几种硫醚类香料诱导HL-60细胞毒性的研究

本研究以19种可食用硫醚类香料为原料,在体外考察其对人白血病细胞HL-60的细胞毒性作用,比较分析其构效关系,并初步探究其肿瘤细胞毒性机理。用19种硫醚类香料处理HL-60细胞,通过DNA片段化来考察其对HL-60细胞的诱导凋亡作用,并对其进行筛选。结果显示,二糠基二硫醚(DFDS)、双-(2-甲基-3-呋喃基)二硫醚(BMFDS)及甲基2-甲基-3-呋喃基二硫醚(MMFDS)具有很强的细胞毒性。利用CCK-8试剂盒检测,添加10μM上述三种硫醚类香料使HL-60细胞的生存率分别下降41.77±1.18%、53.49±2.88%和23.63±0.90%。通过分析19种硫醚类香料的构效关系,明确硫原子数和呋喃基对硫醚类香料的HL-60细胞毒性起重要作用。为了探讨三种硫醚类香料的细胞毒性机理,采用抗氧化剂谷胱甘肽、N-乙酰半胱氨酸预处理细胞,进一步检测硫醚类香料对细胞生存率和DNA片段化的影响。结果表明,抗氧化剂对BMFDS及DFDS诱导的HL-60细胞毒性具有显著的保护作用,说明这两种硫醚类香料可能通过诱导活性氧产生而发挥细胞毒性作用。     

肉桂醛对HL－60细胞的生长抑制及诱导凋亡作用研究

目的：探讨肉桂醛对HL-60细胞的增殖及凋亡的影响。方法：采用台盼蓝细胞计数和四甲基偶氮唑蓝(MTT)法研究肉桂醛对HL-60细胞增殖的抑制作用;采用流失细胞仪(FCM)、荧光显微镜(FM)、扫描电镜(SEM)以及透射电镜(TEM)探讨肉桂醛对HL-60细胞的诱导凋亡作用。结果：台盼蓝细胞计数和MTT法结果表明：肉桂醛对HL-60细胞的增殖有明显抑制作用,抑制作用与肉桂醛质量浓度及作用时间呈依赖关系,其半数致死质量浓度为34.87μg/mL;流式细胞仪检测结果表明：肉桂醛(终质量浓度20μg/mL)作用16h后,HL-60细胞早期凋亡比例为32.6%;该组细胞在荧光显微镜、扫描电镜以及透射电镜观察下均呈现早期凋亡特征。结论：肉桂醛能抑制HL-60细胞增殖并促进其凋亡。     

叶黄素和玉米黄素抑制口腔上皮细胞癌增殖的实验研究

为阐明玉米蛋白粉类胡萝卜素提取物叶黄素和玉米黄素对人口腔上皮癌细胞株KB凋亡的诱导作用,探讨其分子生物学机制。本文采用单细胞凝胶电泳、DNA凝胶电泳、流式细胞术检测叶黄素和玉米黄素诱导口腔癌细胞凋亡的情况。结果发现:单细胞凝胶电泳显示玉米蛋白粉叶黄素和玉米黄素能造成KB细胞DNA损伤、DNA凝胶电泳显示能使KB细胞DNA发生明显的降解,DNA周期分析主要阻断口腔癌细胞由G1向S期转变;流式细胞仪检测明显抑制人口腔癌细胞株KB中bcl-2基因的蛋白表达,明显升高p53、Bax基因的蛋白表达;说明玉米蛋白粉类胡萝卜素提取物叶黄素和玉米黄素具有明显抑制口腔上皮癌细胞增殖、并诱导其凋亡的作用。     

松口蘑菌丝体蛋白质诱导细胞凋亡

采用深层发酵技术培养菌丝体并提取分离出的蛋白质,进行体外抗人子宫颈癌HeLa细胞增殖及诱导细胞凋亡机理的研究.试验发现松口蘑菌丝体水提液中活性蛋白质TMP在体外具有抑制肿瘤细胞增殖的作用,扫描电镜观察到TMP处理细胞产生明显的凋亡小体,TMP对细胞周期的影响是通过抑制细胞从S到G2M期的转化来抑制HeLa细胞增殖,诱导细胞发生凋亡的.DNA电泳出现以200bp左右为单位的DNA碎片.结果表明采用液体培养方法生产的松口蘑菌丝体蛋白质,具有显著的抑制细胞增殖和诱导细胞凋亡作用.     

