新生大鼠海马神经元体外分散培养技术研究

在实验室建立了分散的新生大鼠海马神经细胞体外培养模型。该模型属原代培养,在体外能直接观察神经元的生长发育。研究表明：该模型稳定性好,差异性小;培养神经细胞可存活１个月以上,１４ｄ左右细胞生长最为丰满,且四周晕光明显,神经突起多而粗大,分枝互成网络;１４ｄ以后生长减缓。由于在此模型培养的条件下,既不存在血脑屏障问题,又容易控制条件,便于在细胞和分子水平上深入研究神经细胞的形态学、生理学、生化代谢及药理学等方面的问题,特别适合在体外进行神经药理学的研究。     

牛膝总皂苷体外干预人膝骨关节炎软骨细胞Ⅱ型胶原表达及最佳浓度的实验研究

目的：观察牛膝总皂苷（TSA）体外干预人膝骨关节炎软骨细胞增殖及Ⅱ型胶原（CollagenⅡ）表达水平及最佳浓度。方法：将人膝骨关节炎软骨细胞进行分离、培养、鉴定,并将其分为6组,分别为0、0.01、0.05、0.1、0.5、1 mg/ml组。制备TSA存储液及6组不同TSA浓度DMEM/F12（D/F12）培养基,分别用6组培养基对P3代软骨细胞培养并进行细胞形态学观察;采用CCK-8法检测软骨细胞P1、P2、P3传代增殖情况,观察其生长曲线;采用Western-blot法检测P3代软骨细胞Collagen II蛋白表达、RT-PCR法检测CollagenⅡ mRNA表达。结果：软骨细胞体外分离并培养成功,经甲苯胺蓝染色鉴定细胞为软骨细胞来源;CCK-8法绘制的生长曲线符合Logistic生长曲线规律,P3代软骨细胞增殖速度高于P1、P2代;Western-Blot法检测结果显示,Collagen II蛋白表达量浓度为0.5 mg/ml时达最高,RT-PCR法检测结果显示,各组浓度AST能促进软骨组织Ⅱ型胶原mRNA表达上调,以0.5 mg/ml组最明显（P<0.05）。结论：TSA能体外有效促进软骨细胞增殖和Ⅱ型胶原mRNA表达上调,以0.5 mg/ml浓度最明显,具有促进软骨修复的作用,具体作用机制有待进一步研究。     

南海北部珠江口盆地新生代伸展过程及其区域差异性定量研究

以珠江口盆地为研究对象,自西向东选取了能控制区域构造格局的5条区域性大剖面,运用2D Move软件分别对其进行平衡剖面恢复,并计算出各剖面不同时期的伸展量、伸展率和伸展速率,以此为基础对珠江口盆地的构造演化过程及其区域性差异进行了定量研究。研究结果显示:(1)珠江口盆地新生代以来一直处于一个持续伸展的过程,并于32-30 Ma期间出现一个伸展高峰期,但总体呈现伸展率和伸展速率逐渐降低的趋势;(2)珠江口盆地新生代主要伸展变形时期其伸展的过程具有明显的区域性差异,即自西向东伸展率和伸展速率明显逐步变大。     

芹黄素对脑缺血再灌注损伤保护作用的实验研究

目的：观察芹黄素对脑缺血再灌注损伤沙土鼠脑保护作用及炎症反应的影响。方法：采用双侧颈总动脉夹闭手术法建立脑缺血再灌注损伤沙土鼠模型,将其随机分为模型组、尼莫地平组（2 mg/kg）、芹黄素组（40 mg/kg）、假手术组,建模前1周开始腹腔注射给药,再灌注6 h内记录各组神经症状并做卒中评分,再灌注6 h后以免疫组化法测定脑组织海马CA1区核转录因子-κB（NF-κB）的表达。结果：芹黄素组和尼莫地平组脑缺血再灌注损伤沙土鼠的卒中评分、脑部海马CA1区NF-κB,与模型组相比,差异有统计学意义（P＜0.01）;与假手术组脑部极低的NF-κB表达相比,模型组沙土鼠海马CA1区NF-κB表达明显升高（P＜0.01）。结论：芹黄素可抑制核转录因子的激活,调控炎症因子表达,减轻脑部炎症反应,实现对脑缺血再灌注损伤沙土鼠的脑保护作用。     

