影响薄片法血小板聚集实验的因素

目的:研究常见影响因素对薄片法血小板聚集实验(SPAT)时间的影响.方法:测定下列富含血小板血浆(PRP)的SPAT:①调整血小板计数分别为240,120,60,30和15×109/L的8例健康献血者PRP;②分别在40,35,30,25,20,15,10和5℃条件下恒温30 min的10例健康献血者PRP;③含终浓度为0,10,20,30,40和50g/L的二甲基亚砜(DMSO)并室温放置30 min的10例健康献血者PRP;④在室温放置1,2,3,4 h后的8例健康志愿者服用阿司匹林前后PRP;⑤含浓度分别为0,0.5,1.0,2.0和3.O U/L的肝素或0.1 g/LEDTA的8例健康献血者PRP.结果:①随着血小板计数的降低,SPAT时间逐渐延长;同一供者血小板计数与其SPAT时间呈显著的直线负相关(r=0.996,P=0.004);②温度在20～40℃对SPAT时间无明显影响,但当温度低于20℃时SPAT时间明显延长;③DMSO对SPAT有明显的影响,使SPAT时间明显延长,并呈明显的剂量效应;④血小板标本室温放置1,2,3和4 h SPAT无显著差异(P=0.815);⑤肝素对SPAT时间无明显影响,而0.1 g/L乙二胺四乙酸(EDTA)可完全抑制血小板聚集.结论:血小板计数、所处的温度以及含有DMSO时,均能在一定程度上影响SPAT时间,但是上述因素均不影响血小板最终形成肉眼可见的聚集,且聚集时间仍然在180 s以内.     

表没食子儿茶素没食子酸酯对肝癌细胞增殖的抑制作用研究

目的 探讨表没食子儿茶素没食子酸酯(EGCG)对肝癌细胞HepG2增殖和凋亡的影响及其可能机制.方法 采用不同浓度(0、25、50、100、200、400mg/L)EGCG作用于HepG2细胞,于24、48h后采用MTT方法检测细胞增殖抑制率.以不同浓度(0、50、100、200mg/L)EGCG作用于HepG2细胞,24h后采用流式细胞术检测细胞凋亡率、细胞周期、细胞分裂周期蛋白25A(CDC25A)及Smad3蛋白的表达,RT-PCR检测CDC25A和Smad3 mRNA的表达.结果 MTT检测结果显示,不同浓度EGCG对HepG2细胞均有生长抑制作用,且具有时间和剂量依赖性(P＜0.01).随着EGCG浓度升高,细胞增殖指数(PI)明显降低(P＜0.01),细胞凋亡率明显增高(p＜0.01),CDC25A蛋白和mRNA表达水平下降(p＜0.05),Smad3蛋白和mRNA表达水平上升(p＜0.05).结论 EGCG可能通过下调CDC25A、上调Smad3的表达,抑制HepG2细胞增殖并诱导其凋亡,从而对肝癌细胞起到生长抑制作用.     

“药辅合一”茶多酚-蜂毒肽纳米复合物的制备及抗肿瘤研究

目的制备具有协同抗肿瘤作用的茶多酚-蜂毒肽纳米复合物(EMN),并考察其抗肿瘤活性。方法通过拜耳反应合成了多聚表没食子儿茶素没食子酸酯(poly EGCG),~1H-NMR和MS表征其结构和相对分子质量。通过正交试验优化EMN的制备工艺,并考察在不同p H值条件下蜂毒肽(Mel)的体外释放。通过流式细胞仪比较异硫氰酸荧光素(FITC)标记的Mel和EMN的细胞摄取情况。MTT法考察poly EGCG和Mel在B16F10和A549细胞的协同抗肿瘤效应。结果合成的poly EGCG用~1H-NMR和MS确证了结构,正交试验优化的EMN最佳制备工艺为将0.5 mg/m L poly EGCG溶液逐滴加入到1 mg/m L等体积的Mel溶液中,边滴加边搅拌,室温孵育30 min,即得。体外释放实验表明EMN释放具有酸响应性。摄取实验表明Mel和EMN均能被肿瘤细胞摄取,摄取量相当。MTT实验结果表明,poly EGCG和Mel联合使用的协同系数(CI)小于1.0,具有协同抗肿瘤效果。结论采用自组装的方式制备EMN,工艺简单,粒径适宜,且具有协同抗肿瘤的效果,值得深入研究。     

