ABL-N对L1210细胞增殖和凋亡的影响及其作用机制

发布时间：2018-01-06 21:04

  本文关键词：ABL-N对L1210细胞增殖和凋亡的影响及其作用机制　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： ABL-N Bax Bcl-2 p53 白血病 凋亡

【摘要】：1-O-乙酰基大花旋覆花内酯(ABL)是欧亚旋覆花中含量较高的活性成分之一,为倍半萜内酯类化合物,具有抗肿瘤活性,但其稳定性和溶解性都不太好,本实验所用的药物是ABL的衍生物,简称ABL-N。目的:观察ABL-N对小鼠白血病细胞L1210增殖和凋亡的影响,并初步探讨其作用的机制。 方法: (1) MTT法测定ABL-N对小鼠白血病细胞L1210增殖的影响:取细胞培养于96孔培养板内,培养24 h后,给药组加入不同浓度的ABL-N溶液,对照组加与最高浓度组等量的DMSO。分别培养24 h、48 h、72 h后用Fluo-star在检测波长570 nm条件下测定光密度值(OD value),计算各浓度的ABL-N对L1210细胞的抑制率。(2)台盼蓝拒染法检测ABL-N对L1210存活细胞数的影响。取对数生长期的细胞,给药组给予不同浓度的ABL-N溶液,对照组加与最高浓度组等量的DMSO。培养后的分别于第24 h、48 h、72 h取样,台盼蓝染色,计数活细胞,用活细胞数对时间绘制生长曲线。(3)通过瑞氏-吉姆萨染色和Hochest 33258染色观察ABL-N作用48 h后L1210细胞的形态变化。收集ABL-N(20 mg/L)处理后的细胞,进行染色,观察细胞形态学变化。(4)用流式细胞仪测定ABL-N作用后L1210细胞的凋亡率和周期分布。取对数生长期的细胞,给予ABL-N(10、20 mg/L)作用48 h后,PI染色,在流式细胞仪上测定各组细胞的凋亡率和周期分布。(5)Western-blot法检测ABL-N作用后L1210细胞Bax和Bcl-2蛋白表达的变化。(6)免疫荧光法检测ABL-N作用后L1210细胞p53蛋白表达的变化。 结果: (1)用不同浓度ABL-N(5、10、20 mg/L)分别处理L1210细胞24 h后抑制率分别为14.6%、38.5%、47.4%。作用48 h时抑制率分别为28.8%、63.2%、74.8%。72 h后抑制率分别为35.7%、81.5%、91.6%。表明ABL-N可明显抑制L1210细胞的增殖,并呈现出浓度和时间依赖性。(2)用不同浓度ABL-N(5、10、20 mg/L)作用于L1210后,从生长曲线可以看出明显的浓度效应关系,且高浓度组加药1 d后及中浓度组加药2 d后,活细胞数量减少,说明ABL-N对体外培养的小鼠白血病L1210细胞有较强的抑制作用及杀灭作用。(3)本研究采用Giemsa染色及Hoechst 33258荧光染色方法分别观察了ABL-N作用于L1210细胞48 h后的形态变化,2种染色方法均显示给药组细胞呈现核聚集和碎裂等凋亡特征性形态学变化。(4)不同浓度的ABL-N(10、20 mg/L)作用48 h后凋亡率分别为13.6%、29.9%。溶剂对照组的凋亡率为3.57%。可见ABL-N作用后凋亡率明显的增加。(5)ABL-N(10、20 mg/L)作用48 h后G1期细胞数量由26.8%增加到30.9%、51.4%,S期细胞数量由73.2%下降到69.1%、48.6%。(6)不同浓度的ABL-N(5、10、20 mg/L)作用48 h后,Bax/β-actin比例由0.49分别增加到0.58、0.71(P0.01)、0.82(P0.01)。Bcl-2/β-actin比例由0.66分别下降到0.61、0.57(P0.05)、0.28(P0.01)。Bcl-2/Bax比值由1.36分别下降到1.17、0.80(P0.01)、0.34(P0.01)。(7)对照组p53蛋白表达的荧光强度为63.5。不同浓度的ABL-N(5、10、20 mg/L)作用48 h后的荧光强度分别为83.3(P0.05)、188.0(P0.01)、242.5(P0.01),有了明显的增强。 结论: (1)ABL-N能明显抑制小鼠白血病细胞L1210的增殖。(2)ABL-N对L1210细胞有明显的细胞毒性。(3)ABL-N影响细胞周期动力学,使细胞停滞在G1期。(4)ABL-N抑制L1210细胞增殖作用可能与影响细胞周期动力学有关。使细胞停滞在G1期,影响了DNA的合成。(5)ABL-N能诱导L1210细胞凋亡,这可能与上调p53和Bax,下调Bcl-2蛋白的表达有关。其上调p53蛋白表达可能与使细胞停滞在G1期有关。
[Abstract]:1-O- acetyl flower 1-o-acetylbritannilactone (ABL) is one of the active ingredients of Inula britannica content high, is a sesquiterpene lactone compound, has antitumor activity, but its stability and solubility are not too good, drugs used in this experiment is a derivative of ABL ABL-N. Objective: the effect of ABL-N on proliferation and apoptosis of L1210 mouse leukemia cells, and to explore its mechanism.
Method:
