飞燕草素联合3-MA增敏抗HER-2阳性乳腺癌细胞效应

目的:研究飞燕草素(delphinidin,Dp)联合自噬抑制剂3-甲基腺嘌呤(3-Methyladnine,3-MA)对HER-2+乳腺癌细胞MDA-MB-453的抑制效应。方法:用不同浓度飞燕草素处理MDA-MB-453细胞,CCK-8检测细胞增殖情况;荧光斑点实验和Weston Blot实验检测飞燕草素对细胞自噬的诱导情况;Weston Blot实验检测3-MA对自噬的抑制状况,CCK-8检测飞燕草素联合3-MA对细胞增殖状况的影响。结果:飞燕草素能有效抑制MDA-MB-453细胞增殖,诱导MDA-MB-453细胞自噬并呈剂量依赖效应;3-MA有效抑制MDA-MB-453细胞自噬,同时增强自噬飞燕草素抗HER-2+乳腺癌细胞MDA-MB-453增殖效应。结论:飞燕草素联合自噬抑制剂3-MA,能促进飞燕草素抗HER-2+乳腺癌细胞MDA-MB-453增殖。     

桑叶浸出液抑制MDA-MB-231细胞增殖和抑制Survivin蛋白表达

为了研究桑叶浸出液对乳腺癌MDA-MB-231细胞增殖和对凋亡抑制蛋白Survivin的作用。本实验采用体外培养MDA-MB-231细胞,倒置显微镜观察桑叶浸出液作用后细胞形态变化;CCK-8法检测桑叶浸出液对MDA-MB-231细胞增殖的影响;流式细胞仪分析桑叶浸出液对MDA-MB-231细胞凋亡及周期的影响;Western Blot印迹法检测桑叶浸出液对凋亡抑制蛋白Survivin表达的影响。结果表明桑叶浸出液会引起MDA-MB-231生长形态的变化,抑制细胞增殖,增加细胞凋亡数量,且在桑叶浸出液作用后MDA-MB-231细胞的G0/G1期细胞明显高于正常组(p<0.05),同时桑叶浸出液降低凋亡抑制蛋白Survivin的表达,且表达量随着桑叶浸出液浓度的升高而降低。研究表明,桑叶浸出液能够抑制MDA-MB-231细胞的增殖,促进细胞凋亡及导致G0/G1期阻滞,同时能够降低凋亡抑制蛋白Survivin的表达。     

知母水溶性小分子提取物对胰岛素抵抗HepG2细胞体外糖代谢的影响

目的 研究知母水溶性小分子提取物的制备及其对胰岛素抵抗HepG2 (IR-HepG2)细胞糖代谢的作用.方法 采用水提取,膜分离方式制备知母提取物;采用高浓度胰岛素持续作用于HepG2细胞24 h建立胰岛素抵抗模型,加入知母水溶性小分子提取物保温24 h后,测定对照组和加药组细胞内葡萄糖消耗量、肝糖原含量及己糖激酶(hexokinase,HK)、丙酮酸激酶(pyruvate kinase,PK)的活性.结果 知母水溶性小分子提取物明显增加细胞的葡萄糖消耗、提高IR-HepG2细胞HK、PK活性,增加肝糖原含量.结论 知母水溶性小分子提取物可明显改善胰岛素抵抗状态下HepG2细胞对葡萄糖的利用,提高肝HK、PK活性,促进葡萄糖进入肝细胞,增加肝糖原合成,促进糖代谢.     

