磺化壳聚糖的制备及其对生长因子活性的保护作用

以壳聚糖为基材,分别以氯磺酸、三甲胺.三氧化硫和丙磺酸内酯为磺化试剂,制备了3,6-O-磺化壳聚糖(OCS)、2-N-磺化壳聚糖(NCS)和2-N-磺丙基壳聚糖(PCS)3种磺化壳聚糖.采用红外光谱、核磁共振谱证明了磺化壳聚糖的结构,元素分析测定了磺化壳聚糖的磺化率.以组织修复过程中一种重要的活性因子——碱性成纤维细胞生长因子(bFGF)为目标因子,通过体外成纤维细胞培养实验,评价了不同磺化壳聚糖对bFGF活性的保护能力.结果显示,相对于未改性壳聚糖(CS),磺化壳聚糖对成纤维细胞的形态无显著影响.细胞活性检测结果显示,OCS和PCS对bFGF活性无明显的保护能力;NCS能有效提高bFGF促进成纤维细胞活性的能力,其对bFGF活性的保护能力可达肝素70%左右.磺化壳聚糖的bFGF活性保护能力不随磺化率的改变而变化.

REPARATION OF SULFONATED CHITOSAN AND ITS ABILITY TO PROTECT bFGF ACTIVITES

Three kinds of sulfonated chitosans,i.e.3,6-O-sulfonated chitosan(OCS),2-N-sulfonated chitosan(NCS) and 2-N-sulfopropyl chitosan(PCS) were synthesized.Their chemical structures were verified by FTIR and 1H-NMR.The sulfonation degree of the sulfonated chitosan,which was determined by elemental analysis,could be varied by changing the amount of sulfonation reagent or the reaction time.To evaluate the growth factor-protective ability of sulfonated chitosans,basic fibroblast growth factor(bFGF),which is one of the most important growth factors in tissue repair,was used as the target protein.By in vitro cell culture,the morphology of fibroblasts cultured in different conditions was observed by the costaining of cytoskeleton and cell nuclei,according to which the cell shape indexes such as perimeter,area and aspect ratio were analyzed.The results showed that bFGF/Heparin has the highest values in all cell shape indexes.However,compared to the pristine chitosan(CS),the combination of bFGF with all sulfonated chitosan did not show any effect on the fibroblasts morphology.By cell viability test,the influence of sulfonated chitosan on protecting bFGF activity was studied.Similar to the results of cell morphology,heparin also showed the strongest ability to promote bFGF activity.However,the sulfonated chitosans showed different protecting ability.For OCS and PCS,no significantly effect on bFGF activity was observed.However,NCS could promote the bFGF activity effectively,since the cell viability was significantly higher than that of bFGF/CS,and reached about 70% of bFGF/Hep.Meanwhile,the effect of sulfonation degree was also studied,showing that the cell viability was not affected by the sulfonation degree of chitosan.All these results indicate that the sulfonation position is the key point of the growth factor-protective ability of sulfonated chitosans.