SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

PTP1B抑制剂的制备与构效关系研究

蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase, PTP1B）是蛋白酪氨酸磷酸酶（protein tyrosine phosphatases, PTPs）家族中的一个经典的非受体型酪氨酸磷酸酶，在胰岛素信号通路中起着重要的负调控作用，是目前公认的一个新颖的糖尿病和肥胖症治疗靶点。寻找PTP1B的高活性抑制剂对糖尿病和肥胖症治疗有着重要的应用前景。 双-(2,3-二溴-4,5-二羟基苯基)-甲烷（BDDPM）是从松节藻醇提物中分离鉴定出的溴酚类化合物，体外活性筛选发现，它具有极强的蛋白酪氨酸磷酸酶1B(PTP1B)抑制活性(IC50=2.4μmol/L)。采用高脂饮食-链脲佐菌素诱导的大鼠模型（STZ-DM）对富含BDDPM的松节藻醇提物进行动物实验，发现中、高剂量组同样表现出惊人的活性，降糖效果优于阳性对照临床药物文迪雅，并呈剂量依赖性。于是拟采用STZ-DM大鼠模型对单一组分BDDPM进行药理、药效学等体内降糖活性研究，但体内动物实验需要30g以上BDDPM，所以首先要解决药源的问题。本文尝试从天然海藻提取分离和化学合成两种途径来解决BDDPM制备的问题。 首先本文尝试从松节藻中提取分离BDDPM的制备方法。通过正相硅胶色谱、凝胶Sephadex LH-20色谱和重结晶等纯化手段分离纯化目标化合物BDDPM，并借助IR，MS和NMR等技术确定了其化学结构。最终从常温风干的50kg松节藻干样品中分离得到7.8g BDDPM。由于松节藻藻体构成复杂，给分离纯化BDDPM带来极大困难，致使分离纯化过程耗费大量时间和金钱；并且原材料松节藻的采集也易受季节和原料短缺等自然因素的影响。所以，从天然海藻中分离纯化的方法不适宜用于BDDPM的制备。 本文的重点是对BDDPM（4e）的化学合成途径进行研究。本文通过5步合成法（Friedel-Craftz酰基化反应、苯环逐级溴代、羰基还原、羟基脱保护）成功地合成了BDDPM，合成总产率为23.6%。同时本文采用上述合成路线获得了四个系列共计20个溴酚系列衍生物（4e为目标产物BDDPM，其余19个为溴酚系列衍生物），其中10个为新化合物。合成的20个化合物经1H NMR、13C NMR、MSEI和IR进行了结构鉴定。合成的20个化合物在体外活性筛选中均表现出不同程度的PTP1B抑制活性，其中合成的目标产物4e具有与天然分离纯化获得的BDDPM同等效率的PTP1B抑制作用。 另外，通过比较四个系列化合物PTP1B抑制活性间的差异，对此类溴酚化合物的构效关系作了初步分析，结果表明：1．羰基官能团的存在会明显降低此类化合物的PTP1B抑制率；2．化合物中的羟基官能团被甲氧基保护后，PTP1B抑制活性会得到一定程度的加强；3．化合物苯环上溴原子取代基数目增多时，其PTP1B抑制率也会随之增强。但是，筛选结果中也有少部分化合物的PTP1B抑制作用与上述规则相违背。因此，本文总结的初步构效关系还需要进一步的实验研究加以验证。 最后本文通过化学合成的方法，经过5步反应成功地制备出了30g BDDPM，为后续的药理、药效学研究奠定了基础。

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Immune condition of Chlamys farreri in response to acute temperature challenge

The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 degrees C to 11 degrees C, 23 degrees C and 28 degrees C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 degrees C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 degrees C to 28 degrees C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 degrees C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. (C) 2007 Elsevier B.V. All rights reserved.

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 degrees C, 17 degrees C and 25 degrees C respectively. Thereafter, a recovery period of 24 h at 17 degrees C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immumomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 degrees C and 24 h of air exposure at 17 degrees C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 degrees C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 degrees C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 degrees C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops. (c) 2007 Elsevier B.V. All rights reserved.

Survival, growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay (Yellow Sea, China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 mu m/d) and 20 m (236.9 mu m/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 mu m/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.

