含精氨酰-甘氨酰-天冬氨酰环两亲性肽自组装凝胶与骨髓间充质干细胞相容性研究  

Study on compatibility of self-assembled gel containing RGD amphipathic peptide with bone marrow mesenchymal stem cells

作　　者：肖仁顺 阮宏 蒋星海 吴凯[1] 王晓梅[1] 宋玉林[1] 

机构地区：[1]南昌大学第二附属医院骨科,南昌330006

出　　处：《重庆医学》2017年第35期4912-4916,共5页Chongqing medicine

基　　金：国家自然科学基金资助项目(81360271;30801158);江西省教育厅课题(GJJ14054);江西省自然科学基金计划项目(20151BAB205051);南昌大学研究生创新专项基金(CX2016326)

摘　　要：目的分析骨髓间充质干细胞(BMSCs)与两亲性肽三维凝胶的相容性。方法取3周龄SD健康大鼠3只,分离股骨、胫骨获取BMSCs,采用流式细胞术检测BMSCs表面抗原;将10mg/mL含精氨酰-甘氨酰-天冬氨酰(RGD)环两亲性肽溶液加入等体积DMEM/F12培养基数秒后自组装成三维凝胶,透射电镜显微镜(TEM)下观察三维凝胶结构;1×10~6 cells/mL BMSCs悬液与RGD环两性亲肽混合形成三维培养体系,1×10~6 cells/mL BMSCs悬液与多聚赖氨酸混合形成二维培养体系,无血清培养;CCK-8法观察细胞生长情况,钙黄绿素乙酰氧基甲酯/碘化丙啶(PI)双标染色,荧光显微镜观察RGD环两性亲肽对BMSCs增殖的影响。结果分离培养的细胞高表达CD29、CD90,不表达或低表达CD34、CD45;TEM显示凝胶由多空纳米纤维构成,纳米纤维直径2~5nm,长度100~1 000nm;质谱测得合成多肽相对分子质量为1 256.37,与理论值一致;高效液相色谱分析两性亲肽纯度为95.88%;钙黄绿素乙酰氧基甲酯/PI双标染色显示,三维培养体系中,30min后少数BMSCs出现死亡,12h后细胞开始增殖,增殖较二维培养活跃,差异有统计学意义(P<0.05);CCK-8细胞计数显示,三维培养体系细胞增殖活力高于二维培养体系,差异有统计学意义(P<0.05)。结论 RGD两性亲肽与BMSCs有良好的生物相容性,可能成为组织工程支架材料。Objective To analyze the compatibility of bone marrow mesenchymal stem cells （BMSCs） with amphiphilic peptide three-dimensional gel. Methods Three healthy 3-week-old SD rats were taken for separating femur and tibia to obtain BMSCs, the BMSCs surface antigen was detected by flow cytometry; the 10 mg/mL RGD-cyclic amphiphilic peptide solution was added into the same volume of DMEM/F12 culture medium, after a few seconds, which was self assemble into three-dimensional gel. Three dimensional gel structure was observed by transmission electron microscope （TEM）. 1 × 10 6 cells/mL BMSCs suspension and RGD- cyclic amphiphilic peptide were mixed to form a 3D culture system, 1 × 10 6 cells/mL BMSCs suspension was mixed with polylysine to form a 2D culture system,the serum-free culture was conducted;the CCK-8 method was used to observe the cell growth situation,calcein acetoxy methyl ester/propidium iodide （PI） double standard staining was performed. The effect of RGD-cyclic am phiphilic peptide on the Proliferation of BMSCs was observed by fluorescence microscopy. Results The separated and cultured BM- SCs highly expressed CD29 and CD90,but lowly expressed or did not express CD34 and CD45 ;TEM showed that the gel was com- posed of multiple empty nanofibers with the nanofiber diameter of 2--5 nm and length of 100--1 000 nm; the molecular weight of synthetic peptides detected by mass spectrometry （MS） was 1 256.37,which was consistent with the theoretical value; the HPLC analysis showed that RGD-amphiphilic peptide purity was 95.88 % ;the calcein acetoxyl methyl ester/PI double staining showed that in the 3D culture system,a few BMSCs died after 30 min and the cells began to proliferate after 12 h,the proliferation was more active than that of 2D culture,and the difference was statistically significant （P^0.05） ;CCK-8 cell count showed that the proliferation activity of 3D culture system was higher than that of 2D culture system,and the difference was statistically significant （P〈0. 05）. Conclus

关 键 词：精氨酰-甘氨酰-天冬氨酰 间质干细胞 两亲性肽 细胞相容性 

分 类 号：R318.08[医药卫生—生物医学工程]