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2018隐丹参酮诱导乳腺癌 MDA―MB―231 细胞凋亡的研究

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发表于 2018-8-16 22:57:58 | 显示全部楼层 |阅读模式
  [摘要] 目的 探?隐丹参酮对乳腺癌MDA-MB-231细胞凋亡的影响及其机制。 方法 用不同浓度的隐丹参酮处理MDA-MB-231细胞24 h。采用 MTT、流式细胞术检测不同浓度隐丹参酮对MDA-MB-231细胞活性、凋亡的影响。采用Western blot检测MDA-MB-231细胞中Bcl-2、Bax、Caspase-3蛋白表达水平。 结果 与0 μmol/L对照组相比,10 μmol/L、20 μmol/L、40 μmol/L、80 μmol/L隐丹参酮组MDA-MB-231细胞存活率显著降低,且呈剂量依赖(P0.05)。在40 μmol/L、80 μmol/L浓度时MDA-MB-231细胞凋亡率分别是(18.74±0.65)%、(28.04±3.08)%,与对照组相比有统计学差异(P http://
  [关键词] 隐丹参酮;乳腺癌;MDA-MB-231细胞;凋亡
  [中图分类号] R273 [文献标识码] A [文章编号] 1673-9701(2017)34-0038-05
  Study of cryptotanshinone inducing apoptosis of breast cancer MDA-MB-231 cells
  ZHOU Nanyang1 ZHAO Hong2
  1.Department of Traditional Chinese Medicine, Hangzhou Obstetrics and Gynecology Hospital, Hangzhou 310008, China; 2.Department of Breast Surgery, the First Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine, Hangzhou 310006, China
  [Abstract] Objective To investigate the effect of cryptotanshinone on the apoptosis of breast cancer MDA-MB-231 cells and its mechanism. Methods MDA-MB-231 cells were treated with different concentrations of cryptotanshinone for 24h. The effects of different concentrations of cryptotanshinone on the activity and apoptosis of MDA-MB-231 cells were detected by MTT and flow cytometry. Western blot was used to detect the protein expressions of Bcl-2, Bax and Caspase-3 in MDA-MB-231 cells. Results Compared with that of the 0 μmol/L control group, the survival rates of MDA-MB-231 cells in 10, 20, 40, 80 μmol/L cryptotanshinone groups were significantly decreased(P0.05). The apoptosis rates of MDA-MB-231 cells were (18.74±0.65)% and (28.04±3.08)% respectively at 40 μmol/L and 80 μmol/L, which were different from those of the control group(P    [Key words] Cryptotanshinone; Breast cancer; MDA-MB-231 cells; Apoptosis
  乳腺癌是女性最常见的恶性肿瘤之一,其发病率逐年增加,目前居女性癌死亡率第一位[1]。其中,以雌激素、孕激素及人类表皮生长因子受体2(human epidermal growth factor receptor-2,Her-2)表达阴性的三阴乳腺癌(triple negative breast cancer,TNBC)恶性程度最高,因其对内分泌治疗和抗Her-2靶向治疗不敏感,故生存率比非三阴性乳腺癌差[2,3]。针对抗三阴乳腺癌的新药研究成为目前乳腺癌治疗的重点之一。隐丹参酮(Cryptotanshinone)是从中药丹参根中提取分离的二萜醌类有效单体,具有抗炎、抗菌、抗动脉粥样硬化等多种生物学活性和药理效应[4,5]。近年来文献报道,隐丹参酮可有效抑制多种肿瘤细胞增殖、侵袭,诱导肿瘤细胞凋亡,抑制血管生成,从而发挥其抗肿瘤的作用[6,7]。目前,有关隐丹参酮对乳腺癌细胞凋亡的影响及其机制尚不清楚。本研究以三阴乳腺癌 MDA-MB-231细胞为研究对象,用不同浓度隐丹参酮干预,检测其对细胞活性和凋亡的影响,检测抗凋亡蛋白Bcl-2和促凋亡蛋白Bax、Caspase-3表达水平,从而观察隐丹参酮对MDA-MB-231细胞凋亡的影响,并初步探究其机制。
