摘要
目的探讨壳聚糖-pCrmA纳米粒对白细胞介素（IL）-1β诱导的软骨细胞凋亡的影响。方法制备壳聚糖-pDNA纳米粒并进行表征。荧光显微镜观察壳聚糖介导pIRES2-增强型绿色荧光蛋白（EGFP）在原代软骨细胞中的转染，采用四甲基偶氮唑蓝（MTT）实验分析壳聚糖-plRES2-EGFP纳米粒的细胞毒性，蛋白印迹法分析壳聚糖介导的CrmA在软骨细胞中的表达，用原位末端脱氧核苷酸转移酶标记（TUNEL）法检测壳聚糖-pCrmA纳米粒对IL-1β诱导的软骨细胞凋亡的影响。采用单因素方差分析进行统计学分析。结果壳聚糖-pDNA纳米粒的粒径为50nm，壳聚糖-pDNA纳米粒中的pDNA在pH2．0和pH7．4缓冲液中呈双相释放。壳聚糖能够介导基因CrmA在软骨细胞中表达。壳聚糖-pDNA纳米粒无细胞毒性。壳聚糖-pCrmA纳米粒处理组软骨细胞凋亡率明显低于壳聚糖组（P〈0．05）与磷酸盐缓冲液组（P〈0．01）。结论壳聚糖是一种有效的非病毒基因载体，壳聚糖-pCrmA纳米粒能够显著抑制IL-1β诱导的软骨细胞凋亡。
Objective To study the effect of chitosan-pCrmA nanoparticles on the apoptosis of chondrocytes induced by interleukin-1 beta （IL-Iβ）. Methods Chitosan-pDNA nanoparticles were prepared and characterized. The transfection efficiency of chitosan-mediated pIRES2-EGFP was evaluated using fluorescence microscope. The cytotoxicity of chitosan-pIRES2-EGFP nanoparticles in primary rabbit chondrocytes was analyzed by MTT assay. The expression of chitosan-mediated pCrmA in primary rabbit chondrocytes was verified by Western blotting. The effect of chitosan-mediated CrmA on chondrocytes apoptosis induced by IL-1β were analyzed by TUNEL assay. One-way ANOVA was used to analysis. Results The size of chitosan-pDNA nanoparticles was 50 nm. The pDNA release of chitosan-pDNA nanoparticles appeared as biphasic release at pH 2.0 and pH 7.4 buffer. The expression of CrmA in rabbit primary chondrocytes mediated by chitosan could be detected. The chitosan-pIRES2-EGFP nanoparticles had no cytotoxicity. The apoptosis rate of chondrocytes in the chitosan-pCrmA nanoparticles treated group was significantly lower than that of the chitosan treated group （P〈0.05） and PBS group （P〈0.01）. Conclusion Chitosan is an effective non-viral gene transfer vector. The CrmA mediated by chitosan can significantly inhibit chondrocytes apoptosis induced by IL-lβ, suggesting that chitosan-pCrmA nanoparticles may be the treatment of osteoarthritis.
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2013年第7期477-480,I0002,共5页
Chinese Journal of Rheumatology
基金
基金项目：国家自然科学基金（81071494）
湖北省自然科学基金（2012FFB04302）
