20008/壳聚糖球形多孔微载体支持下培养的肝细胞功能及其代谢活性检测(想明年4月之前发，想申请加急)

摘要 背景：微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。 方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,并对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。 结果：两组培养的细胞数量均呈现前3 d增长,在第3天细胞数量达到最高值;而且实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05),实验组各样本之间差异无显著性意义(P > 0.05);倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。提示,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。 关键词：微载体;壳聚糖;人工肝脏;肝细胞培养;组织工程 doi:10.3969/j.issn.1673-8225.2011.12.018     

微囊化培养对肝细胞增殖生长和功能表达的影响

背景：前期研究证实,肝细胞在微囊化培养过程中结构形态发生变化,细胞的骨架会发生重排。 目的：在前期研究基础上,进一步考察微囊化培养体系对细胞生长和功能表达的影响。 方法：采用海藻酸钠-多聚赖氨酸-海藻酸钠微囊化人肝癌细胞系HepG2细胞,通过显微观察、苏木精-伊红染色、MTT实验、Realtime RT-PCR与Elisa实验,检测微囊内细胞的生长形态、细胞团的形态结构、细胞的活性、以及细胞的功能表达情况。 结果与结论：HepG2细胞在微囊内聚集成团,以三维方式生长,细胞间连接紧密,与传统的平面培养相比,微囊化细胞的生长速率减慢,但微囊化细胞的功能基因表达水平及白蛋白的分泌量增高,说明海藻酸钠-多聚赖氨酸-海藻酸钠微胶囊所提供的微环境有利于肝细胞类组织的体外构建。结果也提示海藻酸钠-多聚赖氨酸-海藻酸钠微胶囊能够实现肝细胞的体外类组织化培养,有望成为一种更具前景的三维培养模式,应用于癌症治疗、高通量药物筛选以及肝组织工程的研究。 关键词：微胶囊;三维培养;细胞聚集体;人肝癌细胞株;生物材料与药物控释 doi:10.3969/j.issn.1673-8225.2010.29.003     

微囊化成年兔许旺细胞的体外培养*

背景：研究发现将体外培养扩增的大量许旺细胞种植到神经导管上来引导周围神经再生效果明显,而目前备受关注的微囊化免疫隔离技术,在克服免疫排斥问题上具有明显的优势与实用性。 目的：微囊包裹兔许旺细胞并进行体外培养,观察许旺细胞形态及增殖变化。 设计、时间及地点：体外细胞水平观察实验,于2005-09/2006-05在南昌大学第二附属医院分子实验室完成。 材料：健康成年家兔由南昌大学医学院动物中心提供,微囊发生器由南昌大学医学院神经生物学研究室研制。 方法：使双侧坐骨神经发生Wallerian变性,经改良后反复差速贴附法进行培养,快速分离纯化许旺细胞。海藻酸钠-氯化钡一步成囊法包裹生长良好的第3代许旺细胞。 主要观察指标：倒置显微镜下观察培养过程中微囊化许旺细胞形态变化,MTT检测不同时期微囊化许旺细胞及游离许旺细胞的增殖改变。 结果：在培养过程中可见微囊及其囊内许旺细胞形态均保持完整,微囊未见破损。微囊化后的许旺细胞总体呈现悬浮生长的趋势。培养0,1,3,5,7 d时 MTT检测微囊许旺细胞组与游离许旺细胞组吸光度值差异有显著性意义(P < 0.05)。 结论：微囊包裹的许旺细胞体外可存活较长时间,微囊化许旺细胞相比游离许旺细胞增殖速度有所减慢。     