转鲑鱼降钙素基因酵母及RANKL对大鼠血管平滑肌细胞体外钙化的影响

观察核因子kB受体激活物配体(RANKL)对大鼠血管平滑肌细胞体外钙化的调节作用,以及转鲑鱼降钙素基因酵母对RANKL调节作用的影响.体外培养大鼠血管平滑肌细胞,建立体外血管平滑肌细胞钙化模型,在培养介质中分别加入RANKL及转鲑鱼降钙素基因酵母蛋白上清液,观察细胞钙沉积、细胞碱性磷酸酶活性及骨钙素的变化情况.结果表明,RANKL促进钙化培养的大鼠血管平滑肌细胞的钙沉积、细胞碱性磷酸酶活性及骨钙素的表达,而转鲑鱼降钙素基因酵母能够抑制RANKL的促钙化作用.RANKL可能通过增加细胞碱性磷酸酶活性及骨钙素的表达从而促进血管平滑肌细胞向成骨细胞转化;转鲑鱼降钙素基因酵母可能通过OPG/RANKL/RANK途径抑制血管平滑肌细胞钙化.     

失荅剌知丸及其拆方对局灶性脑缺血再灌注大鼠海马区ERK1/2通路的影响

目的：探讨失荅剌知丸及其拆方对脑缺血再灌注损伤模型大鼠的作用机制及其有效配伍成分。方法：将204只大鼠随机分为空白对照组（A组）,假手术组（B组）,模型组（C组）,失荅剌知丸组（D组）,巴豆组（E组）,巴豆+三药组（F组）,巴豆+余药组（G组）,其中C、D、E、F、G组制备局灶性脑缺血再灌注模型,并按取材时间点不同分为1、3、7d组。各组大鼠取材前应用Garcia JH法进行神经功能评分;应用HE染色法观察大鼠各时间点脑组织病理学形态改变;免疫荧光法、Western Blot检测细胞外调节蛋白激酶1/2（ERK1/2）蛋白表达水平。结果：1）神经功能评分结果。与A、B组相比,其余各组评分均降低（P＜0.01）;与C组相比,D、E、F、G组评分均增加（P＜0.01）;各给药组中,D组评分高于E、F、G组,且E、F、G组中又以F组评分最高（P＜0.05或P＜0.01）;C、D、E、F、G组评分均随给药时间延长而增加。2）大鼠脑组织海马区HE染色结果。C组大鼠海马CA1区神经细胞较A、B组排列松散、紊乱、模糊,胞核与胞质界限欠清晰;与C组相比,D、E、F、G组神经细胞形态改善明显,且以D组改善最为明显;E、F、G组中以F组改善明显。3）大鼠脑组织海马区免疫荧光结果。C组较A、B组大鼠脑组织海马区p-ERK1/2的阳性细胞计数增加（P＜0.01）;与C组相比,D、E、F、G组p-ERK1/2阳性细胞计数升高（P＜0.01）;各给药组中,D组计数最多,E、F、G组中以F组计数最多（P＜0.05或P＜0.01）;C、D、E、F、G组p-ERK1/2的阳性细胞计数均随给药时间延长而升高。4）Western-bolt结果。与A、B组相比,其余各组大鼠脑组织海马区p-ERK1/2活化程度均升高（P＜0.01）;与C组相比,D、E、F、G组p-ERK1/2活化程度均升高（P＜0.01）;各给药组中C组p-ERK1/2活化程度均优于E、F、G组,E、F、G组中以F组p-ERK1/2的活化程度最显著（P＜0.05或P＜0.01）;C、D、E、F组p-ERK1/2活化程度均随给药时间延长而升高。结论：D、E、F、G组均可改善大鼠脑缺血再灌注后的神经功能损伤,抑制神经细胞凋亡,这可能与其参与调节MAPK-ERK通路中ERK1/2的表达相关;E、F、G组中以巴豆、柴胡、黑诃子、芦荟4味药物的组方治疗效果最佳,考虑该组药物可能是失荅剌知丸能有效抑制局灶性脑缺血再灌注损伤的主要药物组成;巴豆可能是四味药中发挥脑保护作用的主要药物之一。     

