拟南芥生育酚环化酶基因（VTE1）在烟草中的表达及功能研究

维生素E（V.E.）在动物细胞内具有抗氧化等重要作用，但在植物体内的功能却鲜为人知。本研究以烟草为材料，利用根癌农杆菌（Agrobacterium tumefaciens）介导法在烟草中过量表达拟南芥来源的VTE1。通过外源VTE1基因的过量表达提高内源V.E.的含量, 进而研究转VTE1基因植株对胁迫的耐受性反应，以探讨植物体内V.E.含量与植物胁迫耐受性的关系，为植物抗逆机理的研究和利用奠定基础。 本实验利用CaMV35s启动子与拟南芥来源的生育酚环化酶基因（VTE1）构建的嵌合表达载体，以根癌农杆菌介导的叶盘法转化烟草W38。实验结果表明: 1. 具有卡那霉素抗性的再生植株经PCR检测，得到了与阳性对照一致的495bp的目标片段;经RT-PCR检测，其中90%有外源基因表达。 2. 转基因植株的V.E.含量比对照植株高2倍左右，个别株系高达10.16倍。 3. VTE1基因的表达受环境胁迫的影响，不同程度的冷冻、热激、PEG处理均可影响VTE1基因的表达。经过冷冻处理60分钟、热处理20小时、以及PEG处理6小时，该基因表达量均有提高。冷冻处理条件下该基因的表达量是未处理的3倍，热处理条件下是未处理的2倍左右，PEG处理是未处理的3.5倍。在冷冻、热激、PEG胁迫处理过程中，转化苗的V.E.含量变化与外源VTE1基因的表达相对应，表明转化苗的V.E.合成主要由外源VTE1基因的终产物VTE1催化;在冷冻、热激、PEG胁迫处理过程中，V.E.含量与APX、CAT、SOD等抗氧化酶活性之间存在一定程度的正相关性，表明V.E.与这些抗氧化酶共同组成了植物体内的抗氧化网络，保护植株免受氧化损伤;V.E.的变化与MDA之间存在一定程度的负相关性，减轻植物的过氧化损伤; 4. V.E.可提高植物的抗旱性，我们检测了11个转化烟草株系的叶片相对含水量（RWC），在大多数转化烟草植株中，干旱胁迫24小时的RWC都比野生型高，高出0.16-45%（p<0.001）。表明转基因烟草比野生型更抗旱; 5. 在耐盐性实验中转基因植株对盐的抗性明显高于野生型烟草;同时，在不同盐浓度（150、250mM）胁迫下转基因植株V.E.含量比未转化植株增加了1.3-1.8倍。 这些研究结果表明，在植物体内转入V.E.代谢途径中的单个外源基因，可有效提高内源V.E.合成，提高植株对环境胁迫的抗性。

甘蓝型油菜中脯氨酸积累的分子调控机制

　　渗透胁迫下植物中游离脯氨酸的积累被普遍认为对植物有保护作用。本文对甘蓝型油菜（Brassica napus）中的脯氨酸积累及其调控机理进行了研究。正常生长条件下幼苗叶中脯氨酸含量最高，根中脯氨酸含量最低，盐胁迫后不同部位脯氨酸积累程度相近。盐浓度200 mM起，幼苗开始有明显脯氨酸积累,并随着浓度的增加而增加。PEG处理后脯氨酸积累要明显高于ABA和盐胁迫处理后脯氨酸积累。在成株中，生殖器官中脯氨酸含量明显高于营养器官中脯氨酸含量。 　　我们克隆了编码Δ1-二氢吡咯啉-5-羧酸合成酶（P5CS）,鸟氨酸转氨酶(OAT)和脯氨酸脱氢酶(PDH)的四个基因的cDNA。通过Southern杂交检测P5CS基因在甘蓝型油菜及其亲本白菜型油菜和甘蓝中的拷贝数，发现杂交条带在4倍体油菜中并没有显著的多于其两个2倍体亲本，推测有可能是在物种进化的过程中发生了基因丢失。 　　利用实时定量PCR的方法检测了渗透胁迫下甘蓝型油菜中BnP5CS，BnOAT和BnPDH等基因的表达水平。在ABA处理，盐处理和干旱处理时，BnP5CS1和BnP5CS2的表达在脯氨酸积累开始前就开始有明显上调，但是BnP5CS1的上调水平要比BnP5CS2高许多。BnOAT在ABA处理后表达水平没有太大的变化，在盐胁迫后甚至有一点下调，在干旱处理后却表现为一定程度的上调。另一方面，BnPDH的表达在ABA处理，盐胁迫和干旱胁迫后都受到了抑制。在成株不同器官相关基因表达的研究中发现，BnOAT在叶中表达量最高，BnP5CS和BnPDH在花蕾和花中的表达水平都比其他器官中要高。 　　我们的结果说明，渗透胁迫下甘蓝型油菜中的脯氨酸积累是脯氨酸合成途径的诱导和脯氨酸降解途径的抑制共同作用的结果。在轻度的渗透胁迫下，谷氨酸合成途径占主导地位，而在较为严重的渗透胁迫后期，谷氨酸合成途径和鸟氨酸合成途径共同起作用。甘蓝型油菜生殖器官花和花蕾中脯氨酸的代谢非常旺盛，说明脯氨酸可能在花发育中起着一定的作用。