Delphinidin抗乳腺癌EMT体外效应研究

研究飞燕草素(Delphinidin,Dp)抑制HER-2~+乳腺癌细胞(MDA-MB-453)和HER-2~-乳腺癌细胞(MCF-7)上皮细胞-间充质转化(EMT)效应。将不同浓度Dp分别处理MDA-MB-453和MCF-7细胞48 h,于倒置显微镜观察细胞形态及数量变化后,用蛋白质免疫印迹法和逆转录聚合酶链反应检测EMT标志性蛋白E-cadherin、N-cadherin、Vimentin、Fibronectin及关键调控因子锌指转录因子(Snail 1)表达。80μmol/L和100μmol/L的Dp能使MDA-MB-453和MCF-7乳腺癌细胞的E-cadherin标志蛋白表达上调,而降低与细胞迁移和侵袭能力有关的N-cadherin、Vimentin、Fibronectin及Snail因子表达水平。Dp体外具有抗MDA-MB-453和MCF-7乳腺癌EMT的效应,研究结果可为植物黄酮类化合物抗乳腺癌转移及辅助治疗提供新支撑。     

RNA干扰技术抑制乳腺癌细胞系MDA-MB-231中SATB1的表达

构建含有靶向satb1基因的siRNA质粒,体外观察对乳腺癌细胞系MDA-MB-231的SATB1mRNA以及蛋白质表达的影响。设计合成三对靶向人源satb1基因的siRNA,并分别克隆在质粒载体上,在脂质体的介导下转染高表达SATB1的乳腺癌细胞系MDA-MB-231。运用RealTime-PCR方法分析SATB1mRNA的表达情况,Western-Blot方法检测蛋白的表达量。结果表明:经过酶切鉴定和测序证实三个重组质粒psiS823、psiS2567、psiS3373分别构建成功,转染乳腺癌细胞系MDA-MB-231能明显抑制SATB1mRNA及蛋白的表达。说明本试验成功构建的重组质粒psiS823、psiS2567、psiS3373都能有效降低的人乳腺癌细胞MDA-MB-231中SATB1的表达,为SATB1高表达的乳腺癌基因治疗提供了新方法。     

5﹣十七烷基间苯二酚对三阴性乳腺癌MDA﹣MB﹣231细胞增殖的抑制作用

为研究5-十七烷基间苯二酚(AR-C17)对人乳腺癌MDA-MB-231细胞的作用及其潜在机制,采用CCK-8方法检测不同处理下的细胞存活率变化;DCF-DA荧光探针法检测活性氧(reactive oxygen species, ROS)变化情况;流式细胞术测定胞内肿瘤增殖标记物Ki-67的表达,细胞凋亡的变化,线粒体膜电位的变化及自噬体的形成情况;蛋白印迹法检测Bax、Bcl-2、LC3-Ⅱ等凋亡、自噬相关蛋白的表达情况。结果表明,与对照组相比,AR-C17显著抑制了MDA-MB-231细胞存活率,促进胞内ROS的生成,降低肿瘤增殖标志物Ki-67的表达,降低线粒体膜电位,上调Bax/Bcl-2的蛋白表达比率,增加活性Caspase的表达量,诱导MDA-MB-231细胞发生线粒体途径的凋亡;同时,AR-C17能够增加LC3-Ⅱ蛋白表达量,促进自噬体形成,诱导细胞发生自噬;此外,采用自噬抑制剂与AR-C17共同作用能够降低细胞存活率,进一步促进细胞凋亡。研究结果表明,AR-C17能够诱导人乳腺癌MDA-MB-231细胞发生凋亡和自噬,达到抑制乳腺癌细胞增殖的作用。     

FAK对黑米花青素抑制HER-2阳性乳腺癌转移的影响研究

目的:探讨粘着斑激酶(Focal Adhesion Kinase,FAK)在黑米花青素(Black Rice Anthocyanins,BRACs)抑制人表皮生长因子受体2(Human epidermal growth factor-2;HER-2)阳性乳腺癌转移中的作用。方法:以正常永生化乳腺细胞MCF-10A、HER-2阴性乳腺癌细胞MCF-7、HER-2阳性乳腺癌细胞MDA-MB-453为研究对象,使用Cell Counting Kits-8(CCK8)试剂盒检测细胞活性;伤痕愈合实验检测细胞转移状态;免疫印迹实验测定FAK、c Src表达及磷酸化水平;免疫共沉淀实验检测HER/FAK蛋白间相互作用。结果:与对照组相比,分别用50、100、200、400μg/m L BRACs处理MDA-MB-453细胞后,抑制率分别为18.74%、21.06%、22.82%、40.12%;200μg/m L BRACs处理后MDA-MB-453细胞迁移距离减少37%;FAK及c Src磷酸化水平降低,HER-2/FAK蛋白间相互作用减弱。结论:BRACs通过减弱FAK与HER-2受体相互作用抑制FAK及c Src磷酸化从而抑制MDA-MB-453细胞增殖和转移,这可能是黑米花青素抑制MDA-MB-453细胞增殖和转移的分子机制之一。     