我国北部湾球形棕囊藻(Phaeocystis globosa)的表面形态和细胞超微结构的电镜观察

本文首次对分离于我国广西北部湾的一株棕囊藻(Phaeocystis)纯培养进行了形态学研究。通过对其核糖体大亚基序列(LSU rDNA)进行了系统进化分析,并运用光学显微镜、扫描电镜和透射电镜等,对该藻的游动单细胞和囊体细胞表面结构和超微结构进行了详细的观察。结果表明,通过分子序列和形态学特征确证了该纯培养为球形棕囊藻(Phaeocystisglobosa)。通过分子系统进化分析,从北部湾获得的球形棕囊藻与同属的P. antarctica和P. rex的亲缘关系较近,但与P. jahnii的亲缘关系相对较远。我们发现该藻至少有两种类型的细胞:具鞭毛游动细胞和无鞭毛囊体细胞,其大小分别为2.30—3.98μm和3.69—6.49μm。具鞭毛游动细胞表面具有1—2个位置不固定但形状较为规则的圆盘状凸起,细胞表面的鳞片大小无明显差别;囊体细胞均匀地分布在囊体上,囊体细胞表面光滑,有三个较短的附属物。具鞭毛游动细胞和囊体细胞有2个或4个叶绿体,具鞭毛游动细胞的细胞核位于细胞的中下部,而囊体细胞的细胞核则位于细胞的中上部。细胞分泌物在扫描电镜样品处理过程后形成多数与细胞相连的丝状物,且丝状物形成较为规则的五角星形结构,每个角与一根丝相连,有些丝状体上有"瘤状"结构。研究结果填补了我国在球形棕囊藻形态学和超微结构研究上的空白,为深入认识其生物学特征及其成囊机理提供基础。     

2株海马肠道益生菌的筛选与初步鉴定

从正常养殖的健康线纹海马(Hippocampus erectus)肠道中分离纯化到52株细菌,通过溶血性实验对肠道菌群进行初筛,结果发现52株肠道细菌均不产生溶血素,不具有潜在的致病性.以纸片扩散法在胞外蛋白酶选择培养基上对菌株产蛋白酶的能力进行测试,筛选出2种能够分泌胞外蛋白酶的菌株.动物安全性实验证实2株实验菌对海马无明显的毒害作用.海马生长参数测定实验显示投喂菌株的实验组体长、体重的日平均增长率均高于不投喂菌株的对照组,证明所筛选菌株具有一定的生长促进作用,有望对其进行海马养殖微生物制剂的开发.随后测定了2株细菌的16S rRNA基因序列,分析相应细菌序列的同源性,构建系统发育树,结合菌株生理生化特性及其表型特征,分别将Hm7与Hm28鉴定为Bacillus horikoshii和Pseudoalteromonas carrageenovora.     

羚珠散对幼鼠反复热惊厥的防治作用及机制研究

目的：通过评价羚珠散对C57BL/6幼鼠反复热惊厥的减轻作用,探讨其防治热惊厥的药效和可能作用机制。方法：将7 d龄C57BL/6幼鼠30只分为空白对照组、模型对照组和治疗组,每组各10只。采用热水浴法干预模型对照组与治疗组,生成惊厥模型,每次诱导前30 min治疗组灌胃给药羚珠散（1.5 g/kg）,模型对照组灌胃同等体积0.9%氯化钠注射液。观察治疗组与模型对照组幼鼠的惊厥潜伏期和惊厥持续时间的差异,通过HE染色比较各组间幼鼠海马体病理组织学差异,通过Real-time PCR与IHC分析比较空白对照组、模型对照组及治疗组幼鼠海马体中Gabbr1和Kcnt1基因的表达差异。结果：经7次热水浴后,模型对照组幼鼠的惊厥潜伏时间为（32.80±3.27）s,治疗组幼鼠的惊厥潜伏时间为（50.90±6.88）s,治疗组的惊厥潜伏时间长于模型对照组（P＜0.05）;模型对照组幼鼠的惊厥持续时间为（104.40±14.37）s,治疗组幼鼠的惊厥持续时间为（45.40±7.10）s,治疗组的惊厥持续时间显著短于模型对照组（P＜0.01）;HE 染色显示热惊厥状态对模型对照组幼鼠海马神经元损伤明显,细胞肿胀、核皱缩,出现空泡现象,治疗组幼鼠海马神经元损伤减轻,细胞水肿及空泡等现象减轻;治疗组幼鼠中Gabbr1和Kcnt1的 mRNA表达水平高于模型对照组幼鼠（P＜0.05）;免疫组化检测幼鼠海马区中Gabbr1和Kcnt1的阳性细胞比例治疗组高于模型对照组（P＜0.01,P＜0.05）。结论：羚珠散具有显著防治幼鼠热惊厥发作的作用,其机制可能与显著提高Gabbr1与Kcnt1表达,增强GABA抑制神经兴奋作用,减轻海马区神经元损伤有关。     