珠母贝糖胺聚糖PG-Ⅱ诱导人宫颈癌HeLa细胞凋亡的研究

目的:探讨珠母贝糖胺聚糖PG-II诱导人宫颈癌HeLa细胞凋亡,从而抑制其增殖。方法:MTT法检测珠母贝糖胺聚糖PG-II对人宫颈癌HeLa细胞生长的抑制作用。倒置显微镜、荧光显微镜观察HeLa细胞凋亡的形态学特征,琼脂糖凝胶电泳检测DNA断裂片段,流式细胞仪定量检测细胞凋亡的百分率及对其细胞周期的影响。结果:MTT法显示PG-II可抑制人宫颈癌HeLa细胞的增殖。倒置显微镜和荧光显微镜形态学观察可见HeLa细胞凋亡的形态明显变化。琼脂糖凝胶电泳显示PG-II作用HeLa细胞24h,出现分子质量为200bp到2000bp不等的细胞凋亡的特征性DNA梯状条带。流式细胞术测试表明:PG-II可使HeLa细胞阻滞于G1期,剂量为100mg/L作用24h阻滞作用最明显,凋亡指数为42.6%。结论:珠母贝糖胺聚糖PG-II可能通过诱导人宫颈癌HeLa细胞凋亡而抑制其增殖。     

绿茶有效活性成分EGCG诱导肿瘤细胞凋亡及其机制的研究进展

绿茶是亚洲及中东地区最流行的饮品之一,具有抗癌、抗炎、抗菌、抗氧化、治疗心脑血管疾病、改善肥胖和糖尿病等活性。表没食子儿茶素没食子酸酯(Epigallo catechin-3-gallate,EGCG)是绿茶中含量最高的多酚类物质,具有抗肿瘤的功效。已知细胞凋亡是各种抗癌药物引发细胞死亡的主要方式,因此诱导癌细胞凋亡是治疗癌症的一个明确目标。大量研究报道了EGCG的抗肿瘤活性,并揭示了EGCG诱导肿瘤细胞凋亡的作用及机制,因此本文综述了近几年EGCG诱导肿瘤细胞凋亡的作用及机制,包括p53、STAT3、Akt、p38MAPK、Wnt/β-catenin和TRAIL等与凋亡相关的途径,还综述了EGCG与天然产物、抗肿瘤药物及其它化合物协同诱导肿瘤细胞凋亡的作用机制,并对EGCG的生物利用率、体内实验及临床实验的开展进行了展望,为EGCG的临床应用提供理论依据。     

NF-κB和Caspase-3在金雀异黄素诱导人胃癌SGC-7901细胞凋亡中的作用研究

采用MTT法、形态学观察和琼脂糖凝胶电泳法检测了金雀异黄素(genistein,Gen)对体外培养的人胃癌SGC-7901细胞的生长和凋亡诱导作用,并采用免疫组化法和WesternBlot法检测Gen对人胃癌细胞NF-κB、Caspase-3蛋白的表达情况。结果显示:Gen可抑制肿瘤细胞生长,诱导细胞凋亡,抑制NF-κB蛋白表达,增加Caspase-3蛋白表达。结果提示:Gen抑制NF-κB蛋白表达、增加Caspase-3蛋白表达,可能是其诱导胃癌细胞凋亡的机制之一。     

银杏中银杏酸诱导人白血病细胞U937凋亡的研究

目的:研究银杏酸体外对U937细胞生长的抑制作用,探讨其作用机理。方法:采用MTT法检测银杏酸对U937细胞增殖的影响,激光共聚焦显微镜观察细胞形态的变化,DNA琼脂糖电泳检测其生化特征的改变,流式细胞仪分析细胞凋亡率。结果:U937细胞经银杏酸作用24～48h,细胞的生长明显被抑制;银杏酸作用28h出现了细胞皱缩、核浓缩、体积缩小等明显的凋亡的形态学特征;细胞DNA经琼脂糖电泳可见典型的梯形条带;10.0μg/ml的银杏酸作用40h后,细胞凋亡率为14%。结论:银杏酸对U937细胞具有明显的抗肿瘤活性,其作用机理与诱导细胞凋亡有关。     

