耐药的可能机制之一，为PESV联合抗肿瘤作用的进一步研究提供了基础。

[关键词] 蝎毒多肽；多药耐药；BALB/c小鼠模型；P-gp上游信号

[Abstract] Using the BALB/c mouse multidrug resistance model of leukemia，the effect of peptide extract from scorpion venom （PESV） to the upstream signal factors of P-gp of MDR leukemia stem cells on the mouse tumor block was observed，and the mechanism of PESV to reverse the MDR of LSC was studied. At the same time，the expression of P-gp，MDR1 mRNA and PI3-K，NF-κB were respectively detected through flow cytometry，RT-PCR，Western blot and Elisa，and the mouse liver，spleen were examined via histopathological methods. The results of the experiment were as follows： mice of the control group didnt show any obvious changes，while mice of the other six groups all showed arched back，emaciation，liver swell，and inflammation was found in all liver tissue. The expression level of P-gp and PI3K on the LSC membrane of mouse tumor block was down-regulated；the expression of MDR1 mRNA in the cytoplasm was obviously down in the PESV low dose group，and which was inordinately up in the middle dose group and the high dose group. The expression level of NF-κB in the leukemia stem cell nucleus remarkably decreased. PESV had a outstanding role of down-regulating PI3K，NF-κb，MDR1 which were all upstream factors of P-gp，and to a certain degree enhanced the sensitivity of LSC to ADM. Therefore，this experiment explained one of the mighty mechanism of PESV to reverse MDR of LSC，and provided a foundation to further study of combinational anti-cancer effects of PESV.

[Key words] peptide extract from scorpion venom （PESV）；MDR；BALB/c mouse model；upstream signal factors of P-gp

doi：10.4268/cjcmm20162426

白血病细胞对化疗药物产生多药耐药（multidrug resistance，MDR）现象是急性白血病（acute leukemia，AL）化疗失败及复发的主要原因之一[1]。MDR是指肿瘤细胞接触一种化疗药物产生耐药的同时，对多种结构不同、作用机制各异的其他化疗药物也产生交叉耐受性。白血病细胞产生MDR现象较常见的机制是磷脂酰肌醇3激酶（phosphoinositide 3-kinase，PI3K）受到细胞因子、药物等配体活化后，促使生成磷脂酰肌醇（phosphatidylinositol，PI），PI作为细胞内的第二信使活化蛋白激酶B（protein kinase，PKB又称AKT）[2]，AKT磷酸化下游核转录因子（nuclear facter，NF-κB），活化的NF-κB启动多药耐药基因（multidrug resistance 1，MDR1），MDR1转录后的mRNA表达其编码的P-糖蛋白（P-glycoprotien，P-gp），P-gp可以利用水解ATP的能量将各种药物从细胞内转运到细胞外，降低药物在细胞内的浓度，并使其细胞毒作用减弱直至消失[3]，这样形成以P-gp上游信号PI3K/AKT/NF-κB/MDR1介导为主的白血病细胞MDR通路。……