胶原支架复合兔骨髓间充质干细胞体外模拟的微重力培养

背景：骨髓间充质干细胞向软骨细胞诱导的方法包括体外高密度微团培养、体外单层细胞培养、体外三维支架环境诱导、与软骨细胞体外共培养诱导和基因转染诱导培养等。 目的：验证模拟微重力条件下诱导后的兔骨髓间充质干细胞在Ⅰ、Ⅱ型胶原支架上黏附、伸展和增殖情况。 方法：对兔骨髓间充质干细胞体外进行原代和传代培养，按加入诱导条件不同分为2组：实验组(转化生长因子β1+ 胰岛素样生长因子1诱导)组、空白对照组。3周后分别作MTT比色试验、糖胺聚糖检测和免疫组织化学染色。将诱导后的实验组细胞分别种植于Ⅰ、Ⅱ型胶原支架，分为4组培养：复合Ⅱ型胶原支架静置培养、复合Ⅱ型胶原支架模拟微重力培养组、复合Ⅰ型胶原支架静置培养、复合Ⅰ型胶原支架模拟微重力培养组，1周后作苏木精-伊红、甲苯胺蓝染色。 结果与结论：实验组MTT吸光度值和糖胺聚糖水平检测结果均大于空白对照组，且Ⅱ型胶原免疫组织化学检测阳性。苏木精-伊红、甲苯胺蓝染色显示，Ⅱ型胶原支架复合细胞的数量明显高于Ⅰ型胶原支架；模拟微重力培养条件下胶原支架复合细胞的数量明显高于静置培养组。结果说明微重力培养环境有利于高密度细胞的黏附、增殖，有利于细胞之间的信号传递，为维系细胞的生长和代谢提供适宜的微环境；作为诱导后骨髓间充质干细胞的支架材料Ⅱ型胶原支架优于Ⅰ型胶原。     

用新生大鼠肝细胞体外构建工程化肝组织*

目的: 在供体肝短缺的情况下，构建可植入的工程化肝组织具有重要的临床意义。实验尝试以新生大鼠肝细胞为种子细胞体外构建工程化肝组织，以期进一步的体内植入。 方法：实验于2007-04/08在解放军总医院普通外科研究所完成。①实验材料：SPF级、出生24 h以内的雄性SD新生大鼠。②实验过程：采用胰酶消化法获取新生大鼠肝细胞；以2×L-DMEM液（添加地塞米松10 μg/L、表皮生长因子20 μg/L、肝细胞生长因子40 μg/L及胰岛素0.04 U/mL）与液态鼠尾胶原等比例混合；再将肝细胞与胶原凝胶复合构建细胞/凝胶复合物，接种于培养板进行培养。③实验评估：培养后第1，3，5，7，9天，采用相差显微镜观察、四甲基偶氮唑盐比色法、苏木精-伊红染色和免疫组织化学染色分别对工程化组织的生长情况及组织形态特征进行观察，并对工程化组织的白蛋白合成功能及尿素的代谢水平进行评价。 结果：①肝细胞与胶原凝胶复合后，细胞均匀的分布在复合物中。在整个培养过程中，肝细胞保持着稳定的细胞形态。②四甲基偶氮唑盐检测显示肝细胞活性在生长初期平缓下降，直至第5天时仍然保持75%的细胞活性，之后快速下降。③体外培养5 d后，相差显微镜下观察显示肝细胞在胶原凝胶中呈三维立体生长，并保持肝细胞胞体圆形，核大而圆的特异形态；免疫组织化学证实这些肝细胞抗白蛋白抗体染色呈强阳性。④对培养上清中白蛋白和尿素的含量测定表明胶原凝胶中的肝细胞在培养初期保持着稳定的代谢合成功能。 结论：用新生大鼠肝细胞及胶原构建出一种有功能的工程化肝组织模型，这种模型可以应用于今后的工程化肝组织研究。     

