细胞相容性物质的生理功能及其作用机制

以生物界广泛存在的细胞相容性物质甜菜碱为代表,介绍了其生理功能的研究进展,同时对植物中其它细胞相容性物质的一些生理功能,以及这些相容性物质在保护生物大分子结构与功能中的可能作用机制也作了评述。     

壳聚糖基复合材料类新型生物医用材料

壳聚糖是由N-乙酰基-D-葡萄糖胺和D-葡萄糖胺单元通过β-D(1→4)连接组成的碱性线性多糖。壳聚糖由于具有良好的生物相容性、生物可降解性和生物活性,已广泛应用于生物医学领域。壳聚糖由于具有许多的活性基团,能进行各种衍生化反应以及与其他材料进行复合制备各类复合材料。本文主要介绍壳聚糖与各类材料如蛋白质、糖胺聚糖、DNA、可降解的聚酯、无机物、合成(离子型和非离子型)的聚合物、或脂质的复合物及其在生物医学上的应用。     

壳聚糖基温敏水凝胶的研究进展

壳聚糖是一种由甲壳素脱乙酰化得到的氨基多糖,具有生物相容性、低细胞毒性和可生物降解性等特点。壳聚糖/β-甘油磷酸钠溶液温敏水凝胶在组织工程、药物缓释等领域多有报道,其成胶性能取决于凝胶的组分和浓度。针对单纯壳聚糖水凝胶强度较低、降解较快、药物突释等缺陷,通常对壳聚糖进行改性或引入新材料共混,获得更符合实际需要的壳聚糖基温敏水凝胶。对近年来壳聚糖基水凝胶的研究进展进行综述,包括改性壳聚糖、共混体系等,概述了其在组织工程(软骨、血管、神经修复)、药物缓释(癌症药物缓释、糖尿病治疗)领域中研究和应用的新进展,以期为后续温敏水凝胶的进一步研究提供参考。     

浅析壳聚糖基智能水凝胶

煤、石油、天然气等不可再生资源的过度开采和超支使用,给我们带来便利的同时,也给环境和未来资源的利用带来严重的压力,开发一种可再生,可降解高分子材料的意义也愈发重大。壳聚糖来源广泛,具有生物相容性和生物降解性,成为天然高分子材料中极具使用价值和研究价值的材料。本文介绍了壳聚糖基水凝胶对PH、温度、磁场和电场的智能响应,并对未来壳聚糖基智能水凝胶的研究重点进行了展望。     

骨髓基质细胞与交联明胶-聚羟基丁酸酯膜的生物相容性研究

目的研究生物材料交联明胶-聚羟基丁酸酯膜与骨髓基质细胞的生物相容性,探讨新型材料在骨组织工程中的应用前景。方法体外培养兔骨髓基质细胞,分别接种于G-PHB(交联明胶-聚羟基丁酸酯)、PHB(聚羟基丁酸酯)和G(交联明胶)材料膜片。采用MTT法检测细胞增殖活性,体视学方法检测细胞粘附能力,荧光双染法检测细胞完整性,扫描电镜观察细胞-材料界面。结果MTT检测发现G-PHB组增殖活性最强,而且表现为最佳的细胞粘附特性,与对照组比较差异有显著性意义。各组细胞完整性分析没有发现显著性差异。扫描电镜观察显示,G-PHB组细胞粘附及铺展良好,优于其他各组。结论交联后的生物降解膜材料G-PHB与BMSCs细胞的体外相容性明显优于单纯膜材料PHB和明胶,在骨组织工程学领域具有良好的研究价值和应用潜力。     

通过研究改性壳聚糖与细胞的相互作用评价其生物相容性

利用细胞生物学的方法, 研究了四种不同的细胞在经过改性的壳聚糖（ＣＨＩＴＯＳＡＮ） 膜上的生长,测定了细胞相对黏附力、细胞初始黏附率, 并利用ＦＤＡ 实验测定了细胞活力,从而从多个方面评价了这几种不同材料的生物相容性。实验结果表明,与明胶交联的壳聚糖膜明显比其它两种膜有利于细胞的黏附和生长,为进一步对材料进行筛选奠定了基础。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