茶叶中表没食子儿茶素没食子酸酯抑制中波紫外线诱导HaCaT细胞氧化损伤研究

目的研究茶叶中表没食子儿茶素没食子酸酯(EGCG)对中波紫外线(UVB)诱导的人表皮角质形成细胞(HaCaT)光损伤的保护作用。方法用不同浓度的EGCG对HaCaT细胞进行预处理6 h,采用60 m J/cm2的剂量照射细胞,用MTT法检测细胞生存率,吸取细胞上清液检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH–Px)、乳酸脱氢酶(LDH)的活性和丙二醛(MDA)含量变化,用荧光法检测细胞内ROS含量。结果 UVB辐射对Ha Ca T细胞造成严重损伤,与对照组相比,细胞活性下降21.63%;与UVB辐射模型组相比,EGCG尤其高剂量可提高UVB照射后HaCaT细胞的存活率5.89%,显著提高SOD、GSH–Px活性(P0.01),降低LDH活性(P0.01),减少自由基ROS和MDA含量(P0.01)。结论 EGCG可以减少UVB诱导HaCaT细胞的损伤和凋亡,具有光保护作用,其机制与增强细胞抗氧化能力和加速清除氧自由基有关。     

黄参多糖对癌细胞HepG2和P388增殖抑制与细胞凋亡的影响

目的：探索黄参提取物黄参多糖(SGP)对肝癌细胞HepG2和淋巴白血病细胞P388增殖的抑制与诱导细胞凋亡的效应,并初步探讨其对生命延长的作用。方法：应用SGP与肝癌细胞HepG2和淋巴白血病细胞P388共培养,采用MTT比色法测定不同剂量SGP对HepG2和P388细胞增殖的抑制;通过对小鼠皮下移植性肿瘤HepG2生长抑制实验,用流式细胞术分析SGP诱导细胞凋亡和细胞周期分布的变化;探讨SGP对荷淋巴白血病小鼠生命延长的作用。结果：SGP可明显抑制体外培养HepG2和P388细胞的增殖,200mg/(kg ·d)剂量作用48h,对HepG2的抑制率可达37.05%(P＜0.01),而150、200mg/(kg ·d)剂量作用P388细胞48h后抑制率均超过38%(P＜0.01);SGP在小鼠体内也能明显抑制HepG2移植性肿瘤生长,其抑制率超过40%;可诱导移植性肝癌HepG2细胞凋亡,并使细胞时相分布在G1期发生阻滞;对荷P388(腹水)小鼠生命有延长作用,延长率达10%。结论：SGP对小鼠有良好的抗癌变效果以及对小鼠原发性肝癌和淋巴白血病的良好抑制作用。     

Induction of apoptosis in HL-60 cells by skimmed milk digested with a proteolytic enzyme from the yeast Saccharomyces cerevisiae

Bovine skimmed milk digested with cell-free extract of the yeast Saccharomyces cerevisiae was found to exhibit proliferation inhibition activity towards human leukemia (HL-60) cells. The optimum pH for digestion of skimmed milk and production of the proliferation inhibition factor was pH 4.8. Nondigested skimmed milk exhibited little suppressive effect on the proliferation of HL-60 cells. An active enzyme involved in the production of cell proliferation inhibitory materials from skimmed milk was purified from the cell-free extract of S. cerevisiae by a series of column chromatographies: DEAE-Sephacel, D-tryptophan methyl ester-Sepharose 4B, Hiload Superdex G-200 and HPLC Mono Q. The homogeneous purified enzyme and exhibited a molecular mass of 33 kDa in sodium dodeceyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was identified as protease B by N-terminal amino acid sequence analysis. Bovine skimmed milk digested with purified protease B was found to inhibit proliferation activity of HL-60 cells most strongly when digestion was conducted at pH 4.8. The cell proliferation inhibition activity induced by digested skimmed milk was shown to be due to the induction of apoptosis, demonstrated by the formation of apoptotic bodies and fragmentation of DNA in treated cells. The proliferation inhibition factors produced were recovered in the soluble fraction of 92% ethanol, suggesting that the factors were hydrophilic low molecular mass substances derived from skimmed milk.     

冬虫夏草菌丝体水提液对SP2/0细胞周期的作用

用四唑篮比色法试验,琼脂糖凝胶电泳方法及流式细胞仪研究了冬虫夏草菌丝体水提液对小鼠骨髓瘤细胞生长、凋亡及细胞周期的影响。结果发现浓度为100mg/ml水提液抑制瘤细胞的增殖,抑制率为70%,使细胞周期阻滞,在DNA电泳出现明显凋亡带,这提示水提液对瘤细胞抑制作用可能是由于细胞周期阻滞诱导细胞凋亡的结果。     

茶多酚EGCG通过调控miR-16-5p/含铜胺氧化酶1轴发挥对过氧化氢诱导的人心肌细胞凋亡的保护作用

目的:探究茶多酚表没食子儿茶素没食子酸酯(EGCG)对过氧化氢(H2O2)诱导人心肌细胞HCM凋亡的保护作用中的分子机制.方法:利用H2O2建立HCM细胞凋亡模型;通过caspase-3活性检测实验评价细胞凋亡水平;利用CCK-8法检测药物的细胞毒性;qPCR和Western blot用于检测含铜胺氧化酶1(AOC1)...     