种子超干保存种质的研究

当今，生物资源——“物种”正在以地质史上前所未有的速度灭绝或走向濒危。保护生物多样性已是世界范围内环境保护和生物学研究的热点。基于种子具有完善的保护结构和内潜的隐蔽生命以适应逆境的微妙机制，种子库被认为是保护植物种质资源的主要措施。现阶段世界各国的种子库均为低温库或超低温库，可观的建库投资和运转费用的经济负担直接影响到贫困国家和地区植物种质资源及时有效的保护，寻求取代低温库的廉价贮藏种子的策略和技术已列为联合国FAO／IBPGR的全球战略措施。 此乃本文的工作背景和最终目标。早期的研究已查明，通过降低种子含水量可以在适当提高温度的条件下达到在较高含水量在低温下同样的贮藏效果，但是由于干燥技术以及5%为种子安全含水量下限这一经典概念的局限，种子贮藏的含水量一直没有突破5%下限。超干贮藏即以此为依据，摸索以适当的干燥技术将种子含水量降至这一经典下限以下，即采取超低含水量种子密封贮藏的方法，以达到部分或完全取代低温库的目的。 作为种子贮藏的方法首先要保证种子活力的保持。本文选用了不同化学组份以及不同粒度大小的种子为材料，涉及到40个不同的种和品种，针对不同类型的种子摸索了适宜的干燥方法及辅助措施，从种子生活力和活力、细胞形态结构、细胞器发育和功能以及若干生理活性指标等多元角度上确认种子含水量可以安全降至5%以下，除了个别敏感类型种子外，在常温条件下能大幅度增强种子的耐藏性。 本文对种子耐干性差异以及辅助干燥措施提高种子耐干性进行了水分热力学方面的分析，认为种子本身的特性决定了种子内部水分吸附位点对关键水分束缚力量的不同，导致在相同干燥过程中失去关键性水分程度的不同，从而造成种子在耐干程度上有所差异。干燥的辅助措施可以通过改变种子中的化学成份增强种子对关键性水分吸附的力量，继而使得关键性水分不会轻易丢失。超干延缓种子劣变的原因在于，MC不能真实反映种子水分水平，一定MC范围内的超干种子的关键性水分仍然存在，即使在极度干燥的种子中，虽然一部分束缚水已失去，但对导致和抑制脂质过氧化的正负效应折衷的结果使劣变反应从整体上还是受到抑制。内源抗氧剂的活性及含量以及劣变产物MDA的积累量表明种子在超干贮藏过程中脂质过氧化被部分抑制，酶类抗氧剂系统保存完好，非酶类抗氧剂被消耗的量减少，使得种子在结束贮藏进入萌发状态时仍具有较高水平的自由基清除系统的正常运转能力。无氧贮藏，和外源抗氧防护等预处理措施使种子在超干贮藏过程中能更好地清除活性氧的伤害，吸湿回干、PEG等处理使超于种子吸胀时膜系统功能及结构更具完整性。回水预处理诱导的生化修补效应可保留于种子内部，以便在种子再次吸水时及早发挥作用。本文已在干前及干后预处理辅助措施上找到了若干行之有效的方法，其中有些是首次应用于种子技术范畴。 本文在总结系列试验研究结果和纵观近年来国内外在此领域的动态后，认为一般常规型种子在适度超低水分状态下不仅自身不受脂质过氧化的危害，并能保持种子内外形态学和种质遗传性的完整性，而且可使种子内部代谢停顿，更有利于耐藏性的大幅度提高，加上超干技术上的不断完善，为今后建立节能种子库展示了希望。

