壳寡糖对体外培养软骨细胞增殖的影响及对IL-1β诱导凋亡的保护作用

Protective effects of Chitosan oligosaccharides on the proliferation and apoptosis induced by IL-1β of the cartilage cells in vitro

目的体外实验研究不同浓度壳寡糖对软骨细胞增殖及IL-1β诱导细胞凋亡的保护作用。方法从8周龄SD大鼠乳鼠膝关节分离、培养原代软骨细胞,鉴定后选用第2代软骨细胞进行实验。分别加入不同浓度壳寡糖培养48h,通过CCK-8细胞计数法检测软骨细胞的增殖情况;采用IL-1β诱导软骨细胞凋亡,同时加入不同浓度的壳寡糖处理,倒置相差显微镜下观察软骨细胞形态及核分裂象的变化,通过流式细胞仪检测细胞早期凋亡比例,并通过Hoechst33258荧光染色检测软骨细胞凋亡细胞核的形态学变化。结果 CCK-8检测结果表明壳寡糖作用软骨细胞不同浓度、不同时间的软骨细胞增殖率比较均无显著性差异(P>0.05);流式细胞检测结果表明壳寡糖不仅扭转了细胞活力和增殖活性的下降,而且改善了IL-1β诱导的软骨细胞核染色质的损害,同时对软骨细胞凋亡率也有不同程度的降低。结论一定浓度的壳寡糖对软骨细胞增殖无明显影响;壳寡糖能明显抑制IL-1β诱导的软骨细胞凋亡,且呈剂量依赖性。

Objective To investigate the effect of different concentrations of the Chitosan oligosaccharides （COS） on proliferation and apoptosis induced by IL-1β of cartilage cells in vitro.Methods From eight week ages SD rats in the knee joint training and separation of cartilage cells,identified and choose second generation cartilage cells for experiments.Different concentrations of COS were added into culture medium for 24 h for the experimental group,cell counting kit-8 （CCK-8） assay was used to detect the proliferation of cartilage cells.IL-11β were added into the culture medium to induce apoptosis of cartilage cells,add the different concentrations of COS processing,cartilage cell morphology and mitotic changes was observed under inverted phase contrast microscope,cell viability and apoptosis were measured by flow cytometry（FCM）.Hoechst 33258 staining was used to determine the cell apoptosis.Results CCK-8 results showed that COS in various concentrations and at different times had no significant effect on proliferation of cartilage cells in vitro （P ＞ 0.05） ;FCM results showed that COS did not only reverse the decrease of cell viability and proliferation activity,but ameliorated nuclear chromatin damage in IL-1β-induced cartilage cells.Additionally,cartilage cells apoptosis rate was significantly lower.Conclusions A given concentration of COS have no significant effect on proliferation of cultured cartilage cells.COS can inhibit the apoptosis of cartilage cells induced by IL-1β in vitro,and is dose-dependent manner.