不同类型附着基对刺参生长和氮磷收支的影响

采用陆基网围方法,研究了石头(ST)、瓦片(EA)、塑料管(PL)、空心砖(BR)和水泥管(CP)5种类型附着基对仿刺参(Apostichopus japonicus以下简称刺参)生长和系统N、P收支的影响。结果表明:1.收获时刺参产量ST和BR组显著高于其它组(P<0.05),PL、CP和EA组差异不显著(P>0.05);刺参的成活率ST组最高(P<0.05),EA组最低(P<0.05),PL、CP和BR组差异不显著(P>0.05),表明附着基类型对刺参的生长和成活有明显的影响。2.换水是刺参养殖池塘N、P收支的主要途径,附着基类型对N、P各项收支比例未产生明显影响(P>0.05)。3.收获时池塘底泥N、P积累量明显降低,但不同附着基类型间差异不明显(P>0.05),仅ST组N积累显著低于其他组(P<0.05)。4.塑料管附着基具有养殖成本低、收获和清池便利的优势,建议采用塑料管作为刺参池塘养殖的附着基。     

Sorption of lead onto two gram-negative marine bacteria in seawater

Laboratory adsorption experiments performed at environmentally significant lead (Pb) and cell concentrations indicate that the marine bacteria examined have significant binding capacities for Pb. However, the behavior governing Pb sorption onto gram-negative bacteria in seawater may be quite complex. The sorption kinetics appear to involve two distinct phases, i.e., a rapid removal of Pb from solution within the first few minutes, followed by a slow but nearly constant removal over many hours. Also, the average binding coefficient, calculated for Pb sorption onto bacteria and a measure of binding intensity, increases with decreasing sorption density (amounts of bacteria-associated Pb per unit bacterial surface) at low cell concentrations (10⁵ cells ml⁻¹), but decreases with decreasing sorption density at higher cell concentrations (10⁷ cells ml⁻¹). The latter effect is apparently due to the production of significant amounts of extra-cellular organics at high cell concentrations that compete directly with bacterial surfaces for available lead. Lead toxicity and active uptake by marine bacteria did not appear significant at the Pb concentrations used.     

清洁疏浚物对2种海洋微藻种间竞争的影响

采用实验生态学方法,研究了不同起始密度下悬浮清洁疏浚物对中肋骨条藻Skeletonema costatum和赤潮异弯藻Heterosigma akashiwo生长的影响。研究结果表明,在实验室单培养条件下,清洁疏浚物培养液中,起始密度不同,微藻生长状况明显不同。起始密度为0.2×10⁴ cells·mL^-1时,进入指数生长期和静止期的时间延长,但生长所达到的最大细胞密度较高;起始密度为0.8×10⁴ cells·mL^-1时,进入指数生长期和静止期的时间缩短,但生长所达到的种群最大密度较低。混合培养条件下,清洁疏浚物培养液中,起始密度不同,导致了微藻种间竞争关系的变化,中肋骨条藻与赤潮异弯藻具有明显的种间竞争,赤潮异弯藻是竞争的优胜者,中肋骨条藻受到赤潮异弯藻的抑制,整个实验周期内细胞密度下降都非常明显,细胞密度明显低于单培养时的细胞密度,而赤潮异弯藻细胞密度并没有明显的减少。在3种不同质量浓度组清洁疏浚物培养液中,质量浓度最高的1 000 mg·L^-1培养液中藻细胞密度显著低于100 和500 mg·L^-1质量浓度组,而100 mg·L^-1培养液中藻细胞密度最高,说明清洁疏浚物质量浓度越高,对海洋微藻抑制影响越为明显,质量浓度越低,影响越小。     

