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题名	鹰嘴豆豆芽标准提取物对乳腺癌的干预及机制研究
作者	陈花
学位类别	博士
答辩日期	2016-06-03
授予单位	中国科学院大学
授予地点	北京
导师	阿吉艾克拜尔·艾萨
关键词	鹰嘴豆芽 异黄酮 线粒体依赖性细胞凋亡 乳腺癌 化学预防
学位专业	有机化学
中文摘要	鹰嘴豆是豆科鹰嘴豆属的一年生栽培植物。鹰嘴豆作为维吾尔医常用药材，在维吾尔族传统医学中，被用来治疗糖尿病、高脂血症、骨质疏松症和癌症等。异黄酮类植物雌激素是鹰嘴豆及其豆芽中重要的有效成分，主要包括鹰嘴豆芽素A、芒柄花素、染料木素、毛蕊异黄酮、芒柄花苷、印度黄檀苷、三叶豆紫檀苷等，其中豆芽中异黄酮含量相对较多。我们前期研究工作发现，鹰嘴豆豆芽标准提取物对乳腺癌细胞增殖具有双重调节功能。为进一步探索它干预乳腺癌的作用及机制，本文制备了鹰嘴豆豆芽标准提取物（ICS），利用LC/ESI-MS/MS解析其化学成分，在细胞水平和整体动物水平上探讨其诱导乳腺癌细胞凋亡作用及干预大鼠乳腺癌作用。本论文包括下列三个部分：1. LC/ESI-MS/MS分析鹰嘴豆豆芽标准提取物的主要化学成分建立了用HPLC/QTOF-MS/MS快速分离及鉴定鹰嘴豆豆芽标准提取物中的化学成分的方法，通过精确分子量、特征性离子的测定，推测了可能的10种化学成分，并通过与标准品的比对确定该样品中含量较高的4种成分为鹰嘴豆芽素A（Biochanin A）、芒柄花素（Formononnetin）、刺芒柄花苷（Ononin）、印度黄檀苷（Sissotrin）。2. 鹰嘴豆豆芽标准提取物通过线粒体依赖性途径促进乳腺癌细胞凋亡研究研究了ICS对人乳腺癌SKBr3、MCF-7细胞增殖和细胞凋亡的影响及其机制。MTT法观察ICS对人乳腺癌细胞增殖的影响，结果显示10～60 μg/mL的 ICS均能明显抑制两种乳腺癌细胞增殖，且具有量效和时效关系。通过瑞士-吉姆萨染色和Annexin V-FITC/PI双染法观察到ICS能显著诱导两种细胞发生凋亡，甚至出现“凋亡小体”。流式细胞术定量检测细胞凋亡，结果显示ICS诱导两种乳腺癌细胞凋亡作用显著，且呈浓度依赖性。采用DCFH-DA探针检测人乳腺癌SKBr3、MCF-7细胞内活性氧（ROS），结果显示ICS能明显增加两种细胞内ROS的产生。采用DiOC6（3）法测定线粒体膜电位，结果显示ICS处理后两种乳腺癌细胞的线粒体膜电位显著降低。RT-PCR检测结果显示ICS处理后两种乳腺癌细胞中bax基因表达上调，而bcl-2基因表达下调，使bcl-2/bax比值下降。Western blot检测结果显示ICS处理后两种乳腺癌细胞中Caspase-7、Caspase-9、P53和P21表达上调。综上所述，ICS处理ER阳性的MCF-7细胞和ER阴性的SKBr3细胞，可诱导其通过线粒体依赖性途径发生细胞凋亡。3. 鹰嘴豆豆芽标准提取物对大鼠乳腺癌的预防作用研究通过二甲基苯蒽（DMBA）诱导的雌性SD大鼠乳腺癌模型，研究ICS体内的癌化学预防作用及防癌机制。每周记录各组大鼠体重及乳腺癌的发生时间、个数和大小。结果显示，与模型组比较，ICS高剂量组大鼠明显增重；模型组给予DMBA后第7周开始出现可触及肿块，而ICS低、高剂量组推迟到12、14周开始出现；与模型组相比，ICS低、高剂量组的肿瘤发生率均显著降低；ICS高剂量组与在肿瘤平均数目、体积和重量上均明显小于模型组。取胸腺、脾脏、肝脏、肾脏称湿重，计算脏器指数，与模型组比较，ICS高剂量组的胸腺指数明显上升，ICS低、高剂量组脾脏指数升高。检测大鼠血清、肝脏中抗氧化酶系的活力及氧化产物浓度，与模型组相比，ICS低、高剂量组的GSH-PX和CAT活性明显升高，ICS高剂量组的SOD活性显著上升，MDA浓度显著下降。采用ELISA法检测大鼠血清中的TNF-α、TGFβ1及MMP-9含量，与模型组比较，ICS高剂量组TNF-α、TGFβ1、MMP-9浓度明显降低。采用免疫组化方法测定肿瘤组织中PCNA的阳性表达，镜下观察到PCNA与模型组相比，ICS低、高剂量组的阳性细胞百分数明显降低。病理组织学观察结果显示各组大鼠乳腺均发生癌变。综上所述，ICS能明显延迟DMBA诱发的乳腺肿瘤发生时间，降低肿瘤诱发率，且能清除血及肝脏中氧化应激产生的氧自由基，增强乳腺癌大鼠免疫系统的抵抗能力，抑制肿瘤细胞的增殖。提示，ICS具有一定的乳腺癌预防作用。
英文摘要	Cicer arietinum L. (chickpea) is an annual leguminous plant which served as a traditional Uighur herb for over 2500 years in Xinjiang, China and was used to treat diabetes, hyperlipidemia, osteoporosis, cancer, and so on. Iso?avones are the important chemical components of the seeds and sprouts of chickpea. Eight isoflavones, including biochanin A, formononetin, genistein, calycosin, biochanin A-7-O-β-D-glucoside, trifolirhizin, ononin, and sissotrin, are found in chickpea seeds and sprouts. The isoflavone content of chickpea sprouts is substantially higher than that of chickpea seeds. In previous study, we found that the standardized extract from chickpea sprouts (ICS) have dual functions. There have been no reports evaluating the mechanism by which ICS inhibits breast cancer. In the present study, we identified bioactive compounds present in ICS and investigated the effects of ICS on the breast cancer in vivo and in vitro, and explored the related mechanism as follows. The present study included three parts: 1. LC/ESI-MS/MS analysis on the standardized extract from chickpea sproutsThe phytocomponents present in ICS was analyzed by LC/ESI-MS/MS. According to the diagnostic product ions and accurate mass measurements, fourteen components were identified. We confirmed that ICS is a complex mixture mainly containing four isoflavones, including biochanin A, formononetin, ononin, and sissotrin.2. The standardized extract from chickpea sprouts induce mitochondria mediated apoptosis in human breast cancer cellsWe systematically