辛伐他汀通过线粒体途径诱导K562细胞凋亡

目的观察辛伐他汀诱导K562细胞凋亡不同时间的膜电位(Δψm),caspase-3、9和细胞色素C的改变,以推测其凋亡通路。方法采用浓度为20μmol.L-1的辛伐他汀处理K562细胞24、48、72 h,采用流式细胞技术检测细胞凋亡率和线粒体膜电位,分光光度法检测caspase-3、9蛋白活性,免疫组织化学法检测细胞色素C蛋白。结果浓度为20μmol.L-1辛伐他汀作用K562细胞24、48、72 h后,凋亡率分别为(6.1±0.35)%、(14.15±0.42)%(、30.70±0.65)%,随着凋亡率增加线粒体膜电位降低分别为(39.6±4.80)%,(24.4±2.45)%,(6.0±1.62)%;caspase-3、9蛋白活性与对照组相比上调,细胞浆内细胞色素C升高。结论辛伐他汀诱导K562细胞凋亡时线粒体膜电位下降,caspase-3、9活性增高和细胞色素C释放,推测辛伐他汀诱导K562细胞的凋亡可能经过线粒体凋亡途径。     

辛伐他汀诱导K562细胞凋亡过程中对线粒体膜电位的影响

目的:观察辛伐他汀诱导人红白血病K562细胞凋亡时线粒体膜电位(Δψm)的变化及其与时间的关系。方法:常规培养细胞24h,辛伐他汀(20μmol.L-1)处理K562细胞12、24、48、72h,观测细胞形态学改变;MTT法检测细胞增殖抑制情况;流式细胞术检测细胞凋亡率和线粒体膜电位改变,并与溶剂对照组比较。结果:与对照组比较,辛伐他汀作用K562细胞48h后细胞出现核固缩、核碎裂和凋亡小体等形态学改变;作用12、24、48、72h后细胞凋亡率分别增加(2.55±0.35)%、(6.1±0.35)%、(14.15±0.42)%、(30.70±0.65)%,K562细胞凋亡率随着药物作用时间延长而增加;细胞膜电位降低百分率分别为(0.7±0.24)%、(39.6±4.80)%、(24.4±2.45)%、(6.0±1.62)%,24h时膜电位降低百分率最大。结论:线粒体跨膜电位降低是凋亡发生的早期事件,辛伐他汀诱导K562细胞凋亡的可能机制是通过使线粒体跨膜电位崩溃,从而导致细胞凋亡。     

辛伐他汀对K562细胞端粒酶活性及人端粒酶逆转录酶表达的影响

目的探讨辛伐他汀治疗慢性粒细胞白血病可能的作用机制。方法辛伐他汀10和20μmol.L-1与K562细胞作用48或72 h后,用流式细胞术AnnexinⅤ-FITC/PI双染法检测细胞凋亡百分率;提取细胞端粒酶,用PCR-ELISA检测端粒酶活性;实时荧光定量PCR检测人端粒酶逆转录酶(hTERT),c-myc和bcl2-mRNA表达。结果辛伐他汀10和20μmo.lL-1与K562细胞作用48 h后,细胞凋亡率分别为(6.24±0.18)%和(9.41±0.22)%,与对照组(1.88±0.14)%比较明显增加;作用72 h后细胞凋亡率分别为(12.41±0.32)%和(19.08±0.26)%,与对照组(4.20±0.19)%比较明显增加。辛伐他汀10和20μmo.lL-1与K562细胞作用48或72 h后,端粒酶活性,hTERT,c-myc和bcl-2 mRNA表达明显低于对照组。结论辛伐他汀可使K562细胞端粒酶活性降低,其机制可能与下调hTERT mRNA表达有关。     