冠瘤海鞘同种群聚的化学诱因初步研究

研究了不同浓度的冠瘤海鞘成体被囊萃取物和幼体萃取物及不同的幼体密度对冠瘤海鞘(Styela canopus)幼体附着和变态的影响.结果表明,冠瘤海鞘被囊萃取物和幼体萃取物在5%和10%的浓度下均促进冠瘤海鞘幼体的附着和变态,但被囊萃取物的诱导效果明显强于幼体萃取物;幼体密度高于25只/cm3时,促进冠瘤海鞘幼体的附着和变态;幼体密度低于10只/cm3时,幼体的附着和变态不受密度影响.这些结果提示海区中冠瘤海鞘的同种群聚可能与其被囊或幼体内含诱导幼体附着和变态的化学因子有关.     

不同的营养盐浓度对三角褐指藻生长的影响

在温度为２２±１℃,盐度为２８的条件下,用不同的营养盐浓度对青岛海洋大学微藻种质库保存的三角褐指藻Ｐｈａｅｏｄａｃｔｙｌｕｍｔｒｉｃｏｒｎｕｔｕｍ进行培养。实验结果表明,相对生长率最高的最优水平组合为：硝酸钠浓度为１５０ｍｇ／Ｌ,磷酸二氢钠浓度为３５．２ｍｇ／Ｌ,硅酸钠浓度为２４０ｍｇ／Ｌ,微量元素浓度为ｆ／２培养基中微量元素浓度。经过７天的培养,细胞最高密度可达４９６８．７５ｃｅｌｌ／ｍＬ。     

On the advantage of sharing a holdfast: effects of density and occurrence of kin aggregation in the kelp Lessonia berteroana

We investigated the density‐dependent and genetic relatedness that regulate the occurrence of inter‐individual (genet) fusion forming plurigenotypic organisms in the brown alga Lessonia berteroana. Recruitment generally occurs at high densities in the inter‐tidal, allowing contact of neighbouring holdfasts as they grow and expand on the substrate. Algal density, by contrast, is regulated by the effects of herbivory and wave impact, which often lead to low holdfast density. Herein, we investigated whether the occurrence of plurigenotypic organisms and their genotypic composition (number of genotypes per plurigenotypic organism) are density dependent and affected by kin selection in the inter‐tidal kelp L. berteroana. Four microsatellite loci were used to analyse DNA from 260 samples obtained from shared and non‐shared holdfasts, at two sites with high and two site with low holdfast density. Analyses showed that fusions forming plurigenotypic organisms are extremely common. Interestingly, the frequency of fusions was higher in low‐density sites, in which 100% of the plants had at least two genotypes and the average was 3.5. In high‐density sites, 62% of plants were plurigenotypic, with an average of 2.8 genotypes per plant. Additionally, we found that genotypes that shared a holdfast had a significantly higher genetic relatedness than the average in the population, compatible with a kin structure. Density dependence and kin structure suggest that the occurrence of plurigenotypic organisms is linked to environmental quality, and that kin or multilevel selection may be favouring the fusion of genetically related genets.     

Expression of heat shock protein 70 in a permanent cell line (EPC) exposed to sediment extracts from the North Sea and the Baltic Sea

Heat shock proteins 70 (hsp70) are known to be induced by a great variety of chemical stressors. The effects of different environmental contaminants, which were identified in sediments of the North Sea and the Baltic Sea as main contaminants or as contaminants of special toxicological interest, on the extent of expression of hsp70 in a permanent cell line Epithelioma papulosum cyprini (EPC) were investigated. Concentrations of the single contaminants leading to elevated hsp70 levels were compared to the respective concentrations of these contaminants encountered in the sediment extracts thus allowing an assessment of their contribution to the toxicity of these sediments. Analysis of hsp70 was performed using western blotting techniques with subsequent comparative quantification by densitometry. Eleven of thirteen contaminants have shown a dose-dependent increase in hsp70 levels. An expression of hsp70 was observed in most cases between 0.1 and 10 microg/mL test solution. Some of the investigated stressors led to extremely high hsp70 contents even at low concentrations. However, in most cases contaminant concentrations in sediments were too low to cause an effect. For this reason, effects of the tested sediments are attributed to other contaminants or rather to mixtures of stressors.     