investigated the effects of ICS on the human breast cancer cell lines SKBr3 and MCF-7, and explored the related mechanism. MTT assays showed that ICS (10～60 μg/mL) significantly inhibited the proliferation of both cell lines in a time-dependent and dose-dependent fashion. Wright-Giemsa staining as well as Annexin V-FITC staining showed that ICS significantly increased cytoclasis and apoptotic body formation. Quantitative Annexin V/PI assays further showed that the number of apoptotic cells increased in a dose-dependent manner following ICS treatment. Flow cytometry assays using the fluorescent probe 3, 3’-dihexyloxacarbocyanine iodide showed a dose-dependent decrease in mitochondrial membrane potential following ICS treatment. Treatment using ICS also induced a dose-dependent increase in ROS production. Semi-quantitative reverse transcription PCR showed that ICS increased the expression of the apoptosis-promoting gene bcl-2-associated X protein and decreased the expression of the antiapoptotic gene bcl-2. Western blot analysis showed that treatment of SKBr3 and MCF-7 cells with ICS increased the expression of Caspase-7, Caspase-9, P53, and P21 in a dose-dependent manner. In conclusion, we found that ICS induced mitochondria mediated apoptosis in ER-positive and ER-negative human breast cancer cell lines.3. Chemopreventive effect of the standardized extract from chickpea sprouts against mammary carcinoma in ratsWe systematically investigated the cancer chemopreventive effects of ICS and elucidated the related mechanism by testing it against mammary carcinogenesis induced by DMBA in female Sprague-Dawley rats. During the experimental period, body weight were recorded and animals were palpated for the date of appearance, the number and location of tumors weekly. Animals fed high dose ICS diets showed significantly higher weight gains when compared with animals in the model group. Tumor latency was increased from 7 wk after animals received DMBA in the model group to 12 wk and 14 wk in the groups that were fed 250 and 500 mg/kg ICS diets, respectively. Animals treated with DMBA developed mammary tumors with 88.89% tumor incidence. Inhibition of mammary cancer incidence by low dose ICS (42.86%), high dose ICS (25.0%) were observed. ICS also significantly reduced tumor number, weight, and volume at high ICS dose compared with the model group. Moreover, as compared with the model group, the index of thymus in the high dose ICS group was increased significantly, meanwhile, the index of spleen in the low and high dose ICS group were increased dramatically. Histopathology showed that all the groups of animals suffered breast cancer. Administration of ICS and genstein suppressed breast cancer development and was associated with an increase in SOD, GSH-PX, and CAT levels and with a decrease in MDA, TNF-α, TGFβ1, MMP-9 level. Rats administered DMBA developed breast cancer, which was also associated with increased expression of PCNA in mammary tissues. Compared with model group, the percentage of PCNA in the low, high dose ICS group decreased significantly. Our results suggest that ICS is potent antioxidant and offer maximum protection against DMBA-induced mammary carcinogenesis.
内容类型	学位论文
源URL	[http://ir.xjipc.cas.cn/handle/365002/4581]
专题	新疆理化技术研究所_资源化学研究室
作者单位	中国科学院新疆理化技术研究所
推荐引用方式 GB/T 7714	陈花. 鹰嘴豆豆芽标准提取物对乳腺癌的干预及机制研究[D]. 北京. 中国科学院大学. 2016.

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