小白菊内酯诱导耐药白血病细胞K562/ADR凋亡的研究

目的初步探讨小白菊内酯(PTL)对耐药白血病细胞K562/ADR细胞增殖和凋亡的影响及其作用机制。方法MTT法及LDH释放法检测小白菊内酯对K562和K562/ADR细胞增殖抑制作用,流式细胞术检测细胞凋亡和细胞内活性氧(ROS)的变化,Western blot法检测凋亡相关蛋白Bax、Bcl-2、caspase-3、caspase-9的表达变化。结果 PTL抑制K562/ADR细胞的增殖,呈剂量依赖,半数抑制浓度(IC50)值分别为(4.36±0.37)μmol.L-1、(10.6±2.81)μmol.L-1。10μmol.L-1PTL作用24 h诱导K562/ADR细胞的凋亡率为(31.21±0.40)%,其IC50值与凋亡率与K562细胞相比差异无显著性(P>0.05)。经10μmol.L-1PTL处理1 h,K562/ADR细胞内ROS增加;处理24 h,Bcl-2与Bax的比值降低、caspase-3、caspase-9酶源蛋白表达降低。用N-乙酰半胱氨酸(NAC)预处理细胞,能抑制细胞ROS水平升高和细胞凋亡。结论 PTL能诱导耐药白血病细胞凋亡,作用机制与其刺激细胞内ROS升高相关。     

辛伐他汀诱导人红白血病K562细胞凋亡的实验研究

目的:探讨辛伐他汀诱导K562细胞凋亡及其机制。方法:取浓度为0(阴性对照组)、10、20、30μmol·L-1的辛伐他汀作用K562细胞72h后,双染色法检测细胞凋亡率;DNALadder实验检测细胞凋亡条带;用线粒体分离试剂分离出胞浆蛋白、微粒体蛋白、线粒体蛋白;逆转录-聚合酶链反应法检测GRP78、caspase-9、caspase-3、GADD153mRNA表达;Western blot法检测GRP78、caspase-12、caspase-9、caspase-3、GADD153、细胞色素C蛋白水平。结果:与阴性对照组比较,10、20、30μmol·L-1辛伐他汀作用K562细胞72h后出现典型的凋亡条带,凋亡率分别为12.41%、19.08%、23.41%(P<0.01),GRP78、caspase-9、caspase-3和GADD153 mRNA表达上调(P<0.05),caspase-12、caspase-9、caspase-3蛋白水平下降,caspase-12定位于内质网,细胞色素C从线粒体释放,GRP78、GADD153蛋白表达上调。结论:辛伐他汀可通过内质网和线粒体途径诱导K562细胞凋亡。     

木犀草素对K562细胞caspase-3活化的影响

目的:探讨木犀草素对K562细胞增殖和凋亡的影响.方法:采用MTT法检测K562细胞的增殖,流式细胞术和Hoechst33258/PI荧光染色分析K562细胞的凋亡,比色法测定caspase-3的相对活性,半定量RT-PCR检测caspase-3 mRNA水平的改变,Western-blot分析caspase-3酶原的变化.结果:木犀草素处理细胞24 h后的IC50值为(104.6±13.5)μmol·L-1.浓度为10,20,40μmol·L-1木犀草素处理细胞24 h后均可诱导K562细胞发生凋亡,各处理组的细胞凋亡率均明显高于对照组(P＜0.01).随着浓度及时间的增加,各个木犀草素处理组K562细胞的caspase-3活性升高(P＜0.01),其作用具浓度及时间依赖性.而随着木犀草素浓度的增加,K562细胞中caspase-3的mRNA水平逐渐增加,caspase-3酶原的蛋白水平逐渐减少.结论:木犀草素可通过诱导K562细胞凋亡而抑制其增殖,其诱导K562细胞凋亡可能与激活caspase-3有关.     