盐胁迫对光合作用的影响

盐害对植物光合作用的影响机理的研究目前还仅仅处于初步阶段，对于许多关键性问题远没有达成共识。本论文研究了盐胁迫对光合作用的影响途径和原初作用部位，对光合作用的各个分过程影响的先后关系，盐胁迫与光胁迫的协同作用，植物光合作用对盐胁迫的适应等基础理论问题，为盐胁迫下提高植物光合作用速率，改良植物种质提供了一定的理论依据。 1．采用荧光动力学的方法区分盐胁迫中的渗透因素和离子因素。用五种等渗Hogland培养液(分别含NaCl，KCl，NaN03，KN03和PEG)对冬小麦进行处理。结果，与对照相比，NaCl处理引起PSII受体侧电子库含量(CA／Fm)、PSII活性(Fv／Fo)、原初光能转化效率(Fv／Fm)、量子产量(Yield)与荧光光化学猝灭系数(qP)下降;QB-非还原性PSII反应中心含量增加。然而，等渗的PEG处理并不产生类似的伤害。这表明渗透因素不是盐胁迫对光合作用造成伤害的主要原因。鉴于NaCl和NaNO3处理却使QA含量、PSII活性、原初光能转化效率下降和QB-非还原性PSII反应中心的增加，KN03处理对光合作用不产生伤害;而等渗的PEG和KCl处理并不产生类似的伤害，这暗示Na+可能是盐胁迫影响光合作用的主要毒害离子。 2．用荧光动力学的方法研究了不同浓度的NaCl处理对PSII光能利用和耗散的影响。结果表明，与对照、100mmol/L和200mmol/L NaCl处理相比，经300mmol/L和400mmol/L NaCl处理的小麦，其荧光光化学猝灭效率明显降低，荧光非光化学猝灭效率较高，Fo猝灭系数较大，QB-非还原性PSII反应中心含量较大，然而，其荧光非光化学猝灭效率，QB-非还原性PSII反应中心含量和Fo猝灭系数潜在增高能力较弱。随着NaCl处理浓度的增加，PSII吸收过多的光能可能首先通过热耗散和状态转换逸散，而后阻断PSII从QA到QB的电子传递，最后损伤PSII反应中心。 3．研究了轻度(200mmol/L)和重度(400mmol/L)NaCl胁迫对光抑制和光恢复进程的影响。结果表明：1)轻度盐胁迫对光合放氧和碳同化速率的影响相对较小，而重度盐胁迫大大降低了光合放氧和碳同化速率，同时明显降低光合放氧和碳同化的光饱和点。2)碳同化是盐胁迫对光合作用造成伤害的敏感位点。3)轻度盐胁迫对PSII的光抑制进程影响较小;而重度盐胁迫则促进光抑制的产生，同时抑制PSII的修复过程，从而使得PSII受到光抑制的伤害更大。 4 为探讨盐胁迫下植物减轻或避免光抑制的机制，研究了盐胁迫对光能在PSI和PSII之间的分配的影响。结果表明，盐胁迫提高了Chla/Chlb比值和单位鲜重叶片中Chla的含量，降低了单位鲜重叶片Chlb的含量;提高了PSI的电子传递速度，能量储存;同时降低了PSII的电子传递和能量储存。这表明盐胁迫提高了光能在PSI的分配分额和PSI的含量，降低PSII对光能的吸收和利用，这对于植物在盐胁迫下减轻或避免光抑制对PSII的破坏有重要意义。

