壳聚糖微胶囊包埋肝细胞生理功能的维护

背景: 乙酰化率15.3%的壳聚糖在细胞生理条件下的溶解性差,限制了细胞的微囊化过程.目的: 用乙酰化率15.3%的壳聚糖制备乙酰化率50%的壳聚糖,探讨其在细胞生理条件下与异丁烯酸-羟乙基异丁烯-甲基丙烯酸甲酯三元共聚物制备微胶囊包埋肝细胞的可行性.设计、时间及地点: 对照观察实验,于2006-01/10在暨南大学附属第一医院中心实验室完成.材料: 乙酰化率15.3%的壳聚糖由Sigma-Aldrich公司提供,肝细胞采用改良的两步原位胶原蛋白酶灌注法从体质量250～300g的雄性Wistar鼠中分离获取.方法: 微囊化肝细胞利用乙酰化率50%的壳聚糖和三元共聚物在25℃、无菌条件下凝聚制备.主要观察指标: 微胶囊的渗透性以空微胶囊中荧光葡聚糖(FITC-葡聚糖)的渗透率表示.微囊化肝细胞的稳定性通过机械剪切破碎实验测定.微囊化肝细胞白蛋白的合成能力采用ELISA测定.尿素合成能力采用检测试剂盒(Sigma Diagnostic)在540nm波长下比色测定.细胞色素P450活性使用共聚焦激光显微镜测定7-乙氧基试卤灵(细胞色素P450IA1的底物)O位脱烷基化产物的荧光强度来实现定量.测定均以肝细胞平面培养为对照.结果: ①随着乙酰化率50%的壳聚糖质量浓度的增加,Mr20000和40000荧光葡聚糖的渗透率呈下降趋势.Mr70000荧光葡聚糖渗透率很低,随乙酰化率50%的壳聚糖质量浓度变化不明显.②包埋肝细胞密度为2×109>L-1时,震荡24h的破碎率仅为6%左右,未包埋肝细胞微胶囊在相同震荡条件下,4h全部破碎.③培养第1天1×106个细胞中尿素合成达到30μmol/d,明显高于15μmol/d的平面培养实验结果.培养7d后,微囊化细胞和平面培养细胞尿素的合成能力比较接近.④培养前3d 1×106个细胞中白蛋白达到10μg/a左右,培养7d后为5μg/d左右,整个培养过程中均高于平面培养的结果.⑤培养第1天微囊化肝细胞的细胞色素P450活性比平面培养高出近8倍,且在培养1周后细胞色素P450活性仍可以保持第1天培养的50%以上.结论: 生理条件下基于乙酰化率50%的壳聚糖构建的微胶囊在培养过程中有较好的渗透性及结构稳定性.与体外表面培养实验相比,改性后的壳聚糖微胶囊有利于支持肝细胞的体外功能.

Maintenance of physiological function of chitosan microencapsulated hepatocytes

BACKGROUND: Under the cellular physiological condition, 15.3% N-acetylated chitosan has poor solubility which limits cellular microencapsulation process.OBJECTIVE: This study was designed to prepare 50% N-acetylated chitosan with 15.3% N-acetylated chitosan and investigate its feasibility for preparing chitosan microcapsule used for hepatocyte embedding after in conjunction with methacrylic acid-hydroxyethyl methacrylate-methyl methacrylate (MAA-HEMA-MMA) copolymer under the cellular physiological condition.DESIGN, TIME AND SETTING: This study, a control observation experiment, was performed at the Central Laboratory, First Hospital Affiliated to Jinan University, Guagnzhou, Guangdong Province, China between January and October 2006.MATERIALS: 15.3% N-acetylated chitosan was provided by Sigma-Aldrich Company, Singapore. Hepatocytes were acquired from male Wistar rats, weighing 250-300g, by two-step collagenase digestion method.METHODS: Hepatocytes were microencapsulated using 50% N-acetylated chitosan and MAA-HEMA-MMA copolymer at 25℃ under aseptic condition.MAIN OUTCOME MEASURES: Microcapsule permeability was expressed with the diffusion degree of fluorescein isothiocyanate (FITC)-dextran in the empty microcapsule. The homeostasis of microencapsulated hepatocytes was measured by mechanical shearing-crush tests. The albumin-synthesizing capability of microencapsulated hepatocytes was determined by enzyme-labeled immunosorbent assay (ELISA). Urea-synthesizing capability was tested using kits (Sigma Diagnostics, USA) at the wavelength of 540 nm by colorimetric assay. Cytochrome P450 activity was determined using confocal laser microscopes and quantitated by the fluorescence intensity of products of 0-dealkylated 7-ethoxy resorufin, a substrate of cytochrome P450IA1. The plate-cultured hepatocytes were taken as controls.RESULTS: With increasing mass concentration of 50% N-acetylated chitosan, the diffusion degree of Mr 20000 and Mr 40000 FITC-dextran presented with a decreasing tendency, while the diffusion degree of Mr 70000 FITC-dextran was low and did not alter markedly. When the microencapsulated hepatocyte density was 2×109 L-1, the broken rate of microcapsule was only about 6% after 24 hours of shaking. Under the same condition of shaking, empty microcapsules were all broken after 4 hours of shaking. After 1 day of culture, approximately 30μmol/d urea could be synthesized among 1×106 bepatocytes that was markedly higher than the plate culture experimental result, 15μmol/d. After 7 days of culture, urea-synthesizing capability was close between in the microencapsulated hepatocytes and in the plate cultured hepatocytes. In the first 3 days of culture, 10μg/d albumin was acquired from 1×106 hepatocytes, and after 7 days of culture, only about 5μg/d albumin was obtained. The albumin level in the whole culture process was higher than plate culture results. After 1 day of culture, cytochrome P450 activity in the microencapsulated hepatocytes was higher approximately 8 times compared to plate culture results. After 1 week of culture, cytochrome P450 activity still retained at 50% of 1-day culture level.CONCLUSION: Microcapsule prepared by 50% N-acetylated chitosan under the physiological condition has good permeability and structural stability during the process of culture. Compared with in vivo surface culture test, degenerated chitosan microcapsule is better favorable to in vitro function of hepatocytes.