牛磺酸对微囊化肝细胞生长的影响***☆

目的：微胶囊中存在一定程度的高渗透压胁迫，可造成细胞生长代谢减慢，为改善微囊化肝细胞的生长，尝试应用抗渗透压胁迫物质牛磺酸，观察其对微囊化肝细胞生长的影响。 方法: 实验于2006-07/08在中国科学院大连化学物理研究所生物医学材料工程实验室完成。将HepG2细胞悬液(非微囊化细胞)和微囊化HepG2分别接种于牛磺酸浓度为0，0.6，0.8和1.2 mmol/L的MEM培养液中，在37 ℃体积分数为0.05的CO2中培养。MTT法测定非微囊化和微囊化HepG2细胞的生长活性，相差显微镜观察囊内细胞生长状态。 结果：①非微囊化HepG2细胞：0.6，0.8和1.2 mmol/L的牛磺酸对其生长无影响，吸光度值比较差异无显著意义（P > 0.05）。②微囊化HepG2细胞：0.6，0.8 mmol/L牛磺酸对微囊化肝细胞的生长亦无影响（P > 0.05）；但1.2 mmol/L牛磺酸可以显著改善微囊化肝细胞的生长，其吸光度值高于0 mmol/L牛磺酸组（P < 0.05），并促进细胞的聚集。 结论：1.2 mmol/L牛磺酸可以改善微囊化肝细胞的生长并抵制微囊内的高渗透压胁迫对细胞的生长抑制作用。     

微球细胞支架在骨组织工程中的应用*★

摘要：骨组织工程的难点之一在于获得大量表型正确的细胞。作为骨组织工程的核心内容，细胞支架领域发展迅速。微球不仅被广泛用于组织工程种子细胞扩增，还可直接作为载体将细胞传递至骨缺损部位，并作为支架支持新生骨组织生长。目前微球支架材料包括人工合成或天然来源的聚合物和陶瓷两种，微球支架通过微囊包裹或支架贴附与细胞结合，直接植入或与可注射基质结合后注入骨缺损部位，实现骨组织的修复。微球细胞支架以其细胞扩增高效性及细胞传递微创性，在组织工程中显示了良好的应用前景和潜力。 关键词：微球；微囊；细胞支架；骨组织工程     

肝细胞微囊化技术理论进展与临床应用

摘要：肝细胞分离技术日趋成熟，肝细胞移植成为较热门的研究领域，但供体短缺和免疫排斥反应，制约了其广泛应用。肝细胞微囊化有助于为肝细胞移植的推广应用提供大量的具有高度活性和良好功能的肝细胞。文章就肝细胞微囊化技术及其应用进展进行综述，为肝细胞大规模、高活性体外培养及长期冻存提供了新的途径。 关键词：微囊；生物型人工肝；肝细胞移植；肝细胞；综述文献 doi:10.3969/j.issn.1673-8225.2010.03.041     

成骨生长肽缓释微球促成骨细胞的增殖

背景：采用生物可降解聚合物聚乳酸-羟基乙酸共聚物(Polylactide-co-glycolide，PLGA)为支架材料，包裹多肽、蛋白质药物制成缓释微球，使在体内达到缓释目的，是近10多年来各国学者大力研究的新领域。 目的：观察成骨生长肽（osteogenic growth peptide，OGP）缓释微球对成骨细胞的促分裂增殖作用。 设计、时间及地点：对比观察，于2006-06/11在西安交通大学生命科学院完成。 材料：新西兰兔，用于制备成骨细胞；PLGA由山东医疗器械研究所提供。 方法：以PLGA为载体材料，采用复乳法制备OGP-PLGA缓释微球，并对微球的形态学、粒径分布、载药量和包裹率及体外释药情况进行观察。实验分为2组，OGP组：培养液为含体积分数0.1小牛血清+含50 μg/L OGP的DMEM；OGP缓释微球组：培养液为含体积分数0.1小牛血清+含50 μg/L OGP-PLGA缓释微球的DMEM，分别测定成骨细胞的增殖数量。 主要观察指标：①微球的形态、粒径分布、载药量、包裹率及体外释药性。②OGP缓释微球对成骨细胞增殖的影响。 结果：①复乳法制备的OGP-PLGA缓释微球表面光滑圆整，球体均匀度好；微球粒径为(19.6±4.47) μm，载药量和包裹率分别为（83.9±4.21）%，（72.9±5.13）%；微球的体外释药过程较为稳定，56 d 释药率为85.43%。②培养1，2 d 时，OGP组的A值高于OGP缓释微球组，差异有显著性意义(P < 0.05)；培养3，4 d 时，OGP组和OGP缓释微球组间A值差异无显著性(P > 0.05)；培养6，8 d时，OGP缓释微球组的A值高于OGP组，差异有显著性意义 (P < 0.05)。 结论：采用复乳法制备OGP-PLGA缓释微球的工艺可行，微球中OGP的生物活性保存良好，能缓慢持续释放活性OGP，促进成骨细胞的体外分裂增殖。     

