三种羧甲基壳聚糖的初级生物学评价

Primary Biological Evaluation on Three Types of Carboxymethyl Substituted Chitosan

本文旨在对三种不同取代位置的羧甲基壳聚糖(N-羧甲基壳聚糖(CMC),N,O-羧甲基壳聚糖,O-羧甲基壳聚糖)进行初级生物学评价。本文利用化学改性的方法制备的三种羧甲基壳聚糖,其生物学评价试验包括细胞毒性试验和溶血试验。细胞毒性试验:采用浸提液试验中的细胞增殖度法提取材料浸提液培养L929细胞,观察其对细胞生长、增殖的影响,测定加样后2、4、7d的OD值,计算RGR,评定细胞毒性等级;溶血试验:采用溶血率法评价材料的溶血性能,取新鲜兔血与材料直接接触,测定溶血率。毒性试验结果发现:经纯化的壳聚糖和三种CMC的细胞毒性均为0级或1级,符合医疗用途的技术要求。材料浸提液浓度越大,细胞毒性越大,壳聚糖的三种羧甲基化产物中,N-CMC和N,O-CMC的细胞毒性比壳聚糖小,O-CMC的细胞毒性最大。溶血试验结果发现:壳聚糖和三种CMC的溶血率均小于5%,符合医疗用途的技术要求。N-CMC的溶血率最小,O-CMC的最大,这结果与细胞毒性试验一致。经体外细胞毒性试验和溶血试验可知:经初步检验,可认为壳聚糖的羧甲基化衍生物具有良好的生物相容性,为以后更深一步的研究提供了科研基础和实验依据。

The purposes of this article are to make primary biological evaluation on one of chitosan derivatives, soluble carboxymethyl chitosan （ CMC ） , including N-carboxymethyl chitosan, N, O-carboxymethyl chitosan and O-carboxymeth＇yl chitosan. To prepare three types of CMC by chemical modification in this article and their biological evaluation includes cytotoxicity test and hemolysis test. Cytotoxicity test： L-929 fibroblasts were cultured in the medium of extraction of materials. Cell viability and proliferation were tested by cell proliferation assay through values of OD at 2, 4 and 7 days respectively after inoculation. The cell relative growth rate （RGR） was calculated and the cytotoxicity was graded by generally accepted standard. Hemolysis test： hemolysis percentage measurement was used to assay the hemolytic properties of four types of materials through direct contact with rabbit blood. Results of the cytotoxicity showed that purified chitosan and three types of CMC was grade 0 or 1, in accordance with medical standard. We found that the materials represented the more cytotoxicity with the more concentration of extraction of materials and the cytotoxicity of N-CMC was the least. The hemolysis rates of the materials were less than 5% , up to standard purpose. Hemolysis rates of N-CMC was the least and O-CMC the most. The result was in accord with cytotoxicity test. In vitro cytotoxicity test and hemolysis test showed that chitosan and its soluble derivatives had good biocompatibility and the further steps could be taken to study deeply and comprehensively for the medical application.