大鼠颅骨成骨细胞的胶原水凝胶三维立体培养

发布时间：2018-06-01 03:23

本文选题：成骨细胞 + 三维立体培养　；参考：《四川大学学报(医学版)》2016年02期

【摘要】：目的建立大鼠乳鼠颅骨成骨细胞(ROBs)的胶原水凝胶三维立体培养模型。方法取新生SD大鼠颅骨,通过酶消化法获得成骨细胞。将传代培养的第一代(标记为P1代)ROBs与不同浓度Ⅰ型鼠尾胶原(胶原浓度为1、2、3mg/mL)、DMEM培养液、NaOH等混合,调节混合液pH并将其置于37℃使胶原凝固成形,建立细胞的三维立体培养。ROBs立体培养3d后,普通显微镜观察细胞形态及密度,培养6d后二乙酸荧光素/碘化丙啶(FDA/PI)染色法鉴定细胞存活率;培养3d、6d、9d后CCK-8检测法测定细胞活性,筛选出三维立体培养最佳胶原浓度及最佳培养体系。扫描电镜和HE染色观察最佳培养体系中ROBs细胞形态以及细胞与胶原复合物的黏附和分布情况。结果胶原浓度为2mg/mL培养体系,胶原成形最好、硬度最高。ROBs三维立体培养3d后,胶原浓度为1mg/mL、2mg/mL培养体系中细胞饱满、多呈长梭形。胶原浓度为3mg/mL的培养体系细胞多呈圆形并固缩。胶原浓度为2mg/mL培养体系中,细胞存活率最高(P0.05),细胞活性强于1mg/mL组和3mg/mL组(P0.05)。2mg/mL培养体系组扫描电镜结果显示细胞与胶原复合物黏附较好且细胞形态正常,HE染色结果显示细胞在胶原中分布均匀且细胞形态正常。结论胶原浓度为2mg/mL时,能够成功建立ROBs胶原水凝胶三维立体培养模型,该模型中胶原成形较好,硬度较高,细胞生存良好,分布均匀,能够维持正常形态及活性,适用于后期科学研究。
[Abstract]:Objective to establish a three-dimensional culture model of collagen hydrogel in rat calvarial osteoblast (ROBs). Methods the osteoblasts were obtained from the cranium of newborn SD rats by enzyme digestion. The first generation (labeled P1 generation) ROBs was mixed with different concentration of rat tail collagen (1,2,3mg/mL), DMEM culture solution, NaOH and so on. The mixed liquid pH was placed at 37 C to solidify the collagen, and the cell morphology and density were observed by a three-dimensional culture of.ROBs for 3D. The cell viability was identified by two fluorescein / FDA/PI staining method after 6D, and the cell viability was determined by CCK-8 detection after 3D, 6D and 9D, and three of the cells were screened. The optimum collagen concentration and the best culture system were cultured. The morphology of ROBs cells in the optimum culture system and the adhesion and distribution of collagen complex were observed by scanning electron microscopy and HE staining. Results collagen concentration was 2mg/mL culture system, collagen formation was best, and the highest hardness of.ROBs was.ROBs three-dimensional culture 3D, collagen concentration was 1mg/mL, 2 The cells in the culture system of mg/mL were full and long shuttle shaped. The cells in the culture system with collagen concentration of 3mg/mL were mostly round and fixed. The cell viability was the highest in the collagen concentration 2mg/mL culture system (P0.05). The cell activity was stronger than that of the 1mg/mL group and the 3mg/mL group (P0.05).2mg/mL culture body group. The results showed that the cell morphology was normal and the HE staining results showed that the cells were evenly distributed in collagen and the cell morphology was normal. Conclusion when the collagen concentration is 2mg/mL, the three-dimensional culture model of ROBs collagen hydrogel can be successfully established. In this model, the collagen formation is better, the hardness is higher, the cell survival is good, the distribution is uniform, and it can maintain normal form and maintain normal form. The activity is suitable for late scientific research.
【作者单位】：兰州军区兰州总医院骨科研究所;兰州理工大学生命科学与工程学院;
【基金】：国家自然科学基金(No.81270963,No.81471090)资助
【分类号】：R318.08;Q813.11

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