【作者】 余志艳；

【导师】 李平；

【作者基本信息】 安徽医科大学 ， 老年医学， 2008， 硕士

【摘要】 目的:马钱子碱(Brucine)为吲哚型生物碱,是马钱子的主要有效成份之一。本研究旨在观察Brucine对人乳腺癌细胞MDA-MB-231的增殖抑制和诱导凋亡作用,并分析Brucine在诱导细胞凋亡过程中对Bcl-2、Bax和Caspase-3蛋白表达的调控作用,为Brucine用于乳腺癌的临床治疗提供实验依据。方法:人乳腺癌细胞株MDA-MB-231(雌激素受体阴性)以L-15培养基孵育,至对数生长期时,分别以Brucine刺激24h、48h和72h,Brucine浓度为50mg/L、100mg/L、200 mg/L、400 mg/L。用MTT法测定Brucine对细胞的增殖抑制作用;倒置显微镜观察细胞形态;Brucine处理结束后,收集细胞制成单细胞悬液,加入碘化丙啶(PI)后以流式细胞仪分析细胞周期并计算凋亡率的变化:加入AnnexinV-FITC和PI后以流式细胞术分析细胞凋亡。为检测Bcl-2、Bax和Caspase3分子在Brucine诱导乳腺癌细胞凋亡过程中的作用,以中和性抗体进行阻断实验。为检测Brucine作用后Bcl-2、Bax和Caspase3蛋白水平的变化,以50mg/L、100mg/L、200 mg/L、400 mg/L的Brucine处理乳腺癌细胞48h后,裂解乳腺癌细胞后收集蛋白并进行定量分析,经聚丙烯酞胺凝胶电泳分离蛋白后以半干式电转移将蛋白转移到硝酸纤维素膜上,以Western蛋白印迹法检测Bcl-2、Bax和Caspase3蛋白水平。结果:(1)细胞形态学观察结果:空白对照组细胞贴壁均匀,状态良好,各干预组贴壁细胞明显减少,培养液内细胞碎片增多,且随着药物剂量的增加和干预时间的延长更明显。(2)MTT比色试验结果:各剂量组OD值较空白对照组显著降低(P＜0.01),而且与干预剂量、干预时间成反比,有显著统计学差异(p＜0.01)。(3)PI流式细胞仪检测结果:随着Brucine浓度的增加和作用时间的延长细胞凋亡率逐渐增高并呈明显的浓度、时间依赖,与对照组相比差异有显著性(P＜0.05);同时S期细胞逐渐减少,G0/G1期细胞逐渐增多(P＜0.05),细胞被阻滞在G1/S期。(4)Annexin V—FITC/P1分析结果:与空白对照组相比,各干预组早期调亡细胞所占比例显著增加(p＜0.01),各剂量组间也有显著差异(p＜0.01)。(5)Western blotting检测结果:作用48h后,各剂量组与空白对照组相比,抗凋亡基因Bcl-2表达水平明显降低,而促凋亡基因Bax明显增高,凋亡执行因子中Caspase-3表达增加。结论:Brucine能够有效的抑制乳腺癌细胞株MDA-MB-231细胞的增殖,呈明显浓度、时间依赖;Brucine能够诱导乳腺癌MDA-MB-231细胞凋亡,其分子机制可能与激活Bax及抑制Bcl-2等凋亡调控基因有关;Bcl-2的表达降低有可能诱发Caspase-3激活,促使细胞凋亡。更多还原

【Abstract】 Objective: Brucine, a main component of nux vomica, is one of indole alkaloids. In the study, we observe the effect of Brucine on proliferation inhibition and apoptosis induction on breast cancer cell MDA-MB-231. And analyze the regulation of Brucine on the expression of Bcl-2, Bax and Caspase-3 protein, thus provide an experimental basis for the clinic.Methods: The breast cancer cell MDA-MB-231 was cultured in L-15 medium. At the logarithmic growth phase, Brucine was added, the concentration of which was 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L, to stimulate the cells for 24h, 48h, 72h. MTT assay was used to measure the proliferation inhibitory effect of Brucine on the cells. The changes of cellular morphology were observed under inverted microscope. After treatment, cells were collected to prepare single cell suspension. Breast cancer cells were collected and resuspended in PBS. PI was added and the cell cycle was analyzed with flow cytometry while the apoptotic rate was calculated at the same time. PI and Annexin V-FITC were added to analyze the early apoptosis. To analyze the function of Bcl-2, Bax, Caspase-3 protein in the process of apoptosis induction, blocking assay was applied by neutralized antibody. Breast cancer cells were treated with Brucine for 48h at the concentrations of 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L. Then the cells were lysed and protein was quantified and separated with SDS-PAGE and transferred to a nitrocelular membrane with a semi-dry apparatus. The level of Bcl-2, Bax, Caspase-3 protein was detected by Western blotting assay.Results: (1) Cellular morphology: Cell adherence in control group were homogeneous and in good condition. Cell adherence in intervention group fall Off obviously , cell debris increased, and had doses and time dependent. (2)MTT: The optical density degraded significantly in every intervention group(p<0.01), and had doses and time dependent. (3)PI simple staining and flow cytometry analysis: Cell apoptotic rate increased with the increment of the concentration and treatment time, and has doses and time dependent. It had significant deviation to control group (p<0.05). S phase cells decreased while G0/G1 phase cells increased (p<0.05) and cells were blocked at G1/S phase. (4) Annexin V-FITC/PI analysis: In contrast to the control group, the early apoptosis cells increased significantly in all intervention groups (p<0.01) and had significantly differences between each intervention groups. (5) Western blotting: After treatment for 48 hours, Bcl-2 level was decreased, Bax level and Caspase-3 level were increased.Conclusion: Brucine can inhibit the proliferation of MDA-MB-231 cells effectively with doses and time dependent. Brucine can induce the apoptosis of MDA-MB-231 cells, the molecular mechanism of which is related with the activation of Bax and inhibition of Bcl-2. The decreased expression of Bcl-2 stimulates the activation of Caspase-3. The decreased expression of Bcl-2 may stimulate the release of cytochrome C, and activate Caspase-3 and Caspase-3 cascade through a key point in its downstream, then induce apoptosis.更多还原