4-[4″-(2″,2″,6″,6″-四甲基-1″-哌啶氮氧自由基)氨基]-4′-去甲表鬼臼毒素对K562/ADM细胞的抑制作用

目的比较4-[4″-(2″,2″,6″,6″-四甲基-1″-哌啶氮氧自由基)氨基]-4′-去甲表鬼臼毒素(GP-7)对多药耐药人慢性粒细胞白血病K562的多柔比星耐药株细胞(K562/ADM细胞)的抑制作用是否优于依托泊苷。方法以依托泊苷和K562细胞为对照,用不同浓度GP-7处理K562/ADM细胞不同时间,MTT比色法测定细胞增殖,流式细胞仪测定细胞周期和细胞凋亡率,普通光学显微镜观察细胞凋亡形态,琼脂糖凝胶电泳观察细胞DNA凋亡性降解。结果8~128mol.L-1GP-7处理48h或64μmol.L-1GP-7处理24~72h,GP-7对K562/ADM细胞的增殖抑制呈剂量依赖性(r=0.947,P<0.05)和时间依赖性(r=0.999,P<0.01)。GP-7及依托泊苷对K562/ADM的IC50分别为(45.9±1.8)及(68.7±4.6)μmol.L-1;64μmol.L-1GP-7作用48h可使G2/M期细胞明显增多,相同情况下依托泊苷则使S期细胞明显增多;GP-7可引起K562/ADM和K562细胞凋亡,但其引起的K562/ADM和K562细胞凋亡率与依托泊苷无明显差异;GP-7可引起K562/ADM和K562细胞典型的凋亡形态学变化和DNA凋亡性降解,但GP-7引起的K562/ADM细胞DNA凋亡性降解弱于K562细胞;128及256μmol.L-1GP-7或依托泊苷处理K562/ADM和K562细胞48h,GP-7诱导DNA凋亡性降解的作用强于依托泊苷,但32和64μmol.L-1时作用则相反。结论GP-7可抑制多药耐药白血病细胞株K562/ADM的增殖,诱导细胞凋亡。GP-7抑制多药耐药白血病细胞株K562/ADM的作用优于依托泊苷。     

α-维尼非林经线粒体凋亡途径诱导K562细胞凋亡

目的旨在研究α-维尼非林(α-viniferin)对人慢性髓系白血病细胞K562的作用与相关机制。方法 MTT法评价α-viniferin对K562细胞的细胞毒活性。采用细胞形态学和生物化学方法检测细胞凋亡。通过化学荧光法对细胞内线粒体膜电位、caspase-9、caspase-3活性分析。通过半定量RT-PCR表达分析来确定Bcl-2家族相关基因在α-viniferin诱导K562细胞凋亡中的作用。结果α-viniferin能抑制K562细胞增殖,呈剂量和时间依赖性,IC50为13.61 mg·L-1。α-viniferin引起K562细胞出现死亡并伴随有染色质聚集、核破碎、凋亡小体等典型的凋亡形态学特征;此外还伴随有线粒体膜电位显著降低,caspase-9、caspase-3活性升高等现象;α-viniferin(2~32 mg·L-1)引起K562细胞caspase-3 mRNA表达持续升高,Bax、Bad、Bim、Bid促凋亡基因mRNA表达增加,而Bcl-2、Bcl-xL抗凋亡基因mRNA表达持续下降。结论α-viniferin通过线粒体途径诱导K562细胞凋亡。     

二甲氧雌二醇诱导K562细胞凋亡作用及其凋亡机制

目的 研究二甲氧雌二醇(2-ME)对人白血病细胞K562细胞的增殖、凋亡作用及其机制.方法 分别以不同浓度的2-ME处理白血病细胞K562,应用Annexin Ⅴ和PI双染的流式细胞术检测K562细胞凋亡率,应用比色法测定caspase-3及caspase-9的活性变化,应用凝胶蛋白电泳迁移率分析法EMSA检测K562细胞核内NF-KB蛋白的结合活性变化情况.结果 2-ME浓度升高,细胞凋亡率明显增加(P＜0.01);当2-ME浓度为8μmol/L时,细胞凋亡率达64.3％;4μmol/L,2-ME作用K562细胞24h、36h、48 h后Caspase-3、Caspase-9活性明显升高.同时K562细胞核内NF-kappa B的DNA结合活性明显降低(P＜0.05).结论 2 -ME可显著抑制人白血病细胞K562增殖并诱导其凋亡,其机制与Caspase-3、-9活化及NF-kappa B蛋白信号通路有关.     