柠檬皮多酚成分分析及其对胰岛素抵抗HepG2细胞糖代谢的影响

目的:探究柠檬皮多酚（Limon Peel Polyphenols,LPP）的组成成分,并研究其对胰岛素抵抗（Insulin Resistance,IR）的HepG2细胞糖代谢的影响。方法:采用高效液相色谱串联四级杆飞行时间质谱法（HPLC-QTOF-MS）分析LPP组成,利用HepG2细胞建立胰岛素抵抗模型,用LPP作用于IR-HepG2细胞,通过测定细胞葡萄糖消耗量初步探究LPP对糖代谢的影响,再通过测定糖原含量和己糖激酶（Hexokinase,HK）、丙酮酸激酶（Pyruvate Kinase,PK）、磷酸烯醇式丙酮酸羧基酶（Phosphoenol Pyruvate Carboxykinase,PEPCK）、葡萄糖六磷酸酶（Glucose-6-Phosphatase,G6Pase）的活性,探究LPP调节细胞糖代谢的作用途径。结果:经过HPLC-QTOF-MS分析出12种物质,主要为黄酮及其苷类成分;在糖代谢研究方面,与模型组相比浓度为0.1~2 mg/mL柠檬皮多酚可显著提高细胞葡萄糖消耗量（P<0.05）,且当浓度为0.5 mg/mL时其提高IR-HepG2细胞糖原含量和HK、PK活性,降低PEPCK和G6Pase活性的能力最接近于二甲双胍阳性对照。结论:柠檬皮多酚可以缓解IR-HepG2细胞的胰岛素抵抗状态,并能通过促进糖原合成、提高糖酵解关键酶活性、降低糖异生酶活力的方式调节糖代谢水平,为后续的体内研究提供了数据支持,并为未来开发功能性产品提供了理论依据。     

野生苦菜体外抗氧化及抑制肿瘤活性研究

以野生苦菜为原料,采用1,1-二苯基-2-三硝基苯肼(DPPH)清除能力、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)清除能力、羟基自由基清除能力和铁离子还原能力法评定其抗氧化能力,采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法评定苦菜提取物对雌激素依赖性人类乳腺癌细胞MCF-7和雌激素非依赖性人乳腺癌细胞MDA-MB-231的抑制活性。结果表明,苦菜甲醇提取物对ABTS、DPPH都具有较强的清除能力,且具有较强的铁离子还原能力,对雌激素依赖性人类乳腺癌细胞MCF-7和雌激素非依赖性人乳腺癌细胞MDA-MB-231均有显著的抑制活性。苦菜的甲醇提取物对MCF-7和MDA-MB-231半抑制率分别为99、107μg/mL。     

桦褐孔菌提取物抗乳腺癌活性部位筛选及成分分析

以人乳腺癌MDA-MB-231细胞筛选桦褐孔菌抗乳腺癌活性部位,并对其活性部位的成分进行初步分析。采用MTT法检测发酵液不同萃取部位抗乳腺癌活性;通过细胞形态观察进一步了解药物对细胞的影响;划痕试验检测桦褐孔菌活性部位对细胞迁移的影响;采用LC-MS/MS对活性部位的成分进行分析。结果表明,桦褐孔菌石油醚萃取部位对MDA-MB-231细胞增殖的抑制作用最强,且呈量效和时效关系,作用24 h的IC50值为76.17μg/mL;细胞形态观察显示石油醚萃取部位对乳腺癌MDA-MB-231细胞的凋亡作用随着药物质量浓度的升高而加强;划痕实验显示,药物对细胞迁移有较强的抑制作用;LC-MS/MS数据初步表征了其中的14个化学成分,初步鉴定了其中的10种成分,这10种成分首次从桦褐孔菌中得到鉴定,虽然其他4个物质未能鉴定,但亦对其相对分子质量和分子式进行了初步表征。证实了石油醚部位是桦褐孔菌抗乳腺癌MDA-MB-231细胞的主要活性部位,能抑制乳腺癌细胞增殖并诱导细胞凋亡,为进一步开发利用桦褐孔菌食药用资源提供科学依据。     

beta-Sitosterol enhances tamoxifen effectiveness on breast cancer cells by affecting ceramide metabolism

