苦参碱诱导人白血病耐药细胞K562/IM自噬和凋亡的研究

Effect of Matrine on Autophagy and Apoptosis of Human Leukemia K562/IM Cells

目的观察苦参碱对人白血病耐药细胞K562/IM自噬和凋亡的影响。方法 (1)苦参碱及伊马替尼对细胞增殖及自噬情况检测:分别设空白对照组、不同浓度(0.25、0.50、1.00、2.00、5.00mg/mL)苦参碱组和(0.05、0.25、0.50、5.00、20.00μmol/L)伊马替尼组,MTT检测K562细胞和K562/IM细胞抑制率。分别设置空白对照组、0.4 mg/mL苦参碱组和0.5μmol/L伊马替尼组,免疫荧光激光共聚焦检测K562细胞和K562/IM细胞自噬现象,Western Blot检测细胞中自噬标志蛋白Beclin-1、LC3-Ⅱ/Ⅰ、P62的表达。(2)苦参碱联合3-甲基腺嘌呤(3-MA)或雷帕霉素(rapamycin,Rapa)诱导K562/IM细胞自噬及凋亡情况变化的检测:实验分6组,空白对照组、苦参碱组(0.4mg/mL苦参碱处理)、3-MA组(5mmol/L 3-MA处理)、Rapa组(0.5μmol/L Rapa处理)、苦参碱+3-MA组(0.4mg/mL苦参碱+5mmol/L3-MA处理)、苦参碱+Rapa组(0.4mg/mL苦参碱+0.5μmol/L Rapa处理),采用MTT检测细胞的抑制率,流式细胞仪检测细胞凋亡水平;Western Blot检测细胞自噬标志蛋白Beclin-1、LC3-Ⅱ/Ⅰ、P62及凋亡相关蛋白Caspase-3表达。结果 (1)苦参碱和伊马替尼作用K562、K562/IM细胞,细胞增殖受抑制明显(P<0.05),LC3-Ⅱ/Ⅰ的聚集明显增加,Beclin-1及LC3-Ⅱ/Ⅰ蛋白表达升高,P62蛋白表达减少(P<0.05),以K562/IM细胞更明显(P<0.05)。(2)苦参碱组K562/IM细胞增殖抑制率(36.32±2.21)%,凋亡率(15.35±0.39)%。苦参碱+3-MA组K562/IM细胞增殖抑制率升高[(65.25±2.06)%,P<0.01)],凋亡率升高[(21.70±0.48)%,P<0.05],同时Beclin-1及LC3-Ⅱ/Ⅰ蛋白表达下降,P62蛋白表达增强,Caspase-3表达增强(P<0.05);而苦参碱+Rapa组作用则相反。结论苦参碱和伊马替尼均可抑制K562、K562/IM细胞增殖的同时可诱导自噬,以耐药K562/IM细胞株自噬明显。通过3-MA抑制自噬可增强苦参碱对细胞K562/IM作用的敏感性,而雷帕霉素减弱苦参碱作用。

Objective To study effects of matrine on autophagy and apoptosis of human leuke- mia K562/IM cells. Methods （1） Detection of cell proliferation and autophagy by matrine and imatinib ： the K562 and K562/IM cells were divided into the blank control group, the different concentrations matrine （0.25, 0.50, 1.00, 2.00, 5.00 mg/mL） group and imatinib（0.05, 0.25, 0.50, 5.00, 20.00 mol/L） group respectively. The inhibition rate of the cells were detected by methyl thiazolyl tetrazolium （MTT） method. At the same time in K562 and K562/IM cells, the blank control group, 0.4 mg/mL matrine group and 0.5 mol/L imatinib group were set up, the autophagy of K562 cell and K562/IM cell were detected by immunofluorescence confocal laser, autophagy protein expression of Beclin-1, LC3-Ⅱ/I and P62 weredetected by Western Blot.（2） Detection of autophagy and apoptosis of K562/IM induced by matrine, 3-MA and Rapa： the K5623/IM cells were divided into 6 groups as the blank control group, matrine group （0.4 mg/mL matrine）,3-MA group （5 mmol/L3-MA）, Rapa group （0.5 μmol/L）, matrine ＋ 3-MA group （0.4 mg/mL matrine＋5 mmol/L3-MA）and matrine＋ Rapa group（0.4 mg/mL matrine ＋0.5 μmol/L Ra- pa）. The inhibition rate of the cells were detected by MI-I- method. The apoptosis rate was measured by flow cytometry （FCM）.Western Blot was adopted to measure the expression of Beclin-1, LC3-Ⅱ/I , P62 and apoptosis-related Caspase-3. Results （ 1 ） Matrine and imatinib affected K562 and K562/IM cells ; the proliferation was inhibited （P 〈0.05）, LC3- Ⅱ/ I aggregation increased significantly, Beclin-1 and LC3- Ⅱ / I protein expression increased, and P62 protein expression decreased （P 〈0.05）, especially in K562/IM cells（P 〈0.05）. （2） In matrine group, the inhibition rate of K562/IM cell was （36.32 ＋ 2.21 ）%, and the apoptosis rate was （15.35 ±0.39）%. The inhibition rate of K562/IM cells in matrine ＋ 3-MA group in- creased [（65.25 ±2.06）%,P〈0.01],theapoptosisrateincreased [（21.70 ± 0.48）%,P〈0.05],the expression of Beclin-1 and LC3-Ⅱ/I protein decreased, the expression of P62 protein increased, and the expression of Caspase-3 increased （P 〈0.05）; while the effects were opposite in matrine ＋ Rapa group. Conclusions Both matrine and imatinib can inhibit the proliferation of K562 and K562/IM cells and induce autophagy, which is obvious in the drug resistant cell lines. Inhibition of autophagy by 3-MA can enhance the sensitivity of matrine to cell K562/IM, while rapamycin attenuates the role of matrine.