大鼠颅骨成骨细胞的胶原水凝胶三维立体培养  

3D Collagen Hydrogel Culture of Rat Calvarial Osteoblasts

作　　者：谢艳芳[1] 石文贵[1] 周建[1] 高玉海[1] 王鸣刚[2] 陈克明[1] 

机构地区：[1]兰州军区兰州总医院骨科研究所,兰州730050 [2]兰州理工大学生命科学与工程学院,兰州730050

出　　处：《四川大学学报（医学版）》2016年第2期164-168,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.81270963;No.81471090)资助

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(P<0.05),细胞活性强于1mg/mL组和3mg/mL组(P<0.05)。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。Objective To establish a collagen hydrogel three-dimensional culture model with rat calvarial osteoblasts（ROBs）.Methods ROBs were obtained through enzyme digestion of segregated neonatal SD rat skull.The collagen hydrogel three-dimensional culture model was established by mixing ROBs with different concentrations of typeⅠ rat tail collagen（collagen concentration of 1,2,3mg/mL）,DMEM medium and NaOH under adjusted PH and a temperature of 37 ℃.Cell viability and activity were detected by FDA/PI staining and CCK-8 3dafter cell culture.The optimal culture method of 3Dcollagen hydrogel was identified.Cell distribution was observed using scanning electron microscopy and HE staining.Results ROBs collagen was formed firmly at 2mg/mL,which had significantly higher levels of cell viability and activity than those at 1 mg/mL and 3mg/mL.Scanning electron microscopy and HE staining showed that cells under the 2mg/mL collagen culture system adhered with collagen tightly and distributed homogeneously.Conclusion A collagen hydrogel 3D culture model was established successfully by mixing ROBs with collagen at 2mg/mL.

关 键 词：成骨细胞 三维立体培养 胶原水凝胶 

分 类 号：R318.08[医药卫生—生物医学工程] Q813.11[医药卫生—基础医学]