The objective of this study was to investigate the effects of the dietary phytosterol beta-sitosterol (SIT) and the antiestrogen drug tamoxifen (TAM) on cell growth and ceramide (CER) metabolism in MCF-7 and MDA-MB-231 human breast cancer cells. The MCF-7 and MDA-MB-231 cell lines were studied as models of estrogen receptor positive and estrogen receptor negative breast cancer cells. Growth of both cell lines as determined using the sulforhodamine B assay was inhibited by treatment with 16 microM SIT but only MCF-7 cell growth was inhibited by treatment with 1 microM TAM. The combination of SIT and TAM further inhibited growth in both cell lines, most significantly in MDA-MB-231 cells. CER is a proapoptotic signal and CER levels were increased in both MCF-7 and MDA-MB-231 cells by individual treatment with SIT and TAM and the combined treatment raised cellular CER content even further. SIT and TAM raised CER levels by different means. SIT potently activated de novo CER synthesis in both MCF-7 and MDA-MB-231 cells by stimulating serine palmitoyltransferase activity; whereas TAM promoted CER accumulation in both cell types by inhibiting CER glycosylation. These results suggest that the combination regimen of dietary SIT and TAM chemotherapy may be beneficial in the management of breast cancer patients.     

远红外锦纶织物对乳腺肿瘤细胞增殖的影响

为探究远红外织物对乳腺肿瘤患者的辅助治疗效应,使用6种不同的远红外锦纶织物和普通锦纶织物对MCF7、Bcap37和MDA-MB-231 3种乳腺癌细胞进行辐射培养,对比分析了织物材料以及辐射时长对3种乳腺癌细胞增殖的影响。结果表明:不同远红外锦纶织物的远红外光发射率有所差异,其中茶叶碳锦纶的远红外光发射率在6种远红外织物中最强;6种远红外织物可不同程度地抑制MCF7和Bcap37 乳腺肿瘤细胞的增殖,对MDA-MB-231细胞也有抑制作用但不显著;随着远红外光辐射时长的延长,远红外锦纶织物的远红外光发射率越高,其抑制乳腺肿瘤细胞增殖的效果越显著,从细胞生物学角度说明远红外织物可抑制且不促进乳腺肿瘤细胞的增殖,为探究穿戴远红外织物作为乳腺癌患者的辅助治疗提供理论参考。     

酶法制备壳寡糖及其抗肿瘤活性评价

为进一步分析壳寡糖抗肿瘤活性及其机制,采用酶法制备工艺获取组分清晰的壳寡糖,优化酶解条件为酶解时间4 h、加酶量120 U/g,结合乙醇沉淀、超滤和纳滤联用的工艺获得聚合度2～4的壳寡糖。比较评价壳寡糖及其与阿霉素联用对3种肿瘤细胞的抑制效果,其中作用最为明显的人乳腺癌MDA-MB-231的细胞生存率降低20%。利用细胞迁移实验和激光共聚焦分析机制,得出壳寡糖可抑制MDA-MB-231细胞迁移并促进阿霉素入核。由此说明,壳寡糖可增强MDA-MB-231细胞对阿霉素的敏感性。     

