接枝Nogo-A抗体的温敏型壳聚糖水凝胶的制备及其与大鼠骨髓间充质干细胞生物相容性的研究

Preparation of a thermosensitive chitosan/glycerophosphate hydrogel modified with Nogo-A receptor antibody and its biocompatibility with the rat mesenchymal stem cells

目的 制备接枝Nogo-A受体（NgR）抗体的温敏型壳聚糖（C/GP）水凝胶,观察大鼠骨髓间充质干细胞（BMSCs）接种于该水凝胶上的生长及两者间生物相容性.方法 β-甘油磷酸钠（GP）与壳聚糖（C）按1∶3体积比制备C/GP水凝胶,氧化接枝NgR抗体,制备C/GP-NgR抗体水凝胶,观察理化性状及成胶,检测NgR抗体水平.分离、培养BMSCs,流式细胞仪检测CD34、CD45、CD29、CD90的表达;第3代BMSCs分别接种于C/GP水凝胶（A组）及C/GP-NgR抗体水凝胶（B组）上培养,水溶性四氮唑（WST-1）法检测BMSCs细胞增殖效应,吖啶橙（AO）染色及荧光倒置相差显微镜观察BMSCs生长及活性.结果 成功制备C/GP-NgR抗体水凝胶,具有温敏性,NgR抗体水平较对照组增高（P＜0.05）,室温下C/GP溶液pH值平均为7.2.CD34、CD45、CD29、CD90阳性细胞比率分别为0.79％、6.80％、98.90％、99.90％.AO染色细胞核呈绿色,未见橘红色固缩状细胞.两组细胞培养1 ～7d呈时效性增殖,3～5d生长最快;培养5d,B组细胞增殖明显.结论 BMSCs接种于C/GP-NgR抗体水凝胶上培养,细胞生长增殖良好、活性正常,两者间具有良好的生物组织相容性.

Objective To fabricate thermosensitive chitosan/glycerophosphate （C/GP） hydrogels modified with Nogo-A receptor （NgR） antibody,and to observe its biocompatibility and the growth,proliferation of the rat mesenchymal stem cells （BMSCs） planted on C/GP hydrogels.Methods β-sodium glycerophosphate solution was added to chitosan solution in a ratio of 1∶3 to mix C/GP hydrogel.C/GP hydrogels were modified with NgR antibody.The physicochemical properties,gelling and the expression of NgR antibody were observed.The BMSCs were separated from the bone marrow of SD rats,cultured in vitro,and CD34,CD45,CD29,CD90 were detected by flow cytometry （FCM）.The BMSCs of third generation were planted respectively on C/GP hydrogels （group A） or C/GP hydrogels modified with NgR antibody （group B）.The proliferation of BMSCs was observed using water soluble tetrazolium （WST-1） method,and the growth and activity were observed by fluorescent invertedphase contrast microscope and the techniques of acridine orange （AO） staining.Results A thermosensitive C/GP hydrogel modified with NgR antibody was prepared successfully,and it was liquid at room temperature while it became solid hydrogel at 37℃.The expression of NgR antibody in the group B was stronger than that in the group A （P ＜ 0.05）.The pH of C/GP hydrogel was 7.2 on average at room temperature.The expression rate of CD34,CD45,CD29,and CD90 was 0.79％,6.80％,98.90％,and 99.90％ respectively.The nuclei of cells showed green and no orange condensation cells were detected in AO staining.The proliferation of BMSCs planted on the hydrogels from 1 to 7 days showed time-related growth pattern,and the number of cells increased significantly after 3 to 5 days of culture.The number of cells in group B was much greater than in group A after 5 days.Conclusion BMSCs survive and proliferate well in the C/GP hydrogel modified with NgR antibody.The biocompatibility of BMSCs to the hydrogel is very good.