蝎毒多肽对白血病干细胞 MDR1 mRNA和P－gp表达的影响

摘要

目的：探讨蝎毒多肽（ PESV）对多药耐药白血病干细胞MDR1 mRNA和P－gp表达的影响。方法：以K562／A02细胞株为例，免疫磁珠法分选对数生长期细胞后经CCK－8法确认其耐药性，将耐药的K562／A02干细胞接种于BABL／c裸鼠右腋下形成瘤块，再将瘤块皮下包埋建立耐药性稳定的白血病模型。成模裸鼠随机分为6组：模型对照组、ADM组、PESV组、ADM＋高剂量PESV组（高剂量组）、ADM＋中剂量PESV组（中剂量组）和ADM＋低剂量PESV组（低剂量组）。模型对照组给予等体积0．9％氯化钠溶液腹腔注射，其余组予相应剂量ADM和（或） PESV腹腔注射，连续给药14天。第21天观察各组裸鼠移植瘤生长情况，流式细胞术检测各组瘤组织P－gp表达， RT－PCR法检测各组瘤组织MDR－1 mRNA表达水平。结果：K562／A02细胞经免疫磁珠分选前后的CD34＋CD38－细胞比率和耐药率分别为31．5％、（60．33±10．68）μg／mL，92．8％、（58．33±9．72）μg／mL，分选后细胞干性显著提高，而耐药性无差异性损失。各组造模小鼠成瘤率100％， P－gp表达结果显示模型对照组89．8％、ADM组91．9％、PESV组88．4％、高剂量组53．9％、中剂量组78．0％、低剂量组78．7％，PESV＋ADM下调P－gp表达，并与PESV呈现浓度依赖关系。 MDR1 mRNA的表达量PESV组＞低剂量组＞高剂量组＞中剂量组＞ADM组，其中PESV组、中剂量组、低剂量组高表达，P－gp表达与MDR1 mRNA水平具有一定的一致性。结论：PESV降低ADM作用K562／A02干细胞耐药性的强度，并与PESV剂量呈现正相关，其机制可能为PESV通过调控P－gp和MDR－1 mRNA的表达，增强了K562／A02干细胞对ADM的敏感度。%Objective:To investigate the impact of PESV ( peptide extract from scorpion venom ) on the expression level of MDR1 mRNA and P-gp in multi-drug-resistant leukemia stem cells .Methods:Take K562/A02 cell line for exam-ple, immunomagnetic beads were used to select cells in log phase of growth , then confirm the drug resistance with CCK-8 method.The drug-resistant K562/A02 stem cells were inoculated to right subaxillary of BABL/c nude mice to form tumor block , then tumor blocks were subcutaneously embedded into the nude mice to establish stable drug -resist-ant leukemia models.Model mice were randomly divided into six groups:a model control group ,an ADM group,a PESV group(middle dose),an ADM+PESV high dose group (high dose group),an ADM+PESV middle dose group (middle dose group ) and an ADM+PESV low dose group ( low dose group ) .The model control group received an equal volume of 0 .9%sodium chloride solution by means of intraperitoneal injection , and other groups received the corresponding dose of ADM and ( or) PESV in the same way.All groups were continuously medicated for 14 days.On the 21st day,the transplanted tumor growth was observed in nude mice in each group ,and then detect P-gp expression level of the tumor tissues in each group by flow cytometry ,then test MDR-1mRNA expression level of the tumor tissues in each group by RT-PCR method.Results: Before and after K562/A02 cells were selected with immunomagnetic beads , CD34 +CD38-cell ratio and drug-resistant dose were 31.5%(60.33 ±10.68)ug/ml,92.8%(58.33 ±9.72)ug/ml respec-tively.After the selection, commitment of the cells was significantly improved , while no difference of drug -resistance was shown .Tumor formation rate of model mice in each group was 100%,and P-gp expression results were respectively as follows:89.8% in model control group, 91.9% in ADM group, 88.4% in PESV group, 53.9% in high -dose group, 78.0%in middle dose group, 78.7%in the low-dose group.Therefore, we speculated that PESV plus ADM reduced P-gp expression , and the expression level showed a concentration -dependent manner to PESV .The results of MDR1 mRNA expression level were as follows:PESV group>low dose group>high dose group>middle dose group>ADM group,which showed a higher expression level in PESV group , middle dose group and low -dose group.Above all, we supposed that the expression level of P -gp and MDR1mRNA had a certain consistency .Conclusion: PESV re-duces the drug resistance of K 562/A02 stem cell to ADM, and it shows a positive correlation to the dose of PESV .The mechanism may be that PESV regulates the expression of P -gp and MDR -1 mRNA and enhances the sensitivity of K562/A02 stem cells to ADM .