STARVATION-INDUCED CHANGES OF HEMOCYTE PARAMETERS IN THE ZHIKONG SCALLOP CHLAMYS FARRERI

Substantial nutritional and energetic demands m-e associated with immune activation and the maintenance of an efficient immune system. One-year-old Chlamys farreri (Jones and Preston) scallops were maintained ill lantern nets ill different nutritional conditions (satiation and starvation) for 40 days. After the 40-day treatments, the condition index and the total hemocyte count (THC) decreased significantly in the starved group compared with the satiated and initial control groups. The percentage of phagocytic hemocytes also was significantly reduced with starvation. In contrast. no significant effect of starvation was observed oil reactive oxygen species (ROS) production. The acid phosphatase (ACP) activities in cell-free hemolymph increased significantly in scallops in starved and satiated treatments compared with the initial control. whereas ACP activity in hemocyte lysate was significantly lower ill the starved group. These results indicate that starvation stress compromises immunological activities of scallops.

Total synthesis of bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane as potent PTP1B inhibitor

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator and has been proved to be an effective target for the treatment of type 2 diabetes mellitus. Bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane 7 was first reported as a natural bromophenol with significant inhibition against PTP1B which was isolated from red algae Rhodomela confervoides. Intrigued by its astonishing activity (IC50 = 2.4 mu mol/L), compound 7 was synthesized with the overall yield of 24% and evaluated for its PTP1B inhibitory activity compared with natural compound. (C) 2008 Li Jun Han. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Inhibition of bromophenols against PTP1B and anti-hyperglycemic effect of Rhodomela confervoides extract in diabetic rats

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator in insulin signaling pathways. PTP1B is an effective target for the treatment of type 2 diabetes mellitus. Four bromophenol derivatives from red algae Rhodomela confervoides, 2,2',3,3'-tetrabromo-4,4',5,5'-tetra-hydroxydiphenyl methane (1), 3-bormo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl) pyrocatechol (2), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (3) and 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxy-methyldiphenylmethane (4) showed significant inhibitory activity against PTP1B (IC50 were 2.4, 1.7, 1.5 and 0.84 mu mol/L, respectively) as potential therapeutical agents for the treatment of type 2 diabetes mellitus. The anti-hyperglycemic effects of the ethanol extracts from R. confervoides on streptozotocin-induced diabetes (STZ-diabetes) in male Wistar rats fed with high fat diet were investigated. The STZ-diabetic rats treated with medium-dose and high-dose alga extracts showed remarkable reductions in fasting blood glucose (FBG) as compared with the STZ-diabetic control. The results indicate that the in vivo anti-hyperglycemic activity of the R. confervoides extracts can be partially attributed to the inhibitory actions against PTP1B of the bromophenol derivatives and that may be of clinical importance in improving the management of type 2 diabetes mellitus.

空间对接缓冲试验台助推装置研究

设计了一种用于空间对接缓冲试验台的新型高动态响应助推力模拟装置。介绍了助推装置的机构组成和原理,计算了助推气动系统的响应时间,并分析了助推运动过程。研究表明:该助推装置的响应速度快、精度高,助推力方向实时通过模拟飞行器的质心,满足设计要求。

Understanding and Modeling the Behavior of a Harmonic Drive Gear Transmission

In my research, I have performed an extensive experimental investigation of harmonic-drive properties such as stiffness, friction, and kinematic error. From my experimental results, I have found that these properties can be sharply non-linear and highly dependent on operating conditions. Due to the complex interaction of these poorly behaved transmission properties, dynamic response measurements showed surprisingly agitated behavior, especially around system resonance. Theoretical models developed to mimic the observed response illustrated that non-linear frictional effects cannot be ignored in any accurate harmonic-drive representation. Additionally, if behavior around system resonance must be replicated, kinematic error and transmission compliance as well as frictional dissipation from gear-tooth rubbing must all be incorporated into the model.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Separation of compounds interacting with liposome membrane in combined prescription of traditional Chinese medicines with immobilized liposome chromatography

Immobilized liposome chromatography (ILC), the stationary phase of which has been regarded as a mimic biomembranes system was used to separate and analyze compounds interacting with liposome membrane in Danggui Buxue decoction, a combined prescription of traditional Chinese medicines (CPTCMs), and its compositions Radix Astragli and Radix Angelica Sinensis. More than 10 main peaks in the extract of Danggui Buxue decoction were resolved on the ILC column, suggesting that more than 10 components in the prescription have significant retention on ILC column. Ligustilide, astragaloside, TV and formononetin, three main bioactive ingredients in Danggui Buxue decoction, were found to have relatively significant, while ferulic acid, another bioactive ingredient in the prescription, relatively weak retention on ILC column. Effects of the eluent pH and amount of immobilized phosphatidylcholine (PC) on separation of interactional compounds in the extract of Danggui Buxue decoction were also investigated. It was found that these two factors strongly affected the retention of some interactional compounds. In addition, the fractions partitioned with different solvents from water extract of this combined prescription were evaluated with this ILC column system. (c) 2005 Elsevier B.V. All rights reserved.