Hormonal regulation of apoptosis in rabbit granulosa cells in vitro: evaluation by flow cytometric detection of plasma membrane phosphatidylserine externalization

Annexin V and propidium iodide bivariate analysis and the TUNEL method were used to quantify hormonal regulation of apoptosis in rabbit granulosa cells from preovulatory follicles in vitro. The aim of this study was to analyse comparatively the effects of gonadotrophins and their second messenger in the regulation of granulosa cell apoptosis in (i) cultured isolated granulosa cells and (ii) granulosa cells scraped from cultured follicles. The results showed that increasing doses of FSH had no effect on apoptosis of cultured isolated cells but caused a decrease in the number of apoptotic granulosa cells from preovulatory follicles cultured in serum-free conditions. Unlike FSH, addition of hCG did not modify apoptosis of granulosa cells significantly. In contrast, dibutyryl cAMP had an apoptotic effect in the two cellular models in the presence of serum. Moreover, a biphasic effect of dibutyryl cAMP in isolated granulosa cells was observed with an increase in the incorporation of [(3)H]thymidine into DNA at the lowest dose and an increase in apoptotic cell death at the highest dose. It was concluded that, in rabbits: (i) FSH requires follicle integrity to exert its anti-apoptotic effect in granulosa cells; (ii) dibutyryl cAMP induces a dose-dependent apoptotic effect in granulosa cells cultured alone or obtained from cultured preovulatory follicles; and (iii) cAMP signals induce opposite effects on growth and apoptosis in granulosa cells.     

Lycium barbarum polysaccharide inhibits the proliferation of HeLa cells by inducing apoptosis

BACKGROUND: Lycium barbarum polysaccharide (LBP), isolated with boiling water from the famous Chinese medicinal herb Lycium barbarum fruits, is one of the most important functional constituents in Lycium barbarum. In this study the effects of LBP on cell proliferation, cell cycle and apoptosis in human cervical carcinoma cells (HeLa cells) were investigated. RESULTS: LBP could inhibit the proliferation of HeLa cells by changing cell cycle distribution and inducing apoptosis. In addition, the loss of mitochondrial transmembrane potential (Δψ_m) was observed by flow cytometry and the increase of intracellular Ca²⁺ concentration was detected by laser scanning confocal microscope in apoptotic cells. At the same time, the nitric oxide content, nitric oxide synthase and inducible nitric oxide synthase activities were also increased. CONCLUSION: The inhibitory effect of LBP on the proliferation of HeLa cells was caused by inducing apoptosis through the mitochondrial pathway. The results showed that LBP can be developed as a potential chemotherapeutic agent candidate against human cervical cancer. Copyright © 2012 Society of Chemical Industry     

Involvement of theca cells and steroids in the regulation of granulosa cell apoptosis in rabbit preovulatory follicles

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.     

EGCG inhibits protein synthesis,lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells

In the previous studies, (–)‐epigallocatechin‐3‐gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose‐dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP‐activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E‐binding protein‐1 (4E‐BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl‐CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up‐regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti‐lipogenesis agent for human hepatoma cells.     

葛根黄酮提取物对HL-60细胞bc1-2和bax基因表达的影响

目的：探讨葛根黄酮提取物对HL-60细胞bcl—2和bax基因表达的影响。方法：用RT—PCR和蛋白质免疫印迹技术检测葛根黄酮提取物、葛根素和大豆甙元处理HL-60后，对bcl—2和bax基因的mRNA和蛋白表达的影响。结果：葛根黄酮提取物、葛根素和大豆甙元处理HL-60细胞后能够上调bax mRNA表达水平，下调bcl-2蛋白表达和上调Bax蛋白表达，且随着处理时间的延长其诱导下调和上调能力增强。结论：葛根黄酮提取物诱导HL-60细胞凋亡与其下调bcl-2mRNA和蛋白表达水平和其上调bax mRNA和蛋白表达水平有关。