曲古抑菌素A对细胞PANC-1株凋亡和肿瘤转移相关基因表达的影响

目的探讨曲古抑菌素A(TSA)诱导胰腺癌细胞PANC-1细胞凋亡机制。方法 TSA 0.1～0.6μmol.L-1培养PANC-1细胞0～48 h,MTT法检测细胞存活率并计算IC50。TSA 0.4μmol.L-1PANC-1培养0～48 h,Hoechst 33258染色观察细胞核形态变化。TSA 0.4μmol.L-1PANC-1培养0～12 h,检测胱天蛋白酶3活性。TSA 0.4μmol.L-1与PANC-1培养24 h,实时定量PCR检测c-myc,p53,Bcl-2,Bax,存活素,基质金属蛋白酶1(MMP1)和基质金属蛋白酶1组织抑制剂(TIMP-1)和Notch-1基因的表达。TSA 0.4和0.6μmol.L-1与PANC-1培养24 h,细胞免疫化学方法检测Notch-1蛋白的胞内活性形式NICD表达。结果TSA可以明显抑制PANC-1细胞增殖,12,24和48 h的IC50值分别为0.42,0.32和0.19μmol.L-1,具有量效(r=0.640,P=0.01)和时效(r=0.768,P=0.002)关系。Hoechst 33258染色结果表明,TSA 0.4μmol.L-1增加PANC-1细胞核的蓝色荧光并出现凋亡特征。TSA 0.4μmol.L-1作用4,8和12 h后,胱天蛋白酶3的活性分别为正常对照组的1.62±0.12,2.68±0.17和(3.92±0.23)倍。PCR结果显示,TSA 0.4μmol.L-1作用24 h后,p53,c-myc和存活素mRNA表达下降,分别为对照组的(18.3±5.1)%,(24.2±0.9)%和(15.8±1.0)%,Bcl-2和Notch-1 mRNA未见明显变化,而Bax,MMP1和TIMP-1 mRNA升高(P<0.05),Bcl-2/Bax比值降低到正常对照组的(13.0±2.8)%。Notch-1活性分子NICD明显升高(P<0.05)。结论TSA可通过线粒体途径诱导胰腺癌PANC-1细胞凋亡,而且使Notch-1激活,促进转移相关基因表达。     

Safety assessment of poloxamers 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403, and 407, poloxamer 105 benzoate, and poloxamer 182 dibenzoate as used in cosmetics

Poloxamers are polyoxyethlyene, polyoxypropylene block polymers. The impurities of commercial grade Poloxamer 188, as an example, include low-molecular-weight substances (aldehydes and both formic and acetic acids), as well as 1,4-dioxane and residual ethylene oxide and propylene oxide. Most Poloxamers function in cosmetics as surfactants, emulsifying agents, cleansing agents, and/or solubilizing agents, and are used in 141 cosmetic products at concentrations from 0.005% to 20%. Poloxamers injected intravenously in animals are rapidly excreted in the urine, with some accumulation in lung, liver, brain, and kidney tissue. In humans, the plasma concentration of Poloxamer 188 (given intravenously) reached a maximum at 1 h, then reached a steady state. Poloxamers generally were ineffective in wound healing, but were effective in reducing postsurgical adhesions in several test systems. Poloxamers can cause hypercholesterolemia and hypertriglyceridemia in animals, but overall, they are relatively nontoxic to animals, with LD(50) values reported from 5 to 34.6 g/kg. Short-term intravenous doses up to 4 g/kg of Poloxamer 108 produced no change in body weights, but did result in diffuse hepatocellular vacuolization, renal tubular dilation in kidneys, and dose-dependent vacuolization of epithelial cells in the proximal convoluted tubules. A short-term inhalation toxicity study of Poloxamer 101 at 97 mg/m(3) identified slight alveolitis after 2 weeks of exposure, which subsided in the 2-week postexposure observation period. A short-term dermal toxicity study of Poloxamer 184 in rabbits at doses up to 1000 mg/kg produced slight erythema and slight intradermal inflammatory response on histological examination, but no dose-dependent body weight, hematology, blood chemistry, or organ weight changes. A 6-month feeding study in rats and dogs of Poloxamer 188 at exposures up to 5% in the diet produced no adverse effects. Likewise, Poloxamer 331 (tested up to 0.5 g/kg day(-1)), Poloxamer 235 (tested up to 1.0 g/kg day(-1)), and Poloxamer 338 (at 0.2 or 1.0 g/kg day(-1)) produced no adverse effects in dogs. Poloxamer 338 (at 5.0 g/kg day(-1)) produced slight transient diarrhea in dogs. Poloxamer 188 at levels up to 7.5% in diet given to rats in a 2-year feeding study produced diarrhea at 5% and 7.5% levels, a small decrease in growth at the 7.5% level, but no change in survival. Doses up to 0.5 mg/kg day(-1) for 2 years using rats produced yellow discoloration of the serum, high serum alkaline phosphatase activity, and elevated serum glutamicpyruvic transaminase and glutamic-oxalacetic transaminase activities. Poloxamers are minimal ocular irritants, but are not dermal irritants or sensitizers in animals. Data on reproductive and developmental toxicity of Poloxamers were not found. An Ames test did not identify any mutagenic activity of Poloxamer 407, with or without metabolic activation. Several studies have suggested anticarcinogenic effects of Poloxamers. Poloxamers appear to increase the sensitivity to anticancer drugs of multidrug-resistant cancer cells. In clinical testing, Poloxamer 188 increased the hydration of feces when used in combination with a bulk laxative treatment. Compared to controls, one study of angioplasty patients receiving Poloxamer 188 found a reduced myocardial infarct size and a reduced incidence of reinfarction, with no evidence of toxicity, but two other studies found no effect. Poloxamer 188 given to patients suffering from sickle cell disease had decreased pain and decreased hospitilization, compared to controls. Clinical tests of dermal irritation and sensitization were uniformly negative. The Cosmetic Ingredient Review (CIR) Expert Panel stressed that the cosmetic industry should continue to use the necessary purification procedures to keep the levels below established limits for ethylene oxide, propylene oxide, and 1,4-dioxane. The Panel did note the absence of reproductive and developmental toxicity data, but, based on molecular weight and solubility, there should be little skin penetration and any penetration of the skin should be slow. Also, the available data demonstrate that Poloxamers that are introduced into the body via routes other than dermal exposure have a rapid clearance from the body, suggesting that there would be no risk of reproductive and/or developmental toxicity. Overall, the available data do not suggest any concern about carcinogenesis. Although there are gaps in knowledge about product use, the overall information available on the types of products in which these ingredients are used, and at what concentration, indicates a pattern of use. Based on these safety test data and the information that the manufacturing process can be controlled to limit unwanted impurities, the Panel concluded that these Poloxamers are safe as used.     

