N-己烷壳聚糖的制备、细胞毒性与凝血性能  

Preparation of N-hexane chitosan and its cytotoxicity and coagulation property

作　　者：刘梦媛 荆妙蕾[1] 关静[2] 黄姝杰[2] 杨健[2] 李志宏[2] Liu Meng-yuan;Jing Miao-lei;Guan Jing;Huang Shu-jie;Yang Jian;Li Zhi-hong(Department of Textile,Tianjin University of Technology,Tianjin 300387,China;Institute of Medical Equipment,Academy of Military Medical Sciences,Tianjin 300161,China)

机构地区：[1]天津工业大学纺织学院,天津市300387 [2]军事医学科学院卫生装备研究所,天津市300161

出　　处：《中国组织工程研究》2018年第22期3520-3526,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(31570956)~~

摘　　要：背景:尽管壳聚糖是一种天然止血材料,但其对严重大出血创面的止血效果尚不明显,需要进一步的改性研究。且在壳聚糖中引入较短碳链的烷基(己烷)对凝血效果的影响也不明确。目的:合成N-己烷壳聚糖,研究其细胞毒性与凝血效果。方法:还原氨化法制备N-己烷壳聚糖,傅氏转换红外线光谱分析仪、元素分析法完成结构表征;MTT比色法和荧光标记法测试其细胞毒性;全血凝固时间、流变性能和血浆凝固实验评价其凝血性能。结果与结论:(1)傅氏转换红外线光谱分析仪、元素分析法均证实合成的产物是N-烷基化壳聚糖,取代度分别为8.67%,18.06%,32.88%;(2)MTT结果显示N-己烷壳聚糖的毒性分级是0级和1级。荧光染色表明与各组材料作用后,L929细胞生长旺盛,几乎无死细胞,提示明材料毒性作用小;(3)与壳聚糖相比,N-己烷壳聚糖具有明显促凝作用,且取代度越大其促凝作用越强。血浆凝固时间结果发现各组间活化部分凝血活酶时间、凝血酶原时间、凝血酶时间相比均无显著性差异(P>0.05),提示N-己烷壳聚糖不能促进凝血因子活化,其促凝机制有待于深入研究;(4)结果表明,通过还原胺化法合成了毒性较低、取代度介于8.67%-32.88%的N-己烷壳聚糖。与纯壳聚糖相比,N-己烷壳聚糖具有明显的促凝作用,且取代度越大,其促凝效果越好。BACKGROUND： Chitosan is a natural hemostatic material, but its hemostatic effect is limited and needs to be improved. Introducing alkyl groups into chitosan can modify its coagulation property. However, the coagulation mechanism of short alkyl groups that are grafted on chitosan powder is unclear until now. OBJECTIVE： To study the cytotoxicity and coagulation property of the prepared N-hexane chitosan.METHODS： N-hexane chitosan was prepared by reductive reaction and characterized by Fourier transform infrared spectroscopy and elemental analysis. Cytotoxicity of N-hexane chitosan was tested by MTT and fluorescence microscopy. Whole blood coagulation time, rheological properties and blood plasma coagulation time were evaluated for its coagulation effects. RESULTS AND CONCLUSION：（1） A series of N-hexane chitosans with substitution degree of 8.67%, 18.06%, and 32.88% were synthesized successfully.（2） MTT results showed that the toxicity of N-hexane chitosan was graded 0 and 1. Fluorescence staining results showed that L929 cells grew well on the materials and dead cells were scarcely visible, indicating the low toxicity of the materials.（3） N-hexane chitosan could promote blood clotting, and its blood coagulation efficiency was increased with its substitution degree. No significant difference was found in activated partial thromboplastin time, prothrombin time and thrombin time of N-hexane chitosans with different substitution degrees. N-hexane chitosan could not promote activation of clotting factors. Its mechanism for accelerating coagulation needs in-depth investigations. N-hexane chitosan, compared with pure chitosan, shows better clotting effect, and this effect is enhanced with the increasing of the substitution degree.

关 键 词：壳聚糖 细胞毒性试验 免疫 血液流变学 止血 烷基化 FTIR测试 MTT 荧光染色 全血凝固 静态流变 动态流变 生物材料 血浆凝固 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]