重建胶原纤维对细胞生长的影响*★

背景：胶原种类很多，而不同细胞只在特定类型的胶原环境中才表现出最佳的增殖和表型。 目的：观察Ⅰ型胶原和Ⅱ型胶原对小鼠皮肤成纤维细胞、人牙周膜成纤维细胞和兔软骨细胞生长的影响。 设计、时间及地点：对比观察，细胞学体外实验，于2007-09/2008-09在四川大学生物材料工程研究中心胶原研究室完成。 材料：自制达到中试水平的猪皮Ⅰ型胶原和猪软骨Ⅱ型胶原。 方法：分别将3种细胞在猪皮Ⅰ型胶原和猪软骨Ⅱ型胶原环境中进行体外培养，比较不同细胞在材料上的行为差异。 主要观察指标：傅里叶变换红外光谱、示差扫描热分析和凝胶动力学分析比较2种胶原的热变性温度及在结构和性质上的差异；MTT法观察细胞在2种胶原环境中的黏附、增殖情况。 结果：猪软骨Ⅱ型胶原和猪皮Ⅰ型胶原的热变性温度分别为105.8，87.5 ℃，2种胶原的官能团区相似。凝胶化曲线的对比中猪皮Ⅰ型胶原的成纤维过程较猪软骨Ⅱ型胶原迅速。MTT值显示细胞生长初期，小鼠皮肤成纤维细胞在软骨Ⅱ型胶原环境表现了较好的黏附性，但是更适于在猪皮Ⅰ型胶原上生长；人牙周膜成纤维细胞与小鼠皮肤成纤维细胞对胶原纤维的反应恰好相反。兔软骨细胞第2天在两种胶原上的黏附相似，第4天时在猪软骨Ⅱ型胶原环境中表现出较大的细胞生长密度。 结论：不同细胞对Ⅰ，Ⅱ型胶原纤维表现了不同的时间依赖性，但猪软骨Ⅱ型胶原环境更接近于软骨生长的真实环境，更有利于软骨细胞朝特定方向分化，使软骨细胞维持稳定的表型。     

Histoenzymatic profile of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord

We examined histoenzymatic characteristics of human muscle fibers grown in monolayer culture and innervated de novo in culture for 60-90 days by fetal rat spinal cord neurons. Serial cryostat cross sections were obtained using a freshly frozen sandwich of adult rat muscle and cultured human muscle. An advanced degree of morphologic and histoenzymatic maturation of cultured human muscle was reached after innervation. In contrast to aneurally cultured human muscle fibers, the innervated muscle fibers were smaller in diameter and had myonuclei preferentially located at the periphery of the fiber. The innervated fibers contained a well-developed intermyofibrillar network revealed by the NADH-TR and SDH reactions. Phosphorylase activity was strong to moderate in most muscle fibers. Although most of the innervated cultured muscle fibers were still not fully differentiated into two histochemical fiber types because they had strong ATPase activity after both alkaline and acid preincubation, a few of them had an ATPase profile similar to type 2 fibers in human adult muscle and had reciprocal staining with phosphorylase and NADH-TR reactions. This is the first evidence of differentiation into different histochemical fiber types of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord.     

