从白血病干细胞水平研究蝎毒多肽提取物逆转多药耐药机制  

Reversion Mechanism Study of PESV to Multidrug Resistance at Leukemia Stem Cell Level

作　　者：杨向东[1] 史哲新[1] 闫理想 刘宝山[2] 陈化禹[1] 颜田赅[1] 张伟锋[1] 杨曦[1] 秦浩人 刘超[3] 冀虹霞 

机构地区：[1]天津中医药大学第一附属医院血液科,天津300381 [2]天津医科大学总医院中医科,天津300052 [3]天津中医药大学研究生院,天津300193

出　　处：《中国中西医结合杂志》2016年第7期867-874,共8页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：天津市科委资助项目(No.12JCQNJC09000)

摘　　要：目的探讨蝎毒多肽(peptide extract from scorpion venom,PESV)对白血病干细胞(leukemic stem cell,LSC)在体内多药耐药(multidrug resistance,MDR)的影响。方法培养K562/A02细胞,在对数生长期收集并经免疫磁珠法分选出K562/A02干细胞备用;40只BABL/c裸鼠,取5只注射K562/A02干细胞形成皮下瘤块备用;采用随机数字表法将35只裸鼠分为正常对照组、模型组、耐阿霉素(Adriamycin,ADM)组、PESV组和ADM+PESV高、中、低剂量组,每组5只。正常对照组不作处理,其余各组包埋瘤块组织,造模后模型组给予1 m L生理盐水腹腔注射,每天1次;ADM组腹腔注射ADM 0.05 mg,隔天1次;PESV组腹腔注射PESV 2μg,每天1次;ADM+PESV高、中、低剂量组腹腔注射ADM 0.05 mg,隔天1次,同时给予PESV(5、2、1μg)腹腔注射,每天1次。给药14天。流式细胞术检测P-糖蛋白(P-glycoprotein,P-gp);RT-PCR检测乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)、多药耐药基因1(multidrug resistance 1,MDR1)m RNA表达;免疫组化法检测乙醛脱氢酶1(aldehyde dehydrogenase 1,ALDH1);Western blot法检测磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)蛋白表达;ELISA检测核转录因子NF-κB含量。结果 K562/A02细胞经免疫磁珠分选前后的CD34+CD38-细胞比率分别为31.5%、92.8%,耐药率(IC50)分别为(60.33±10.68)μg/m L、(58.33±9.72)μg/m L,造模裸鼠成瘤率为100%。与模型组比较,ADM组各项检测指标比较,差异无统计学意义(P>0.05);PESV组BCRP、MDR1 m RNA和NF-κB因子水平比较,差异均有统计学意义(P<0.05);ADM+PESV高、中、低剂量组P-gp值降低,PI3K蛋白表达下调(P<0.05),ADM+PESV高、中剂量组BCRP m RNA表达降低,MDR1 m RNA表达升高,差异均有统计学意义(P<0.05);ADM+PESV高剂量组PI3K蛋白表达下调,差异有统计学意义(P<0.05)。分别与ADM组和PESV组比较,ADM+PESV高剂量组P-gp、BCRP m RNA、MDR1 m RNA、PI3K和NF-κB水平明显均下调,差异有统计学意义(P<0.05)。各组ALDH1阳性率�Objective To explore the effect of peptide extract from scorpion venom（PESV） to multidrug re-sistance（MDR） of leukemic stem cell（LSC） in vivo. Methods K562/A02 cells were cultured and collected in the logarithmic phase. K562/A02 stem cells were screened using immunomagnetic beads for reserve. K562/A02 LSC was injected to 5 of 40 BABL/c nude mice for preparing subcutaneous tumor. The rest 35 nude mice were then randomly divided into 7 groups, i.e., the normal control group, the model group, the Adriamycin（ADM） group, the PESV group, the ADM＋ high dose PESV group, the ADM ＋ middle dose PESV group, the ADM ＋ low dose PESV group, 5 in each group.Tumor tissue was embedded in all groups except the normal control group. One milliliter normal saline was peritoneally injected to mice in the model group after modeling, once per day. ADM 0. 05 mg was peritoneally injected to mice in the ADM group, once per other day. PESV 2 μg was peritoneally injected to mice in the PESV group, once per day. Mice in 3 ADM ＋ PESV groups were peritoneally injected with ADM 0. 05 mg（once per other day） plus PESV（5, 2, and 1 μg respectively, once per day）. All medication lasted for 14 days. P-glycoprotein（P-gp） was detected using flow cytometry.Breast cancer resistance protein（BCRP） and m RNA expression of multidrug resistance 1（MDR1） were measured using RT-PCR. Aldehyde dehydrogenase 1（ALDH1） was detected using immunohistochemistry. Phosphoinositide 3-kinase（PI3K） was detected using Western blot. NF-κB content was detected using ELISA. Results CD34＋CD38-ratio was31. 5% and IC50was（60. 33 ± 10. 68） μg/m L before K562/A02 cells were screened with immunomagnetic beads, while they were 92. 8% and（58. 33 ± 9. 72） μg/m L after screen. The tumor formation rate was 100% in modeling mice. Compared with the model group, no statistical difference of each index occurred in the ADM group（P 〈0. 05）. There was statistical difference in BCRP, MDR1 m RNA, or NF-κB factor between the m

关 键 词：蝎毒多肽 K562/A02 BABL/c裸鼠 多药耐药 

分 类 号：R733.7[医药卫生—肿瘤]