根茎在羊草响应短期盐胁迫和干旱胁迫过程中的作用

羊草 (Leymus chinensis (Trin.) Tzvel.) 又称碱草，隶属禾本科，赖草属，因其营养价值高，富含蛋白质，适口性好，抗旱，耐盐碱，耐贫瘠，抗逆性强，适应广等优点，对我国发展草原畜牧业和退化草地、荒漠化治理方面具有举足轻重的作用。近年来，由于自然环境变劣，荒漠化加剧，以及过渡放牧等不利影响，已对我国草地构成了严重威胁，严重制约了我国人工草地建设和天然草地的改良和沙化治理的步伐。目前, 国内外关于羊草的报道大多集中在草原生态等宏观领域，在羊草根茎生理学方面的研究却十分匮乏。作为一种典型的根状茎型克隆植物，羊草根茎在无性繁殖和克隆基株间的克隆整合、形态可塑性和分工合作等方面具有重要的作用,但羊草根茎在羊草感应环境胁迫中的作用却研究甚少。为了探讨根茎在植物感应环境胁迫中的作用, 本文以羊草为实验材料，通过分别处理羊草根茎和根，研究和比较了短期NaCl、PEG胁迫根、根茎、根和根茎三种处理方式下羊草对盐胁迫和干旱胁迫的生理响应。主要研究结果如下： 1、200 mmol• L-1 NaCl处理羊草根、根茎、根和根茎24小时显著降低羊草叶片净光合速率和蒸腾速率，增加叶片渗透浓度与脯氨酸含量;其中同时处理根和根茎，叶片蒸腾速率和净光合速率的降低程度显著高于分别处理根和根茎。在分别处理根与根茎的情况下，叶片含水量、脯氨酸含量、净光合速率、蒸腾速率均无显著性差异。不论单独胁迫根、根茎还是同时胁迫根和根茎，羊草根、根茎和叶片内钠离子（Na+）含量增加，而钾离子（K+）含量和K+/Na+ 比降低。 这些结果表明：（1）羊草根茎在感应和响应盐胁迫的生理过程中与根系具有类似的功能;（2）羊草根茎在盐胁迫条件下参与Na+ 吸收和向地上部的转运;（3）鉴于根茎的生物量和表面积都明显的低于根系，在盐胁迫下羊草根茎吸收Na+ 的效率高于根系。 2、15％ 聚已二醇（PEG－6000）处理羊草根、根茎、根和根茎24 小时显著降低羊草叶片含水量、净光合速率、蒸腾速率和水分利用效率，增加叶片脯氨酸含量。在分别胁迫根与根茎两种处理下，处理根茎对叶片的净光合速率影响程度更大，表明根茎对干旱胁迫反应更敏感。

大豆种子低温吸胀相关基因的分离与蛋白变化的研究

大豆 (Glycine max (L.) Meer.)是人们日常生活中不可缺少的食品，也是一种非常重要的油质、蛋白资源。目前根据大豆种子吸胀阶段对低温敏感性的不同，可将其划分成3种生态型：低温非敏感型、低温敏感型及中间型。对于低温非敏感型的种子来讲，4℃下吸胀24小时对其发芽率影响很小，而敏感型种子萌发率不超过5%。我国属于温带大陆性气候，大豆播种后由于温度波动而造成一部分种子不能萌发，最终导致减产甚至绝产的现象普遍存在。高产是育种工作的主要目标，提高逆境胁迫的适应能力是高产的前提和基础，所以从分子角度研究种子吸胀非常必要，一方面能够挖掘新的基因资源，另一方面为今后育种工作提供必要的理论依据。 本试验以此为立足点，低温吸胀非敏感型大豆品种 (Z22)为材料，利用cDNA-AFLP方法及蛋白质技术分离与低温吸胀相关的基因及蛋白，得到结果如下： 第一，本试验成功的分离出4个受低温诱导的基因，半定量RT-PCR方法进一步验证了这4个基因在种子吸胀24小时内受低温诱导。 第二，利用RACE方法成功的得到2个完整的全长基因，在NCBI数据库中查找后发现其中1个基因为新基因，命名为SCHI基因 (SCHI：Soybean chilling-induced gene)。SCHI全长为390bp，编码分子量大约为14.2KD的蛋白；另外一个基因是已知基因，其同源序列已经在其他的物种中得到分离。由于此基因与核糖体蛋白L34高度同源，所以把把这个基因命名为SOL34 (Soybean L34)。 第三，利用半定量RT-PCR方法对基因表达模型进行分析，结果表明：SCHI在种子低温吸胀18～24小时期间诱导表达量最高，而当种子低温吸胀24小时后转入常温下，其表达量在常温下18小时左右迅速下降；ABA (100μM)、PEG (30%，10000)及NaCl (250mM)能够诱导SCHI的表达，在诱导表达量上，ABA和PEG诱导效果最明显，而NaCl能够微弱的诱导此基因表达；对不同生态型的大豆品种而言，低温吸胀过程中，SCHI在非敏感型种子中的表达量高于敏感型种子，但非敏感型和中间型之间没有差别；另外，SCHI在大豆胚轴中是诱导型表达，在叶片和根尖中则是组成型表达。SOL34的表达在萌发前24小时内被低温诱导，但在不同生态型之间没有差别。SOL34在胚轴和根尖中受低温诱导，在叶片中是组成型表达。 第四，SCHI能够在原核生物中表达出相应蛋白，诱导表达蛋白的分子量在26-29KD，大约为理论值的2倍，说明在大肠杆菌中被表达的蛋白以2聚体形式存在。另外低温试验结果表明SCHI能够提高菌落忍耐短时间-20℃低温的能力。 第五，利用双元表达载体把SCHI转入拟南芥植株，经过低温、干旱和盐胁迫后，转基因植株的成活率均高于野生型植株。超表达SOL34的拟南芥植株降低了对低温的耐性；而抑制拟南芥中L34的表达反而提高了植株对低温的抗性。 第六，本试验利用蛋白质等有关试验检测了大豆种子低温吸胀时蛋白质发生的变化。从吸胀 (4℃和22℃下24h)后的大豆胚轴中成功鉴定出上调蛋白点25个，下调蛋白点15个。其中有参与能量代谢反应 (占10%，例如柠檬酸脱氢酶和苹果酸脱氢酶等)、细胞生长与分裂相关反应 (20%，例如LEA蛋白和种子成熟蛋白PM26)、胁迫反应 (10%，如乙醇脱氢酶)、种子宿命和贮藏蛋白 (20%，大豆球蛋白)等蛋白在此过程中发生了变化，暗示种子萌发前期低温吸胀过程中多种代谢发生变化。细胞生长变缓、能量代谢增强、胁迫代谢蛋白的高表达以及贮藏蛋白降解速度减慢等变化都有利于种子在吸胀过程中度过低温环境，为以后的生长作好准备。

