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
发布时间：2018-10-22 08:09
【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(P0.05);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(P0.05)。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。
[Abstract]:Objective to analyze the compatibility of bone marrow mesenchymal stem cells (BMSCs) with amphiphilic peptide 3 D gel. Methods three healthy 3-week-old SD rats were used to isolate femur and tibia to obtain BMSCs,. BMSCs surface antigen was detected by flow cytometry. 10mg/mL containing arginyl-glycyl-aspartic (RGD) ring amphiphilic peptide solution was added to the DMEM/F12 medium of equal volume for several seconds and self-assembled into a three-dimensional gel. The three-dimensional gel structure was observed under transmission electron microscope (TEM), 1 脳 10 ~ (6) cells/mL BMSCs suspension and RGD ring amphiphilic peptide were mixed to form a three-dimensional culture system, 1 脳 10 ~ (6) cells/mL BMSCs suspension and polylysine were mixed to form a two-dimensional culture system without serum, and the cell growth was observed by CCK-8 method. The effect of RGD cyclic amphiphilic peptide on the proliferation of BMSCs was observed by double labeling staining of calcium xanthocyanin acetyl methyl ester / propanidine iodide with fluorescence microscope. Results the high expression CD29,CD90, or low expression CD34,CD45;TEM of the cultured cells showed that the gel was composed of multi-empty nanofibers, the diameter of the nanofibers was 2 ~ 5 nm, the length was 100 ~ 1 000 nm, and the relative molecular weight of synthetic polypeptides was 1 256.37, which was consistent with the theoretical values. The purity of amphophilic peptide by HPLC was 95.88.The double labeling staining of calcium xanthophyllin acetyl methyl ester / PI showed that a few BMSCs cells died after 30min, and the cells began to proliferate after 12 hours, and the proliferation was more active than that in two-dimensional culture. The difference was statistically significant (P0.05); CCK-8 cell count showed that the proliferation activity of three-dimensional culture system was higher than that of two-dimensional culture system, the difference was statistically significant (P0.05). Conclusion RGD amphophilic peptide has good biocompatibility with BMSCs and may be used as scaffold material for tissue engineering.
【作者单位】： 南昌大学第二附属医院骨科;
【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)
【分类号】：R318.08
本文编号：2286602
[Abstract]:Objective to analyze the compatibility of bone marrow mesenchymal stem cells (BMSCs) with amphiphilic peptide 3 D gel. Methods three healthy 3-week-old SD rats were used to isolate femur and tibia to obtain BMSCs,. BMSCs surface antigen was detected by flow cytometry. 10mg/mL containing arginyl-glycyl-aspartic (RGD) ring amphiphilic peptide solution was added to the DMEM/F12 medium of equal volume for several seconds and self-assembled into a three-dimensional gel. The three-dimensional gel structure was observed under transmission electron microscope (TEM), 1 脳 10 ~ (6) cells/mL BMSCs suspension and RGD ring amphiphilic peptide were mixed to form a three-dimensional culture system, 1 脳 10 ~ (6) cells/mL BMSCs suspension and polylysine were mixed to form a two-dimensional culture system without serum, and the cell growth was observed by CCK-8 method. The effect of RGD cyclic amphiphilic peptide on the proliferation of BMSCs was observed by double labeling staining of calcium xanthocyanin acetyl methyl ester / propanidine iodide with fluorescence microscope. Results the high expression CD29,CD90, or low expression CD34,CD45;TEM of the cultured cells showed that the gel was composed of multi-empty nanofibers, the diameter of the nanofibers was 2 ~ 5 nm, the length was 100 ~ 1 000 nm, and the relative molecular weight of synthetic polypeptides was 1 256.37, which was consistent with the theoretical values. The purity of amphophilic peptide by HPLC was 95.88.The double labeling staining of calcium xanthophyllin acetyl methyl ester / PI showed that a few BMSCs cells died after 30min, and the cells began to proliferate after 12 hours, and the proliferation was more active than that in two-dimensional culture. The difference was statistically significant (P0.05); CCK-8 cell count showed that the proliferation activity of three-dimensional culture system was higher than that of two-dimensional culture system, the difference was statistically significant (P0.05). Conclusion RGD amphophilic peptide has good biocompatibility with BMSCs and may be used as scaffold material for tissue engineering.
【作者单位】： 南昌大学第二附属医院骨科;
【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)
【分类号】：R318.08
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