(1) the effect of ABL-N on the proliferation of leukemia cells in L1210 mice was determined by MTT assay: cells cultured in 96 well plates, cultured 24 h, the drug group added different concentrations of ABL-N solution, the control group was treated with the highest concentration of equal amounts of DMSO. were cultured for 24 h, 48 h, determination of optical density values in the the detection wavelength of 570 nm under the condition of 72 h Fluo-star (OD value), to calculate the concentration of ABL-N the inhibition rate of L1210 cells. (2) effect of trypan blue dye exclusion assay for the detection of ABL-N L1210. The number of viable cells in logarithmic growth phase cells, ABL-N solution treatment groups were given different concentrations. The control group was treated with the highest concentration of equal amounts of DMSO. cultured in twenty-fourth h, 48 h, 72 h sampling, trypan blue staining, counting live cells, draw the growth curve of time with the number of living cells. (3) by Wright Giemsa staining and Hochest staining was observed in 33258 ABL-N 48 h L1210 cell morphology Change. Collect ABL-N (20 mg/L) treated cells, staining, morphological changes were observed. (4) with the rate of apoptosis and L1210 cell cycle distribution was determined by ABL-N flow cytometry. After logarithmic growth phase cells treated with ABL-N (10,20 mg/L) 48 h, PI staining, apoptosis the rate and cycle distribution of cells was measured by flow cytometer. (5) the expression changes of L1210 cell Bax detection ABL-N Western-blot method and Bcl-2 protein. (6) the expression changes of L1210 cells p53 ABL-N protein was detected by immunofluorescence assay after effect.
Result:
(1) with different concentrations of ABL-N (5,10,20 mg/L) respectively. The treatment rate was 14.6%, suppressed after 24 h L1210 cells 38.5%, 47.4%. 48 h when the inhibition rates were 28.8%, 63.2%, 35.7% respectively, the inhibition of H 74.8%.72 81.5%, 91.6%. showed that ABL-N can inhibit the proliferation of L1210 cells, and showed a time and concentration dependent. (2) with different concentrations of ABL-N (5,10,20 mg/L) in L1210, see the obvious concentration effect relationship from the growth curve, and the high concentration group after 1 D and the concentration of drug dosing group after 2 D, the number of living cells decreased, indicating that ABL-N has strong inhibition and killing effect the in vitro murine leukemia L1210 cells. (3) the study of morphological changes by Giemsa staining and Hoechst were used to observe the effect of ABL-N on L1210 cells after 48 h 33258 fluorescence staining method, 2 kinds of staining methods showed that drug treated cells showed nuclear accumulation and broken 瑁傜瓑鍑嬩骸鐗瑰緛鎬у舰鎬佸�﹀彉鍖�.(4)涓嶅悓娴撳害鐨凙BL-N(10,20 mg/L)浣滅敤48 h鍚庡噵浜＄巼鍒嗗埆涓，

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