Antioxidant and pro-oxidant challenge of tannic acid in mussel hemocytes exposed to cadmium

The present study investigates the antioxidant and pro-oxidant behavior of tannic acid (TA) in hemocytes of mussel Mytilus galloprovincialis, in the presence or the absence of cadmium (Cd). TA at concentrations up to 20 μM, primarily found to be no toxic (in terms of cell viability, superoxide anions, nitric oxide and lipid peroxidation products currently estimated), significantly diminished the cytotoxic and oxidative effects induced by the metal (50 and/or 100 μM) in all cases. On the other hand, higher concentrations of TA (40 and 60 μM) were toxic, thus enhancing Cd-mediated cytotoxic and oxidative effects. The present study showed TA beneficiary properties in hemocytes of mussels, at least at low concentrations, while TA at concentrations higher than 20 μM could serve as an excellent oxidized substrate, thus enhancing toxic effects either alone or with the presence of micromolar concentrations of non transition metals, such as Cd.     

Distribution of cyanobacteria and other picophytoplankton in the western North Pacific Ocean,Summer 1989

Cell densities of phycoerythrin-fluorescing cyanobacteria and other chlorophyll-fluorescing picophytoplankton in the 0.2–2.0 µm size fraction were investigated, using an epifluorescence microscope, in the western North Pacific Ocean (36.5–44.0 °N, 155.0°E) in the summer of 1989. Cyanobacteria were most abundant in the surface of the subtropical water (36.5–38.0°N) and less in the northern sea area (39.5–44.0°N). The cell density of other picophytoplankton was, however, high in the northern part and low in the subtropical water. Results showed that algae other than cyanobacteria may significantly contribute to the picophytoplankton community under the low water temperature conditions of open waters. Chlorophylla concentration represented well the abundance of picophytoplankton other than cyanobacteria, but had no significant correlation with the cyanobacteria cell density. Chlorophylla-based data must be interpreted with caution, since the abundances of cyanobacteria were often considerably different even though the chlorophylla concentrations were the same level.     

2000年夏季南极普里兹湾及邻近海域浮游植物研究

报道了2000年夏季(1月)在62°~69°S,70°30'~75°30'E的南极普里兹湾邻近海域浮游植物种类组成、丰度分布、叶绿素a浓度及其环境调控.结果表明,调查海区共有浮游植物4门33属65种、变种和变型.浮游植物平均细胞丰度为32.67×103个/dm3.浮游植物细胞丰度和叶绿素a浓度高值均出现在水体较稳定的近岸海湾和冰间湖及毗邻陆架,在大陆坡和深海区它们的值明显减小.浮游植物细胞丰度和叶绿素a浓度在0~50m水层最大,100m以下水层随深度的增加而减小.浮游植物丰度呈昼夜变化,在13:00丰度最高.浮游植物优势种为短菱形藻(Nitzschiacurta),占总丰度的24.8%.浮游植物种类组成和细胞丰度与浮冰和水团稳定性有关.经回归分析,浮游植物细胞丰度与水温、溶解氧成显著正相关,与无机磷(PO_{43--P)和无机氮(NO3--N)成显著负相关.}     

Surfactant production by marine phytoplankton

Electrochemical methods based on adsorption of organic molecules at the mercury electrode-solution interface were used to investigate surfactant production by marine phytoplankton. Six species of marine phytoplankton, representing the classes of Bacillariophyceae, Haeptophyceae, Chlorophyceae and Cryptophyceae, were studied in batch cultures.Our experimental results showed that surfactants were produced in culture media by healthy exponential growing cells. The measured response was found to depend on the particular species and the age of the culture.Total surfactant content in culture media generally increased with cell density, while surfactants per cell showed an inverse relation to cell density. However, we found that in Cryptomonas culture medium, during the exponential growth, excretion of the insoluble surfactant material per cell was independent of cell concentration.In addition to culture experiments, surfactant activity at several northern Adriatic stations was measured during various stages of phytoplankton bloom. It was concluded that a significant part of surfactant activity in a seawater column is due to phytoplankton production.