Glial culture on artificial capillaries: electron microscopic comparisons of C6 rat glioma cells and rat astroglia

The functional association of astroglial footplates with blood vessels is important because astrocytes may provide a channel between the blood and neurons deeper in the brain parenchyma for the passage of ions and metabolites. This hypothesized function is very difficult to study in vivo or in monolayer cultures. We have produced a three-dimensional cell culture model of perivascular astroglia by means of an artificial capillary system. Conventional primary cultures of astroglia were first prepared from neonatal rat cerebral hemispheres in 75-cm2 tissue culture flasks. After 25 days, the cells were seeded in Amicon Vitafiber hollow fiber culture vessels. Direct seeding of brain cell suspensions was not successful. A culture unit consists of a bundle of hollow, semi-permeable polysulfone fibers encased in a plastic shell. The fibers were coated with fibronectin and bovine serum albumin, and astroglia were seeded on their outer surfaces. Warmed medium was pumped through the lumina of the fibers. After 13 days the cells were fixed with paraformaldehyde and examined. Scanning electron microscopy revealed the tubes to be uniformly covered with astroglia with short processes that contacted nearby cells. Transmission electron microscopy showed glial filaments and gap junctions. Astrocyte cultures were compared morphologically to C6 rat glioma cells in hollow fiber culture. The astrocytes formed a monolayer, whereas C6 cells formed a stratified culture. Furthermore, C6 cells did not form gap junctions. Astrocytes have been hypothesized to take up K+ discharged to the extracellular space by depolarizing neurons and move it to areas of low concentration, i.e., to act as a K+ spatial buffer. Our culture system should permit direct testing of this hypothesis.     

壳聚糖球形多孔微载体的血液相容性评价****☆

摘要 背景：文献报道的微载体大多为实心的、大孔的,虽然较二维微载体的比表面积有明显的增加,但距理想的三维微环境相差甚远。 目的：构建壳聚糖球形多孔微载体,通过溶血实验、凝血实验、血小板计数及聚集实验评价其血液相容性。 方法：利用液氮冷冻干燥技术成功构建浓度为1%,2%,3%的壳聚糖球形多孔微载体。选择健康成年新西兰兔为宿主,采用溶血实验、凝血实验、血小板计数及其聚集实验评价壳聚糖球形多孔微载体的血液相容性。 结果与结论：浓度为1%,2%,3%的壳聚糖球形多孔微载体的溶血率分别为1.56%,2.07%,2.31%,均小于5%,均无致溶血性;3种浓度壳聚糖球形多孔微载体样本材料对兔血时间无明显影响,三者与生理盐水阴性对照组间也无明显差别(P > 0.05);3种浓度壳聚糖球形多孔微载体样本材料对兔血小板计数无明显影响,注入浸提液前后比较和组间比较差异均无显著性意义(P > 0.05)。证实壳聚糖球形多孔微载体无致溶血性、无凝血性和无血小板聚集性,表明壳聚糖球形多孔微载体具有良好的血液相容性。 关键词：人工肝脏;壳聚糖;微载体;血液相容性;组织工程 doi:10.3969/j.issn.1673-8225.2011.03.006     

A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.

With our new "explant-reexplanting" technique, abundant growth of mature human muscle in long-term tissue culture was achieved,and with the "sandwich" technique several histochemical reactions were obtained on serial cross sections of the cultured fibers. An advanced degree of maturation but lack of differentiation into reciprocally staining fiber-types was demonstrated. For electron-microscopic and electronmicroscopic-histochemical study, a method was developed in which the embedded fibers of greatest potential interest were identified by light microscopy and punched out by our specially-designed hollow drill. This selection procedure is critically important when the goal is to study in cultured diseased human muscle: (1) successive stages of development and (2) certain structural changes that often occur only in some fibers and only in certain regions of those fibers. The electronmicroscopic-histochemical appearance of developing cultured muscle fibers correlated well with the fresh-frozen light microscopic histochemical cross-sections and longitudinal whole preparations of similar fibers.     

