蝎毒多肽提取物对白血病细胞株K562/A02荷瘤鼠耐药性的影响

doi:10.3971/j.issn.1000-8578.2016.03.003

蝎毒多肽提取物对白血病细胞株K562/A02荷瘤鼠耐药性的影响

Reversion Impact of Peptide Extract from Scorpion Venom on Multidrug-resistance of Leukemia Stem Cell Line K562/A02 in vivo

摘要

摘要: 目的探讨蝎毒多肽（peptide extract from scorpion venom,PESV）逆转白血病干细胞（leukemiastem cells, LSC）在体内多药耐药（multidrug resistance, MDR）的分子机制。方法以多药耐药的K562/A02细胞株成模白血病BALB/c裸鼠为例，成模鼠随机分为6组：模型对照组、阿霉素（ADM）组、PESV组、ADM+PESV（H）组、ADM+PESV（M）组、ADM+PESV（L）组。模型对照组给予等体积0.9%氯化钠溶液腹腔注射，其余各组予相应剂量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，分别检测瘤块中LSC：细胞膜上P-gp的表达，细胞质中ALDH、PI3K的变化及细胞核中MDR1、NF-κB的活性。结果 K562/A02细胞经免疫磁珠分选前后的CD34+CD38-细胞比率和IC50值分别为31.5%、（60.33±10.68）μg/ml和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；各组造模裸鼠成瘤率100%。瘤体中LSC：流式细胞仪检测细胞膜上P-gp表达结果：检测对照组89.8%、ADM组91.9%、PESV组88.4%、ADM+PESV（H）组53.9%、ADM+PESV（M）组78.0%、ADM+PESV（L）组78.7%；半定量RT-PCR检测MDR1 mRNA的表达：PESV组＞ADM+PESV（L）组＞ADM+PESV（M）组＞ADM+PESV（H）组＞ADM组；免疫组织化学检测ALDH，显示灰度值ADM组＞PESV组＞ADM+PESV（H）组＞ADM+PESV（M）组＞ADM+PESV（L）组；Western blot检测PI3K分子与Elisa检测NF-κB因子结果一致，在ADM组、PESV组表达上调，在ADM+PESV组中表达下调，下调强度与PESV剂量呈正相关。结论 PESV具有下调白血病干细胞膜上P-gp，细胞质内ALDH、PI3K及细胞核中MDR1、NF-κB的表达水平，增强了白血病K562/A02干细胞在体内对ADM的敏感度，逆转其多药耐药特性。

Abstract: Objective To investigate the molecular mechanism of the reversion impact of peptide extract from scorpion venom(PESV) on multidrug resistance(MDR) of leukemia stem cells(LSC) in vivo. Methods Took leukemia BALB/c nude mice modeled by K562/A02 cell line for example, they were randomly divided into six groups: Model control group, ADM group, PESV group, ADM+PESV high-dose(H) group, ADM+PESV middle-dose(M) group, and ADM+PESV low-dose(L) group. 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, and all groups were continuously medicated for 14d. On d21, the transplanted tumor growth were observed in nude mice of each group, and we respectively detected the LSC in the tumor block: the expression levels of P-gp on the membrane, the content of ALDH and PI3-K in the cytoplasm, and the activities of MDR1 and NF-κB in the nucleus. Results Before and after K562/A02 cells were selected with immunomagnetic beads, CD34+CD38- cells ratio and IC50 were 31.5% and (60.33±10.68) μg/ml, 92.8% and (58.33±9.72) μg/ml, respectively. After the selection, the stemness of the cells was significantly improved, while no difference of drug-resistance was showed. Tumor formation rate of modeled mice in each group was 100%. The testing results of LSC in the tumor block were as follows: the expression of P-gp on the membrane tested by flow cytometry were 89.8% in model control group, 91.9% in ADM group, 88.4% in PESV group, 53.9% in ADM+PESV(H) group, 78.0% in ADM+PESV(M) group, and 78.7% in ADM+PESV(L) group; the expression levels of MDR1 mRNA detected by semiquantitative RT-PCR were PESV group>ADM+PESV(L) group> ADM+PESV(M) group> ADM+PESV(H) group>ADM group; the gray values of ALDH tested by immunohistochemistry were ADM group>PESV group> ADM+PESV(H) group>ADM+PESV(M) group> ADM+PESV(L) group; PI3-K protein and NF-κB factor were respectively detected by Western blot and Elisa, and both expression levels were up-regulated in ADM group and PESV group, down-regulated in ADM+PESV groups, moreover, the down-regulated levels showed a positive correlation with the dose of PESV. Conclusion PESV could reduce the expression levels of P-gp on the membrane, ALDH and PI3-K expression in the cytoplasm, and MDR1 and NF-κB expression in the nucleus, which result in enhancing the sensitivity of LSC K562/A02 to ADM and eventually reversing its multidrug resistance.

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