HPLC法测定抗妇炎胶囊中木兰花碱、黄柏碱、药根碱、巴马汀、小檗碱、槐果碱、苦参碱、氧化槐果碱、槐定碱和氧化苦参碱

目的 采用高效液相色谱（HPLC）法同时测定抗妇炎胶囊中木兰花碱、黄柏碱、药根碱、巴马汀、小檗碱、槐果碱、苦参碱、氧化槐果碱、槐定碱和氧化苦参碱10种活性成分。方法 采用InerSustain AQ-C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相A：乙腈–无水乙醇（80∶20）,流动相B：0.1%磷酸溶液,梯度洗脱,检测波长220 nm,体积流量1.0 mL/min,柱温30℃,进样量10 μL。结果 木兰花碱、黄柏碱、药根碱、巴马汀、小檗碱、槐果碱、苦参碱、氧化槐果碱、槐定碱和氧化苦参碱分别在2.69～134.50、1.95～97.50、0.63～31.50、0.86～43.00、11.95～597.50、0.59～29.50、6.08～304.00、4.85～242.50、1.66～83.00、19.79～989.50 μg/mL线性关系良好（r≥0.999 3）;平均回收率分别为99.11%、98.23%、96.95%、97.78%、100.02%、97.21%、99.66%、99.52%、98.81%、100.08%,RSD值分别为1.04%、1.23%、1.37%、1.65%、0.70%、1.28%、0.65%、0.81%、1.11%、0.63%。结论 建立的HPLC法可用于抗妇炎胶囊中10种活性成分的测定,作为抗妇炎胶囊质量控制方法。     

Arformoterol: (R,R)-eformoterol, (R,R)-formoterol, arformoterol tartrate, eformoterol-sepracor, formoterol-sepracor, R,R-eformoterol, R,R-formoterol