Use of a three-dimensional in vitro model of the rat blood-brain barrier to assay nucleoside efflux from brain

Extracellular adenosine is produced in brain during physiological and pathophysiological conditions. Once produced, this adenosine can undergo one or more of the following fates: it can interact with its receptors, it can be scavenged by astrocytes and/or neurons for ATP resynthesis, it can be transported across the blood–brain barrier and lost from the central nervous system, or it can be metabolized to inosine and hypoxanthine. The present study used a three-dimensional in vitro cell culture model of the rat blood–brain barrier, in which forebrain astrocytes and microvascular endothelial cells were cultured in cartridges containing multiple parallel polypropylene hollow fibers. Endothelial cells were cultured on the inner surfaces and astrocytes on the outer surfaces of these fibers. Growth medium was constantly perfused through the lumen of the fibers to mimic blood flow across endothelial cells in vivo. This co-culture system was used to examine the permeation of adenosine, and its metabolite inosine, from the astrocyte compartment to the endothelial cell compartment. Dipyridamole was added to the media perfusing the endothelial cell compartment to test whether it could decrease permeation of adenosine and inosine across the in vitro blood–brain barrier. Our results indicate that dipyridamole decreased permeation of total purines, especially inosine, across the barrier. Furthermore, permeation of fluorescein isothiocyanate-labeled albumin and radiolabeled sucrose, markers of the paracellular permeation pathway, were also decreased by dipyridamole. In conclusion, these data indicate that in addition to inhibiting nucleoside efflux across the barrier, dipyridamole can also improve blood–brain barrier function in this model.     

Effects of anticancer drugs on multicellular spheroid of 9L rat brain tumor

The effects of the anticancer drugs Nimustine (ACNU), Aclacinomycin A (ACR), Adriamycin (ADM), Bleomycin (BLM), Cisplatin (CDDP), and 5-Fluorouracil (5-FU) on the multicellular spheroid of a chemically-induced 9L rat glioma was studied. The multicellular spheroid in which cells grow in vitro as three-dimensional aggregates represents a biological model, which is intermediate between monolayer cells in vitro and solid tumors. Spheroids were initiated in bacteriological grade petri dishes seeded with 10(6) 9L rat glioma cells, cultured for four days and thereafter transferred and further developed in a spinner flask. Spheroids of 200-400 micron diameter were sorted and exposed for 24 hours to 5-FU and one hour for other drugs. After treatment both cytotoxic effect and growth delay were analyzed. Following disaggregation using collagenase, pronase and DNAase, cytotoxic effect on multicellular spheroids was measured by colony forming assay and were compared with those effects on 9L monolayer culture cells in the exponential growth. For growth delay assay, multicellular spheroids were individually transferred to 16 mm well containing 0.4 ml agarose base and 2 ml culture medium. Spheroid size was measured twice a week and growth curves were drawn. The growth delay was determined as the treated group vs. control differences in time required to a size four times that of the initial volume. For cells both in the monolayer culture and the multicellular spheroid, the dose response curve for ADM, BLM and 5-FU was "biphasic" and that for ACNU, ACR and CDDP "shoulder-threshold" type.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new model of the blood--brain barrier: co-culture of neuronal, endothelial and glial cells under dynamic conditions

Developing in vitro blood-brain barrier (BBB) models that closely mimic the natural state is important for theoretical and practical applications, including drug development. We previously developed an in vitro BBB model based on co-culturing endothelial cells with glia in the presence of flow on hollow fiber tube culture substrates. We now report that this dynamic in vitro BBB (DIV-BBB) can be successfully used to co-culture differentiated serotonergic neurons in the presence of a BBB. These neurons demonstrated fluoxetine-sensitive serotonin (5HT) uptake and depolarization-induced release of [3H]5HT. Our results demonstrate that the DIV-BBB is a suitable model for culturing of neurons in a quasi-physiological microenvironment and in the presence of a high-resistance, stereoselective BBB.     

Effects of RPE-cell factors secreted from permselective fibers on retinal cells in vitro.