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

Evaluation of multi-sensory feedback on the usability of a virtual assembly environment

Virtual assembly environment (VAE) technology has the great potential for benefiting the manufacturing applications in industry. Usability is an important aspect of the VAE. This paper presents the usability evaluation of a developed multi-sensory VAE. The evaluation is conducted by using its three attributes: (a) efficiency of use; (b) user satisfaction; and (c) reliability. These are addressed by using task completion times (TCTs), questionnaires, and human performance error rates (HPERs), respectively. A peg-in-a-hole and a Sener electronic box assembly task have been used to perform the experiments, using sixteen participants. The outcomes showed that the introduction of 3D auditory and/or visual feedback could improve the usability. They also indicated that the integrated feedback (visual plus auditory) offered better usability than either feedback used in isolation. Most participants preferred the integrated feedback to either feedback (visual or auditory) or no feedback. The participants' comments demonstrated that nonrealistic or inappropriate feedback had negative effects on the usability, and easily made them feel frustrated. The possible reasons behind the outcomes are also analysed. © 2007 ACADEMY PUBLISHER.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Chemical surface modification of poly-ε-caprolactone improves Schwann cell proliferation for peripheral nerve repair.

Poly-ε-caprolactone (PCL) is a biodegradable and biocompatible polymer used in tissue engineering for various clinical applications. Schwann cells (SCs) play an important role in nerve regeneration and repair. SCs attach and proliferate on PCL films but cellular responses are weak due to the hydrophobicity and neutrality of PCL. In this study, PCL films were hydrolysed and aminolysed to modify the surface with different functional groups and improve hydrophilicity. Hydrolysed films showed a significant increase in hydrophilicity while maintaining surface topography. A significant decrease in mechanical properties was also observed in the case of aminolysis. In vitro tests with Schwann cells (SCs) were performed to assess film biocompatibility. A short-time experiment showed improved cell attachment on modified films, in particular when amino groups were present on the material surface. Cell proliferation significantly increased when both treatments were performed, indicating that surface treatments are necessary for SC response. It was also demonstrated that cell morphology was influenced by physico-chemical surface properties. PCL can be used to make artificial conduits and chemical modification of the inner lumen improves biocompatibility.

Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a β1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that β1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

鱼类细胞融合中助融剂效应和特异性

在鱼类细胞融合中,微量的甘油可使PEG作用显著下降。DMSO可以极大提高PEG诱导鱼类细胞融合的能力。在低分子量和较低浓度的PEG中,DMSO的作用更加突出。但PEG浓度不能低于40%的临界,否则,细胞融合就失去了DMSO浓度依赖效应。在三组鱼类细胞交叉融合中,我们发现同核体数目远远超过异核体数目(P<0.05)。这表明,PEG诱导的鱼类细胞融合具有物种和组织特异性。

Development of a nanoporous and multilayer drug-delivery platform for medical implants.