羧甲基壳聚糖膜对皮肤成纤维细胞相容性研究

通过玻璃流延法制备不同分子质量的壳聚糖及羧甲基壳聚糖膜,以体外培养的人皮肤成纤维细胞作为对象,利用膜的浸渍液培养及膜表面直接培养法研究比较了两种多糖膜的细胞相容性.实验发现两种多糖膜的浸渍液对细胞均无毒性效应,生物安全性是小分子质量膜好于大分子质量膜,羧甲基壳聚糖膜好于壳聚糖膜.皮肤成纤维细胞在壳聚糖膜上的生长受到抑制,生长一段时间后细胞有聚集和脱落现象,而羧甲基壳聚糖膜上细胞能很好地贴附、生长,没有聚集和脱落现象.结果表明羧甲基壳聚糖膜具有比壳聚糖膜更优越的细胞相容性.     

角膜上皮细胞代谢的研究进展

角膜上皮层位于角膜表面,外邻泪膜,内与角膜前弹力层相连。角膜上皮细胞代谢所需营养及氧分主要通过泪膜、房水和角膜缘毛细血管运送。正常的角膜上皮细胞代谢是维持角膜上皮细胞正常增殖与分化状态的关键。角膜上皮细胞代谢异常可导致上皮损伤或变性,是多种角膜疾病的病理基础。本文就近年来关于角膜上皮细胞代谢相关的组织结构、营养来源、细胞增殖分化以及相关疾病的研究进展进行综述。     

壳聚糖载体的制备及脲酶的固定化研究

以甲壳素为原料,制备出壳聚糖载体,并对脲酶进行固定化。通过测量悬挂醛基探讨了交联条件对载体性能的影响,优化了脲酶的固定化条件,研究了固定化酶的酶学性质,并与游离酶进行了比较。结果表明。制备载体的最优条件是将微球用6％的戊二醛活化2h,最佳联酶条件是载体与脲酶共反应1h。该固定化酶的最适温度为65℃,最适pH值为6．6,米氏常数为0．009mol／L,较游离酶均有较大改善。热稳定性较游离酶有很大的提高,且具有良好的操作稳定性。     

人胚胎角膜上皮细胞系的建立和生物学特性

为构建角膜基础研究的稳定载体和操作平台,通过合法渠道获得人胚胎,并对角膜上皮细胞进行了分离培养。经反复传代,初步建立了人胚胎角膜上皮细胞系,并对其生物学特性进行了研究。结果显示,培养细胞具有较典型的上皮细胞特征。细胞生长较快,最快时两天可传代一次。K19和PCNA在各代胚胎角膜细胞均有表达,K19的表达部位位于胞浆,而PCNA的表达部位位于胞核。处于细胞周期中S期的细胞比例大约为11%￣23%。染色体核型分析表明各代细胞染色体的数目和形态相似。因此,人胚胎角膜上皮细胞适合于建立相对稳定的细胞系。培养细胞具有分化上的幼稚性和较强且稳定的增殖能力,细胞生长状态良好,而且该细胞系的遗传特征较为稳定。     

兔角膜缘干细胞的研究进展

目前,角膜移植是临床上治疗角膜疾患的最有效途径,但供体角膜非常有限。新近兴起的干细胞技术,为组织工程角膜的研制和应用提供了契机。对于以角膜缘干细胞缺乏或功能障碍为特征的疾病也有治疗效果。采取角膜缘干细胞移植应是一种合理有效的治疗手段。本文主要介绍了兔角膜缘干细胞的体外分离、培养、鉴定及一些生长因子对其增殖的影响。     