This study was undertaken to determine if retinal pigment epithelial (RPE) cells encased in permselective hollow fibers survive in a tissue culture environment and secrete a diffusible trophic factor(s) that may affect retinal cell survival in vitro. In this study, RPE cells were isolated from 6- to 8-day-old Long-Evans rats, then loaded into hollow fibers. The RPE-cell fibers were then cultured for at least one week in serum-containing medium. These RPE-cell fibers were subsequently co-cultured with cells isolated from retinas of day 2 Long-Evans rats in a defined medium. For at least 6 days in culture, opsin-positive cells were observed on the surface of larger flat cells. Over 80% of the small, round cells immunostained for opsin. However, opsin-immunostained cells were seldom seen in cultures with control fibers, that lacked RPE cells. In addition, conditioned medium collected from either the RPE-cell fibers or cultured RPE cells affected survival of opsin-positive retinal cells in culture in a manner similar to that of the RPE-cell fibers. Furthermore, selected growth factors such as epidermal, nerve and fibroblast growth factors, were unable to sustain retinal cell survival and affect morphological development as seen in RPE-CM supplemented cultures. In vivo companion developmental studies demonstrated that few opsin-positive cell bodies were observed in retinas of day 2 Long-Evans rats, the age corresponding to the stage of retinal cell isolation. In retinas of day 5 Long-Evans rats, the age corresponding to the end point of the in vitro assay, a dramatic increase in the number of opsin-immunostained cell bodies was noted, which corresponds to the developmental sequence also seen in culture. Light and electron microscopic examination revealed that the RPE cells cultured in the hollow tubes maintained an RPE-like structure for several months, in that these cells contained melanosomes and extended microvilli from their apical border and formed junctional complexes with adjacent cells. Results of this study confirm our earlier findings that RPE cells secrete an apparent novel factor(s) that affects retinal cell survival in vitro and, most significantly, the described encapsulation/secretion mechanism may provide a convenient method to deliver such factors for further in vivo testing of this phenomenon.     

Endothelial glycocalyx as an important factor in composition of blood-brain barrier

The blood-brain barrier is a dynamic and complex neurovascular unit that protects neurons from somatic circulatory factors as well as regulates the internal environmental stability of the central nervous system. Endothelial glycocalyx is a critical component of an extended neurovascular unit that influences the structure of the blood-brain barrier and plays various physiological functions, including an important role in maintaining normal neuronal homeostasis. Specifically, glycocalyx acts in physical and charge barriers, mechanical transduction, regulation of vascular permeability, modulation of inflammatory response, and anticoagulation. Since intact glycocalyx is necessary to maintain the stability and integrity of the internal environment of the blood-brain barrier, damage to glycocalyx can lead to the dysfunction of the blood-brain barrier. This review discusses the role of glycocalyx in the context of the substantial literature regarding the blood-brain barrier research, in order to provide a theoretical basis for the diagnosis and treatment of neurological diseases as well as point to new breakthroughs and innovations in glycocalyx-dependent blood-brain barrier function.     

Barrier functions of the leptomeninges: a study of normal meninges and meningiomas in tissue culture

The anatomical arrangement of the pia mater suggests that it may act as a regulatory interface between cerebrospinal fluid and the surface of the brain and between arterioles within the brain and the surrounding neural tissue. However, the functional aspects of such a barrier are difficult to evaluate in vivo. In the present study, the enzymic content and endocytotic capacities of normal leptomeningeal cells in situ and meningioma cells in confluent tissue culture are examined in relation to barrier functions of meningeal cells. Growth of cells in culture was obtained from human fetal and newborn rat leptomeninges and from 9/13 meningiomas. But, in only two meningiomas were the cultured cells characterized as meningeal in origin by using the strict criteria of desmosomes identified by immunocytochemistry or by electron microscopy. These two tumours had high (8-8.7%) Ki-67 labelling indices. Glutamine synthetase activity is present in normal meninges and in meningioma cells in culture; this enzyme together with catechol-O-methyltransferase could play a role in limiting the diffusion of neurotransmitters into brain tissue. A steady rate of endocytosis of carbon particles and fluorescent latex beads, 0.2-1 microns in diameter, was observed in cultured meningioma cells. Such endocytosis was inhibited by cytochalasin B indicating the active participation of intracellular microfilaments. Similar endocytosis has been observed in normal leptomeninges in vivo. The results of this study suggest that meningioma cells in culture reflect the barrier functions of the pia mater and may be used as a model to further investigate the functions of the pia mater.