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医学免费论文:蝙蝠葛酚性碱诱导多药耐药细胞系K562/MDR1凋亡及逆转耐药性的研究
【摘要】 目的： 探讨蝙蝠葛酚性碱(phenolic alkaloids from Menispermum dauricum,PAMD)诱导多药耐药(multidrug resistance,MDR)细胞系K562/MDR1凋亡及逆转耐药性的作用。方法: 四甲基偶氮唑蓝(MTT)比色法检测K562/S及K562/MDR1细胞对不同浓度PAMD的敏感性,并计算半数抑制浓度(IC50)。膜联蛋白V (Annexin V)异硫氰酸荧光素(FITC)+碘化丙啶(PI)双参数检测细胞凋亡百分率变化,分析在PAMD的作用下,两种细胞对伊马替尼(IM,STI571)敏感性的变化。结果: PAMD可诱导两种细胞凋亡,其低剂量72 h时对K562/S和K562/MDR1细胞的凋亡率分别为(10.92±1.03)%和(8.12±0.98)%,并可提高伊马替尼对K562/MDR1的凋亡率。PAMD可显著逆转K562/MDR1细胞对伊马替尼的耐药性,其逆转倍数为2.22。结论: PAMD对K562/S和K562/MDR1细胞具有诱导凋亡作用,同时具有逆转白血病细胞株K562/MDR1多药耐药性、回归靶位的作用。
【关键词】 蝙蝠葛酚性碱; 慢性粒细胞白血病; 多药耐药细胞系; 凋亡
Effects of phenolic alkaloids from Menispermum dauricum on inducing
the multidrug resistance cell line K562/MDR1 to apoptosis and reversing
their multidrug resistance
HE Zhiyi, LIU Xianghui, GANG Honglin
(Harbin Food and Drug Inspection Center, Harbin Heilongjiang 150525, China)
[Abstract] Objective: To study the effects of phenolic alkaloids from Menispermum dauricum(PAMD) on inducing the multidrug resistance (MDR) cell line K562/MDR1 to apoptosis and reversing their MDR. Methods: MTT colorimetric assay was employed to detect the sensitivity of K562/S and K562/MDR1 cell lines treated with PAMD of different concentration for 72 h. IC50 values of PAMD were analyzed by MTT assay. The apoptosis rates of the two cell lines were detected by Annexin V/PI to analyze the sensitivity of two cell lines treated with PAMD on imatimib mesylate (IM,STI571). Results: PAMD can induce the apoptosis of the two kinds of cells and the apoptosis rates of the two cell lines were (10.92±1.03)% and (8.12±0.98)% respectively. It was also able to enhance the apoptosis rates of K562/MDR1 on IM. PAMD could reverse MDR of K562/MDR1 on IM and its reversal multiple was 2.22. Conclusion: PAMD could induce K562/S and K562/MDR1 to apoptosis and reverse MDR of K562/MDR1 on IM and recur its target医.学.全.在.线www.lindalemus.com.
[Key words] phenolic alkaloids from Menispermum dauricum(PAMD); chronic myelogenic leukemia; multidrug resistance cell line; apoptosis
多药耐药(multidrug resistance,MDR)是指肿瘤细胞对一种化疗药物产生耐药的同时,对其他结构和作用机制不同的化疗药物也产生交叉耐药的现象,它是导致肿瘤化疗失败的原因之一[1]。克服MDR的方法之一是使用逆转剂,一些具有钙拮抗作用的逆转剂(异搏定、维拉帕米)作用较温和,能在逆转MDR中提高化疗效果,是一类具有很好应用和开发前景的药物。本实验选用具有钙通道阻滞作用的中药蝙蝠葛酚性碱(phenolic alkaloids from Menispermum dauricum,PAMD)进行逆转耐药性的研究,以期寻找克服MDR、提高化疗疗效和对耐药肿瘤细胞进行更有效杀伤作用的新型药物。
1 材料与方法
1.1 材 料
1.1.1 细胞培养 人红白血病敏感细胞株K562/S购自中科院上海细胞所,多药耐药细胞株K562/MDR1由江苏血液研究所陈子兴教授惠赠。两种细胞于含10%小牛血清、100 U/ml青霉素和100 μg/ml链霉素的RPMI1640培养基中,在37 ℃、5%CO2饱和湿度培养箱内传代培养。
1.1.2 主要试剂与仪器 蝙蝠葛酚性碱由黑龙江中医药大学药学院王栋教授提供,RPMI1640培养基(Gibco公司),小牛血清(杭州四季青生物材料工程公司),四甲基偶氮唑蓝(MTT,Sigma公司),二甲基亚砜(DMSO,北京化工厂);酶标仪(DG3022,华东电子管厂),EPICSXL型流式细胞仪(Beckman Coluter公司);膜联蛋白V (Annexin V)-异硫氰酸荧光素(FITC)和碘化丙啶(PI)试剂盒购于BD Pharmingen公司。
1.2 方 法
1.2.1 流式细胞术(FCM)检测细胞的凋亡百分率 取对数生长期的K562/S和K562/MDR1细胞,调整细胞的初浓度,实验组分别加入PAMD至终浓度为40,20,10,5,2.5,1.25,0.625 mg/L,阴性对照组加入等体积的RPMI 1640培养基。置37 ℃、5%CO2培养箱培养72 h,收集细胞,用预冷的1×PBS洗2次,每管用1×结合缓冲液把细胞调整成1×106浓度,每管取100 μl加入5 μl的AnnexinVFITC溶液进行染色,轻轻混匀,再加入5 μl的PI溶液染色,双染后在室温避光静止15 min后每管再加入1×结合缓冲液400 μl,在1 h内上流式细胞仪进行凋亡检测。凋亡后继发坏死细胞既有膜生化改变又有膜通透性改变,AnnexinVFITC+,PI+;坏死细胞呈AnnexinVFITC-,PI+;正常细胞呈AnnexinVFITC-,PI-;AnnexinVFITC+,PI-为早期凋亡细胞。每份标本设一个复管,重复1次,计算凋亡细胞百分数。