兔机械性角膜上皮损伤对结膜杯状细胞及结膜上皮细胞的作用研究

目的探讨机械性角膜上皮损伤对结膜杯状细胞及结膜上皮细胞的作用。方法选取雄性新西兰大白兔12只,建立机械性角膜上皮损伤模型（角膜中央直径8 mm上皮刮除）,建模后使用盐酸林可霉素滴眼,用法为3次/日,1滴/次,观察时间为7 d。在模型建立后第1、4、7天共3个时间点进行结膜印迹细胞学检查、结膜组织透射电镜检查,对结膜上皮细胞及杯状细胞数量及形态进行分析。结果成功建立机械性角膜上皮损伤模型。结膜印迹细胞学检查显示,造模前结膜杯状细胞数量平均值为66.367±2.466（个/每200μm×150μm面积）,Nelson 0级;造模后第1天,结膜杯状细胞数量明显下降,平均值为2.933±0.242（个/每200μm×150μm面积）,Nelson 3级;造模后第4天,结膜杯状细胞数量开始恢复,平均值为17.350±0.991（个/每200μm×150μm面积）,Nelson 2级;造模后第7天,结膜杯状细胞数量已明显恢复,平均值为32.467±2.244（个/每200μm×150μm面积）,Nelson 1级。结膜组织透射电镜检查可见到造模后结膜杯状细胞大量减少,分泌颗粒排空,细胞凋亡,结膜上皮细胞脱落坏死,胞核固缩,胞质中可见溶酶体,上皮下及上皮细胞间炎症细胞浸润;随时间推移,结膜杯状细胞数量及形态逐渐恢复,初期细胞形态欠规则,结膜上皮细胞胞间隙大,连接松散;后期杯状细胞数量明显恢复,形态饱满,分泌功能开始恢复。结膜上皮细胞分化好,细胞连接较为紧密。结论机械性角膜上皮损伤可造成结膜杯状细胞的数量下降及分泌增加,同时可造成结膜上皮细胞凋亡增加,炎症细胞浸润。结膜杯状细胞的数量、功能以及结膜上皮细胞正常结构可在一定时间内自行修复。     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

尺寸均一的壳聚糖微球的制备及其作为胰岛素控释载体的研究

采用新型微孔膜乳化技术制备了载胰岛素的壳聚糖微球。研究表明,要制备粒径均一的壳聚糖微球,必须将亲水性膜修饰成疏水性;制得的微球粒径和所采用的膜孔径之间存在很好的线性关系,使得微球粒径可控;以胰岛素为模型药物,主要考察了交联剂用量和交联时间对微球表面形态、药物包埋率和微球体外释药特性的影响。结果表明当氨基与醛基的摩尔比为1∶0.7、交联时间为1h时,所得载药微球的包埋率最高,随着戊二醛用量的增加和交联时间的延长,药物体外释放速率减慢。     

Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.     

Application of Preserved Human Amniotic Membrane for Corneal Surface Reconstruction

Objective: To evaluate the efficacy of preserved human amniotic membrane transplantation for reconstruction of the corneal surface diseases. Methods: Preserved human amniotic membrane transplantations were performed in 84 eyes of 78 patients for corneal surface reconstruction. The indications were limbal stem cell deficiency from Steven–Johnson syndrome, chemical burn and herpes keratitis (27 eyes), bullous keratopathy (26 eyes), persistent epithelial defect and dellen (17 eyes), band keratopathy (11 eyes), preparing for prosthesis (1 eye), corneal ulcer (1 eye) and acute chemical burn (1 eye). Results: Success was noted in 83.3% (70/84) eyes, partial success in 13.1% (11/84) eyes, and failure in 3.6% (3/84) eyes for an average follow-up of 10.5 months (3 – 29 months). No patient developed major immediate post-operative complications. Conclusion: Amniotic membrane transplantation can reduce inflammation, promote corneal epithelial healing, and decrease irritation in corneal surface problems.     

白内障超声乳化术对糖尿病患者角膜内皮细胞和中央角膜厚度影响

目的:评估糖尿病患者和非糖尿病患者在行白内障超声乳化手术后,角膜内皮细胞和中央角膜厚度变化。方法:观察于我院行白内障超声乳化吸出联合人工晶状体植入术的年龄相关性白内障患者各100例(100眼),于术前1天、术后1周、1个月、3个月、6个月和1年随访记录其角膜内皮细胞密度(endothelial cell density, ECD)、六角形细胞百分比(percentage of hexagonal cells,PHC)、变异系数(coefficient of variation, CV)和中央角膜厚度(central corneal thickness, CCT)等指标,并对结果进行统计学分析。结果:非糖尿病组在术前各指标无明显差异(P0.05),术后1年ECD, PHC在两组均下降,CV升高(P0.05),CCT出现明显波动,糖尿病组在术后一周达到峰值。ECD,CV和PHC在术后各时间点出现明显的组间差异(P0.05)。结论:白内障超声乳化术后远期内,糖尿病组的角膜内细胞和中央角膜厚度较非糖尿病组发生显著变化。