大鼠骨髓间充质干细胞与树枝状两亲性多肽自组装凝胶支架的生物相容性

Compatibility between rat bone marrow mesenchymal stem cells and self-assembling branched peptide-amphiphile nanofiber hydrogel scaffolds

目的 研究大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）与树枝状两亲性多肽自组装凝胶支架的生物相容性。方法 获取大鼠BMSCs,传至第3代行流式细胞术表面抗原检测及成脂分化诱导鉴定干细胞。设计含双链-IKVAV活性集团的树枝状两亲性多肽,用固相合成法合成多肽,质谱仪（MS）测其分子量,高效液相色谱仪（HPLC）测其纯度;将10mg/mL树枝状两亲性多肽溶液在兔膝关节滑液作用下自组装为凝胶支架,透射电镜观察凝胶超微结构;1×10^9/L的BMSCs悬液分别接种于凝胶内部（三维培养体系）及多聚赖氨酸包被的盖玻片表面（二维培养体系）,CCK-8法绘制细胞生长曲线。DEAD/LIVE双标染色,荧光显微镜观察BMSCs在三维多肽凝胶中成活及增殖情况。结果 分离获取的细胞高表达CD29、CD90,不表达CD34、CD45,经成脂诱导2周出现脂滴,油红O染色成红色。MS测得合成多肽相对分子质量为2 344.2,与其理论值一致;HPLC分析其纯度为95.15%;透射电镜观察凝胶由多孔纳米纤维构成,纳米纤维直径为5-7nm,长度为数百纳米至数微米;CCK8试验示三维体系中细胞增殖率明显高于二维培养体系（P〈0.05）。DEAD/LIVE双荧光染色后显示三维培养体系中细胞生存、增殖情况良好,未出现明显的细胞毒性导致细胞死亡。结论 含-IKVAV活性基团的树枝状两亲性多肽与BMSCs具有良好的细胞相容性并能促进其增殖。

Objective To study the compatibility between bone marrow mesenchymal stem cells （BMSCs） and self-assembling branched peptide-amphiphile nanofiber hydrogel scaffolds. Methods First, we obtained BMSCs from rats by whole marrow adherence method and then detected surface antigens using flow cytometry, and induced adipogenic differentiation to identify these stem cells. We designed the branched peptide-amphiphile containing double-IKVAV epitopes. The peptide whose molecular weight （MW） and purity were detected by mass spectrometer （MS） and high performance liquid chromatograph （HPLC）, respectively, was synthesized with solid phase method. The 10mg/mL peptide solution was triggered to form self-assembling branched peptide-amphiphile nanofiber hydrogel under the effect of the rabbit knee joint synovial fluid. The self-assembly hydrogel was examined with transmission electron microscope （TEM）. 1×10^9/L BMSCs were seeded in three-dimensional （3D） hydrogels （experimental group, EG） or surface of coverslips （control Group, CG）. A growth curve of the cells was obtained according to CCK-8. After double-labeling with DEAD/LIVE, the survival and proliferation of the BMSCs living in three-dimensional （3D） hydrogels were observed by fluorescence microscope. Results The cells we obtained highly expressed CD29 and CD90, but did not express CD34 or CD45. Lipid droplets were generated after induction to adipogenic differentiation for two weeks, and oil red O staining was positive. MS showed that molecular weight of the peptide was 2344.2, which was identical to that in theory. HPLC testified that the peptide purity was 96%. TEM showed that the hydrogel was composed of nanofibers with 5-7nm in diameter and hundreds of nanometers to several microns in length. CCK8 test showed that BMSCs proliferated more actively in 3D hydrogel than in 2D culture （P〈0.05）. Observed under fluorescence microscope after DEAD/LIVE kit staining, the cells survived and proliferated in good condition, the hydrogel had no cytotoxicity to the cells. Conclusion The self-assembled hydrogel containing double-IKVAV epitopes can provide survival environment for BMSCs and promote their proliferation.