Biodegradable polymers can be applied to a variety of implants for controlled and local drug delivery. The aim of this study is to develop a biodegradable and nanoporous polymeric platform for a wide spectrum of drug-eluting implants with special focus on stent-coating applications. It was synthesized by poly(DL-lactide-co-glycolide) (PLGA 65:35, PLGA 75:25) and polycaprolactone (PCL) in a multilayer configuration by means of a spin-coating technique. The antiplatelet drug dipyridamole was loaded into the surface nanopores of the platform. Surface characterization was made by atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). Platelet adhesion and drug-release kinetic studies were then carried out. The study revealed that the multilayer films are highly nanoporous, whereas the single layers of PLGA are atomically smooth and spherulites are formed in PCL. Their nanoporosity (pore diameter, depth, density, surface roughness) can be tailored by tuning the growth parameters (eg, spinning speed, polymer concentration), essential for drug-delivery performance. The origin of pore formation may be attributed to the phase separation of polymer blends via the spinodal decomposition mechanism. SE studies revealed the structural characteristics, film thickness, and optical properties even of the single layers in the triple-layer construct, providing substantial information for drug loading and complement AFM findings. Platelet adhesion studies showed that the dipyridamole-loaded coatings inhibit platelet aggregation that is a prerequisite for clotting. Finally, the films exhibited sustained release profiles of dipyridamole over 70 days. These results indicate that the current multilayer phase therapeutic approach constitutes an effective drug-delivery platform for drug-eluting implants and especially for cardiovascular stent applications.

Evaluation of the functionality of biodegradable polymeric platforms for drug delivery systems

We present the development of a drug-loaded triple-layer platform consisting of thin film biodegradable polymers, in a properly designed form for the desired gradual degradation. Poly(dl-lactide-co-glycolide) (PLGA (65:35), PLGA (75:25)) and polycaprolactone (PCL) were grown by spin coating technique, to synthesize the platforms with the order PCL/PLGA (75:25)/PLGA (65:35) that determine their degradation rates. The outer PLGA (65:35) layer was loaded with dipyridamole, an antiplatelet drug. Spectroscopic ellipsometry (SE) in the Vis-far UV range was used to determine the nanostructure, as well as the content of the incorporated drug in the as-grown platforms. In situ and real-time SE measurements were carried out using a liquid cell for the dynamic evaluation of the fibrinogen and albumin protein adsorption processes. Atomic force microscopy studies justified the SE results concerning the nanopores formation in the polymeric platforms, and the dominant adsorption mechanisms of the proteins, which were defined by the drug incorporation in the platforms. © 2013 Elsevier B.V. All rights reserved.

New data on the morphology and systematic status of Spinitectus petrowi and Spinitectus gigi (Nematoda : Cystidicolidae) parasitic in catfishes in central China

Two little-known nematode species of the genus Spinitectus Fourment, 1883, S. petrowi Belous, 1965 (prevalence 25%, intensity 1-8) and S. gigi Fujita, 1927 (prevalence 10%, intensity 2-3), were collected from the gastrointestinal tract of the yellow catfish, Pelteobagrus fulvidraco (Richardson), from Liangzihu Lake, Hubei Province, central China, in September of 2002. The light and scanning electron microscopical examination of this material, supplemented by a few museum specimens of S. gigi collected from the catfish Clarias fuscus (Lacepede) in southern China, made it possible to study in detail the morphology of these parasite species and to redescribe them. The first species, whose correct name is S. petrowi Belous, 1965, exhibits some morphological features (e.g., unusually short vestibule, shape of pseudolabia and of the left spicule) not found in most other congeners; a unique feature is the presence of peculiar pairs of transversely oriented peg-like cuticular spines with rounded ends on the ventral surface of the female tail. Spinitectus gigi was found to have 28-31 cuticular spines in the first ring, relatively long distances between the 2nd-7th rings of spines, and anterior rings divided into 2 sectors; the excretory pore is located at the level of the 4th ring of cuticular spines; males posses 4 pairs of preanal- and 6 pairs of postanal caudal papillae and a pair of small phasmids. Spinitectus bagri Wang, Wu et Yu, 1993 and S. wulingensis Yu et Wang, 1997 are considered junior synonyms of S. petrowi, whereas S. clariasi Ky, 1971, S. ophicephali Ky, 1971 and S. yuanjiangensis Wang, Wit et Yu, 1997 are regarded to be junior synonyms of S. gigi. Spinitectus petrowi was not previously reported from China.