鲍鱼肽的制备及其抑制人乳腺癌MDA-MB-231细胞增殖作用

将鲍鱼内脏进行蛋白酶解、超滤和纳滤制备鲍鱼肽,研究不同质量浓度鲍鱼肽对体外培养人乳腺癌MDA-MB-231细胞增殖和凋亡的影响。结果表明：鲍鱼肽中除含有71.62%（质量分数,下同）蛋白以外,还含有3.38%碳水化合物和11.48%灰分;鲍鱼肽的分子质量主要分布在350～1 000 Da,丰度较高的蛋白肽分子质量分布在350 Da 附近,表明鲍鱼肽中由2～3 个氨基酸组成的寡肽占比较高。根据四甲基偶氮唑蓝法检测的结果,发现不同质量浓度（1、2、4、8 mg/mL）鲍鱼肽均能抑制MDA-MB-231细胞的生长增殖,并呈现出明显的剂量依赖性。伴随鲍鱼肽质量浓度的增加,贴壁细胞数量逐渐减少,细胞变圆,且部分细胞核出现浓缩。流式细胞术分析结果表明,鲍鱼肽主要将MDA-MB-231细胞阻滞在S期和G2期,诱导MDA-MB-231细胞凋亡和坏死,进而抑制肿瘤细胞增殖。因此,利用鲍鱼内脏制备的蛋白肽是一种具有发展潜力的辅助抗肿瘤功能性食品。     

Delphinidin inhibits a broad spectrum of receptor tyrosine kinases of the ErbB and VEGFR family

Delphinidin has been reported to inhibit EGFR signalling. To determine whether other receptor tyrosine kinases (RTKs) are also influenced by delphinidin, we examined its ability to inhibit the kinase activity of EGFR, ErbB2, VEGFR‐2, VEGFR‐3 and IGF1R in a cell‐free test system. We found that delphinidin strongly inhibited the protein tyrosine kinase activity of all tested RTKs at low micromolar concentrations. In A431 and PAE cells, ligand‐induced phosphorylation of the receptors was also potently suppressed, with a preference for the suppression of the activity of ErbB3 (IC₅₀ ? 100 nM) and VEGFR‐3 (IC₅₀ < 50 μM). Thus the inhibition of RTKs by delphinidin is not limited to cell‐free assays but is also of relevance in the cellular context. The results indicate that delphinidin acts as a broad‐spectrum inhibitor of RTKs. Given the crucial role of the receptors in tumour growth and metastasis, we conclude that delphinidin has the potential to act directly against tumour cells as well as to interfere with key tumour–host interactions, although the suitability of delphinidin as a drug in cancer management may be compromised by its limited stability. Nevertheless, delphinidin may represent a novel lead compound for the development of chemopreventative and chemotherapeutic intervention strategies.     

Pinolenic acid inhibits human breast cancer MDA-MB-231 cell metastasis in vitro

Pinolenic acid (PNA), a naturally-occurring polyunsaturated fatty acid (PUFA), is found mainly in pine seeds. Although many studies have demonstrated beneficial effects of pine seed oil, there are no reports of the biological effects of PNA on cancer metastasis. The objective of this study was to investigate the effect of PNA on human breast cancer MDA-MB-231 cell proliferation and metastasis in vitro. We found that PNA did not affect cell viability and cell-matrix adhesion, but it inhibited cell metastasis by suppressing cell invasiveness and motility. Suppression could in part be associated with the modification of the n−6 PUFA composition of cells by PNA which significantly decreased the percentage of arachidonic acid (AA) in phospholipids from 12.6% to 4.9%. The lower AA content of the cancer cells might result in less synthesis of prostaglandin E₂ (PGE₂), and subsequent down-regulation of inducible cyclooxygenase (COX-2) expression. Thus, PNA represents a potential anti-cancer agent.     