Compounds that confer thermal stress resistance and extended lifespan

The observation that long-lived and relatively healthy animals can be obtained by simple genetic manipulation prompts the search for chemical compounds that have similar effects. Since aging is the most important risk factor for many socially and economically important diseases, the discovery of a wide range of chemical modulators of aging in model organisms could prompt new strategies for attacking age-related disease such as diabetes, cancer and neurodegenerative disorders [Collins, J.J., Evason, K., Kornfeld, K., 2006. Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Exp. Gerontol.; Floyd, R.A., 2006. Nitrones as therapeutics in age-related diseases. Aging Cell 5, 51-57; Gill, M.S., 2006. Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans. Aging Cell 5, 23-30; Hefti, F.F., Bales, R., 2006. Regulatory issues in aging pharmacology. Aging Cell 5, 3-8]. Resistance to multiple types of stress is a common trait in long-lived genetic variants of a number of species; therefore, we have tested compounds that act as stress response mimetics. We have focused on compounds with antioxidant properties and identified those that confer thermal stress resistance in the nematode Caenorhabditis elegans. Some of these compounds (lipoic acid, propyl gallate, trolox and taxifolin) also extend the normal lifespan of this simple invertebrate, consistent with the general model that enhanced stress resistance slows aging.     

HPLC测定绿茶浸出液灌胃动脉粥样硬化大鼠血浆中4个有效成分的含量

目的:建立HPLC测定绿茶浸出液中没食子酸、咖啡因、表儿茶素没食子酸酯、山柰酚在动脉粥样硬化大鼠体内的血药浓度,为动脉粥样硬化的治疗提供参考。方法:动脉粥样硬化大鼠灌胃绿茶浸出液后,分别在不同时间点取血,采用依利特Hypersil ODS2色谱柱(4.6 mm×250 mm,5μm),流动相甲醇(A)-0.03%磷酸水溶液(B)梯度洗脱(0~30 min,10%~43%A;30~43 min,43%~45%A;43~44 min,45%~51%A;44~60 min,51%~53%A;60~70 min,53%~90%A),流速0.7 mL·min~(-1),检测波长278 nm的色谱条件进样分析。结果:没食子酸、咖啡因、表儿茶素没食子酸酯、山柰酚分别在0.255~20.40,0.430~86.00,0.245~19.60,0.270~21.60 mg·L~(-1)内呈良好线性关系,日内和日间精密度RSD均9.0%,提取回收率和方法回收率都87%;酸化后主要药代动力学参数药时曲线下面积(AUC0-t)分别为(8.902±1.235),(198.5±12.5),(7.367±0.412),(4.426±0.251)mg·h·L~(-1),表观分布容积(Vd)分别为(0.990±0.134),(1.409±0.201),(0.501±0.243),(0.029±0.009)L·kg~(-1),达峰时间(T_(max))均为1.5 h。结论:该方法对所测成分的分离度好,适用于测定绿茶浸出液中4个有效成分的血药浓度,为临床预防此类疾病提供一定理论依据。     