Sepracor in the US is developing arformoterol [R,R-formoterol], a single isomer form of the beta(2)-adrenoceptor agonist formoterol [eformoterol]. This isomer contains two chiral centres and is being developed as an inhaled preparation for the treatment of respiratory disorders. Sepracor believes that arformoterol has the potential to be a once-daily therapy with a rapid onset of action and a duration of effect exceeding 12 hours. In 1995, Sepracor acquired New England Pharmaceuticals, a manufacturer of metered-dose and dry powder inhalers, for the purpose of preparing formulations of levosalbutamol and arformoterol. Phase II dose-ranging clinical studies of arformoterol as a longer-acting, complementary bronchodilator were completed successfully in the fourth quarter of 2000. Phase III trials of arformoterol began in September 2001. The indications for the drug appeared to be asthma and chronic obstructive pulmonary disease (COPD). However, an update of the pharmaceutical product information on the Sepracor website in September 2003 listed COPD maintenance therapy as the only indication for arformoterol. In October 2002, Sepracor stated that two pivotal phase III studies were ongoing in 1600 patients. Sepracor estimates that its NDA submission for arformoterol, which is projected for the first half of 2004, will include approximately 3000 adult subjects. Sepracor stated in July 2003 that it had completed more than 100 preclinical studies and initiated or completed 15 clinical studies for arformoterol inhalation solution for the treatment of bronchospasm in patients with COPD. In addition, Sepracor stated that the two pivotal phase III studies in 1600 patients were still progressing. In 1995, European patents were granted to Sepracor for the use of arformoterol in the treatment of asthma, and the US patent application was pending.     

欧洲刺柏（Juniper）

活性成分与药理作用欧洲刺柏药用部位是其浆果，具有促水排泄、防腐、抗胃肠胀气和抗风湿作用，还可改善胃功能。用作促水排泄药可增加尿量（水丢失），但不增加钠排泄。成分萜品烯-4-醇可增加肾小球滤过率，但刺激肾。欧洲刺柏浆果对单纯疱疹病毒体外显示抗病毒活性，并具抗真菌活性。动物实验显示，欧洲刺柏浆果提取物具有堕胎、抗生育、抗炎、抗胚胎植入、降血压、升血压和降血糖作用。欧洲刺柏浆果油具有兴奋子宫的活性，以及利尿、胃肠道抗菌和刺激作用，该油对平滑肌有阻止解痉作用。     

Oral Presentations (LDD, LPES, LNM, LPAT, LNT, LPPT, LPM)

Probiotic microorganisms have been shown to provide specific health benefits when consumed as food supplements or as food components. The main problem of such products is the poor survival of the probiotic bacteria in the low pH of gastric fluid. However the use of synthetic excipients for enteric coating to prevent the exposure of microorganisms to gastric fluid is limited in food supplementary industry. Therefore the aim of this study was to develop an enteric coating formulation containing shellac as a natural polymer. Shellac possesses good resistance to gastric juice; the major disadvantage of this polymer is its low solubility in the intestinal fluid [1, 2]. Thus films containing different ratios of shellac and water-soluble polymers (sodium alginate, hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidon (PVP)) or plasticizers (glycerol and glyceryl triacetate (GTA)) were prepared in order to analyse the films’ melting temperatures (T_m), the changes in enthalpy (ΔH), their capability of taking up water, and their solubility in different media. The release characteristics of the films were studied by loading pellets with Enterococcus faecium M74 and coating them with formulations containing different amounts of shellac and polymer or plasticized shellac. Using dissolution tests, performed according to USP XXXI paddle method, the resistance of the coatings to simulated gastric fluid (SGF, pH 1.2) and the release of cells in simulated intestinal fluid (SIF, pH 6.8) was investigated.The trials showed that an increasing amount of plasticizer results in a decrease of T_m and ΔH of the films whereat glycerol had a superior plasticization effect to GTA. The compatibility of films made of water-soluble polymers and shellac was also concentration dependent. HPMC and PVP showed superior compatibility with shellac compared to sodium alginate, since films containing shellac and more than 10% [w/w] sodium alginate tended to separate into two phases. In the end five formulations containing shellac and either 5% [w/w] glycerol, 10% [w/w] PVP, 20% [w/w] PVP, 10% [w/w] HPMC, or 5% [w/w] sodium alginate emerged as feasible for enteric coating purposes.