朝鲜族大酱中异黄酮的提取工艺优化及其抗肿瘤活性研究

为优化朝鲜族大酱异黄酮的提取工艺,并研究朝鲜族大酱中异黄酮的抗肿瘤活性,本实验用超声波法提取朝鲜族大酱中的异黄酮,通过单因素实验和正交实验优化其提取工艺,并通过CCK-8法对正常乳腺细胞、MDA-MB-231和MCF-7两种乳腺癌细胞的增殖抑制情况进行了测定,用Western Blot法检测了朝鲜族大酱中异黄酮处理后的两种乳腺癌细胞凋亡相关蛋白的表达情况。通过单因素和正交试验确定其最佳提取条件为提取时间80 min、料液比1:24、温度50 ℃,此时得率最高,为0.802%。CCK-8结果显示,不同浓度的朝鲜族大酱异黄酮对正常细胞无毒副作用,对MDA-MB-231和MCF-7两种乳腺癌细胞的增殖抑制情况均呈剂量-时间依赖性,且均有显著性差异(p<0.05)。对不同浓度朝鲜族大酱异黄酮处理过的两种细胞凋亡相关蛋白的检测结果显示,两种细胞随着异黄酮浓度的增加,其促凋亡蛋白Bax,caspase-3的表达量与空白对照组相比均显著上升(p<0.05),而抗凋亡蛋白Bcl-2的表达量显著下降(p<0.05),且呈现剂量依赖性。表明朝鲜族大酱异黄酮对乳腺癌细胞的生长能够起到抑制作用。     

Cell proliferation inhibitory and apoptosis-inducing properties of anacardic acid and lunasin in human breast cancer MDA-MB-231 cells

The combination of two or more chemopreventive agents is currently being used to achieve greater inhibitory effects on breast cancer cells. Anacardic acid and lunasin are two plant-derived compounds that have been associated with anti-carcinogenic properties. These compounds show inhibitory effects on cell proliferation and inducing properties of apoptosis in human breast cancer MDA-MB-231 cell line. Both lunasin and anacardic acid exert their effects through the modulation of expression of several genes involved in cell cycle, apoptosis and signal transduction. Their combination arrests the cell cycle in S-phase and induces apoptosis at higher levels than that observed when each compound is used individually. This combination also promotes the inhibition of ERBB2, AKT1, JUN and RAF1 signalling gene expression, whose up-regulation has been reported as responsible for breast cancer cells growth and resistance to apoptosis. Our results introduce these two compounds as a promising strategy to prevent/treat breast cancer.     

南瓜硒多糖的制备表征及活性分析

对南瓜多糖进行硒化修饰,并对南瓜硒多糖的体外抗氧化和抑制人乳腺癌细胞MDA-MB-231生长等活性进行研究。以提取、分离、纯化的南瓜多糖为前体物,用Na2SeO3硒化修饰制备南瓜硒多糖,并用紫外光谱、红外光谱、原子荧光光谱、热重分析对产物结构进行表征,采用邻苯三酚自氧化法、水杨酸法、四甲基偶氮唑蓝比色法测定其清除超氧阴离子自由基（O2－•）、羟自由基（•OH）的能力以及对人乳腺癌细胞MDA-MB-231生长的抑制作用。结果表明：制备产物结构中含有Se＝O键和Se-C键,即实现了南瓜多糖的硒化。南瓜硒多糖对O2 － ·、·OH的清除作用显著强于南瓜多糖,与样品量呈正相关;南瓜硒多糖对人乳腺癌细胞MDA-MB-231生长有抑制作用,比南瓜多糖具有更好的抑制效果。     

羊肚菌多糖抑制人乳腺癌细胞MDA-MB-231增殖和诱导细胞凋亡研究

以黑脉羊肚菌多糖为原料,研究羊肚菌多糖对人乳腺癌细胞MDA-MB-231（以下简称MDA）增殖和凋亡的影响。结果表明：在无细胞毒性范围内,羊肚菌多糖能显著抑制人乳腺癌细胞MDA的增殖,半数有效浓度（median effective concentration,EC50）值为0.096 mg/mL,同时人乳腺癌细胞MDA表现出多种细胞凋亡的形态学变化。免疫印迹检测实验结果显示,羊肚菌多糖能抑制B淋巴细胞瘤-2（B-cell lymphoma-2,Bcl-2）蛋白表达,促进Bcl-2相关X蛋白（Bcl-2 associated X protein,Bax）表达,同时增加Bax/Bcl-2蛋白表达比值,并呈现出剂量依赖效应。说明羊肚菌多糖能抑制人乳腺癌细胞MDA增殖,促进细胞凋亡。