EGCG和PC-B2 联合用药对AFB1 致肝细胞损伤的保护作用及其机制

目的:探讨多酚类植物化学物表没食子儿茶素没食子酸酯(EGCG)与原花青素B2(PC-B2),对黄曲霉素B1(AFB1)起的肝细胞损伤保护作用及机制。方法:选用人胚肝细胞(L-02细胞)进行体外实验研究,实验分为6组,分别为空白组,溶剂对照组,AFB_1染毒组,AFB_1染毒+EGCG组,AFB_1染毒+PC-B_2组和AFB_1染毒+EGCG+PC-B_2组。采用MTT检测细胞存活度,通过单细胞凝胶电泳试验(SCGE)检测细胞DNA损伤,通过流式细胞法检测细胞凋亡,采用Western blot法检测凋亡信号通路相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Caspase-9,p53蛋白表达的水平。结果:EGCG与PC-B_2均能降低AFB_1所致L-02细胞的生长抑制,使AFB_1对肝细胞的生长抑制率从61.12%降至42.18%和46.72%;EGCG与PC-B_2联用则效果更佳。SCGE实验结果表明EGCG与PC-B_2单用与联用均能减小AFB_1引起的L-02细胞DNA损伤(P0.05)。EGCG与PC-B_2单用与联用均能显著降低AFB_1所致L-02细胞的早期凋亡率(P0.05)。Western blot实验结果表明,AFB_1染毒后,EGCG和/或PC-B_2处理均能显著降低Caspase-3,Caspase-9的表达(P0.01),提高Bcl-2/Bax比值,而对p53表达影响不明显。结论:EGCG与PC-B_2通过降低L-02细胞DNA损伤与细胞凋亡率,从而降低AFB_1对人肝细胞的损伤作用,其作用机制可能与下调Caspase-3,Caspase-9的表达,升高Bcl-2/Bax有关。     

表没食子儿茶素没食子酸酯烷基化衍生物合成及其体外抗癌活性

目的:设计合成表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)烷基化程度较高的衍生物,对其进行简单构效分析,并对衍生物抑制肝癌细胞增殖的药理活性进行初步筛选,旨在选出结构较稳定药效较好的EGCG衍生物。方法:以EGCG,(CH_3CH_2O)_2SO_2为原料,通过乙基化反应合成EGCG衍生物,采用噻唑蓝(MTT)法,细胞密度1×10~8/m L,将EGCG衍生物3,4稀释成4个浓度,对SMMC-7721细胞其质量质量浓度分别为60,80,100,150 mg·L~(-1)和30,40,50,60 mg·L~(-1),对Hep G2细胞其质量浓度为60,80,100,150 mg·L~(-1)和20,30,40,50 mg·L~(-1),作为药物组,并设空白组,每个浓度组设4个复孔,加药后继续培养24 h,测定乙基化EGCG对肝癌细胞SMMC-7721,Hep G2的影响。结果:合成4个EGCG衍生物,其结构通过~1H-NMR,~(13)C-NMR,~2DNMR,质谱等方法进行结构鉴定,均为未见文献报道的新化合物,乙基化EGCG产物3,4对肝癌细胞SMMC-7721,Hep G2增殖均有抑制作用。结论:乙基化EGCG产物3,4对肝癌细胞SMMC-7721,Hep G2均有抑制作用,其中4的抑制作用比EGCG明显增强。     

Evaluation of epigallocatechin gallate and epicatechin gallate effects on acrylamide-induced neurotoxicity in rats and cytotoxicity in PC 12 cells

The neurotoxicity of acrylamide (ACR) monomer occurs through different mechanisms such as oxidative stress. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) are green tea catechins which are known as powerful antioxidants. In this study, we examined the possible protective effects of ECG and EGCG on ACR neurotoxicity in both in-vitro and in-vivo models. PC12 cells were exposed to different concentrations of ECG and EGCG. After 24 and 48?hours, ACR was added to the cells (IC₅₀?=?4.85?mM) and cell viability was measured through MTT assay after 24?hours. Male Wistar rats were pretreated with ECG, EGCG (10, 20 and 40?mg/kg, i.p) and vitamin E (200?IU/kg i.p.) for 3?days. Afterwards they were treated with ACR (50?mg/kg, i.p.) for 11?days. After the treatment period, gait score examination was performed and molondialdehyde (MDA) and reduced glutathione (GSH) were measured in cerebral cortex. ACR reduced the cell viability in a concentration-dependent manner. Both ECG and EGCG (20?μM) showed inhibitory effects on ACR cytotoxicity. ACR significantly induced gait abnormalities, decreased GSH level and increased lipid peroxidation in cerebral cortex. ECG and EGCG (20?mg/kg) improved all ACR toxic effects. Although the food intake was increased in pretreated groups compared to the ACR-treated group, intensive weight loss was observed due to the green tea’s different weight loss mechanisms. ECG and EGCG inhibited the cytotoxicity of ACR in PC12 cells and increased GSH level and decreased lipid peroxidation in rat cerebral cortex.