  1 材料?c方法
  1.1 材料来源
  乳腺癌MDA-MB-231细胞购自中国科学院上海生命科学研究院细胞库,本实验室传代保存。胎牛血清和 RPMI 1640 培养基购自Hyclone 公司。隐丹参酮购自成都曼思特生物科技有限公司(纯度大于98%)(图1),将隐丹参酮溶于 DMSO配制成等20 mmol/L的储备液,分装后于-20℃储存;使用时用无血清 RPMI 1640 培养液稀释为10 μmol/L、20 μmol/L、40 μmol/L、80 μmol/L的工作液,DMSO 终浓度为 0.1%,对照组为含0.1% DMSO的细胞培养液。兔抗人Bcl-2、Bax、Caspase-3和 β-actin抗体和辣根过氧化物酶标记山羊抗兔 IgG购自 Cell Signaling Technology公司。Bradford蛋白浓度测定试剂盒购自美国Bio-Rad公司。流式抗体 Annexin V-FICT/PI双染细胞凋亡检测试剂盒购自南京凯基生物。
  1.2 方法
  1.2.1 细胞培养 乳腺癌 MDA-MB-231 细胞在37℃,5% CO2条件下,常规培养于含 10%胎牛血清的 RPMI 1640 培养基。当细胞密度达 90%并处于对数生长期时,用 0.25%胰酶消化传代培养。
  1.2.2 细胞活性检测 取对数生长期的MDA-MB-231细胞,接种于96孔板,每孔100 μL细胞悬液,细胞数1×104/孔,每组设5个复孔。培养 24 h后,吸弃原培养液,各孔分别加入 200 μL 含有不同浓度隐丹参酮的工作液(0、10 μmol/L、20 μmol/L、40 μmol/L、80 μmol/L),同时设调零孔。继续培养24 h后,利用 MTT 法在波长490 nm 处检测各组样品吸光度值(A490)。细胞存活率=(实验组A值-对照组A值)/(对照组A值-空白组A值)×100%,对照组定义为100%。实验重复3次。
  1.2.3 细胞凋亡检测 取对数生长期的MDA-MB-231细胞,接种于6孔板,3×105/孔,每组设3个复孔。培养24 h后,吸弃原培养液,以不同浓度隐丹参酮(0、20 μmol/L、40 μmol/L、80 μmol/L)处理细胞。继续培养24 h后,收集1×105细胞,加入500 μL Binding Buffer悬浮细胞,加入 5 μL Annexin V-FITC和5 μL Propidium Iodide 混匀后,温室避光孵育染色10 min。用流式细胞仪进行检测。实验重复 3 次。
  1.2.4 Western blot 检测蛋白表达 取对数生长期MDA-MB-231细胞,接种于6孔板,1×106/孔,待细胞融合至 80% 时,以不同浓度隐丹参酮(0、40 μmol/L、80 μmol/L)处理细胞。继续培养24 h,收集细胞,4℃预冷的PBS洗涤3次,加入75 μL细胞裂解液,冰上裂解30 min,提取细胞总蛋白,bradford法测蛋白质浓度。取 25 μg蛋白样品,10% SDS-PAGE 电泳分离蛋白后转移至 PVDF 膜,用5% BSA/TBST封闭液室温下封闭1 h,洗膜后加入抗Bcl-2、Bax、Caspase-3和 β-actin 一抗(1∶1 000稀释 ),4°C孵育过夜,TBST洗膜 3 次,加入二抗(1∶1 000稀释)室温下孵育2 h,TBST 洗膜 3 次,将化学发光增强液A和B等体积混匀涂抹于PVDF膜上,用Bio-Rad凝胶成像系统获取图像。实验重复3次。
  1.3 统计学方法
  采用 SPSS 17.0 统计学软件分析,计量资料以(x±s)表示,多组间比较采用单因素方差分析,两两比较采用 Bonferroni 校正的t检验,P    2.2 隐丹参酮对MDA-MB-231细胞凋亡的影响
  本研究用Annexin V-FICT/PI双染流式细胞术检测隐丹参酮对MDA-MB-231细胞凋亡的影响,结果显示,隐丹参酮在20 μmol/L浓度时对MDA-MB-231细胞凋亡率为(6.34±0.52)%,与对照组(6.09±0.76)%相比无统计学差异;隐丹参酮在40 μmol/L、80 μmol/L浓度时??MDA-MB-231细胞凋亡率分别是(18.74± 0.65)%,(28.04±3.08)%。与对照组(6.09±0.76)%相比差异具有统计学意义(P参考文献]
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  (收稿日期:2017-09-29)
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