构建三维培养体系诱导脐血干细胞向血小板分化的实验研究

发布时间：2018-11-11 17:57

【摘要】：目的： 通过构建壳聚糖-明胶三维支架，并用三维支架与旋转培养系统(RCCS)形成的三维培养体系(3D)来培养脐血分离的CD34+细胞，观察脐血干细胞向血小板分化的情况。 方法： 1、冷冻干燥法制备不同浓度配比的壳聚糖-明胶支架，扫描电镜观察支架表面特征，测量支架的孔径大小、并计算孔隙率、吸水率以及相容性测定，选取适合脐血干细胞生长的支架。 2、用磁珠分选的方法对人脐血CD34+细胞进行分离鉴定，将脐血CD34+细胞附着在壳聚糖-明胶支架上，放入RCCS系统进行3D培养，倒置显微镜观察细胞形态，扫描电镜观察细胞在支架上的粘附生长状况，MTT法检测细胞活性，流式细胞仪检测细胞标志物，以及对分化所得的血小板进行形态及功能鉴定，并与二维培养体系(2D)进行比较。 结果： 1、成功构建壳聚糖-明胶支架，选用壳聚糖、明胶各0.5g，预冻温度为-30℃条件下制备支架，孔径为(86±21)μm、吸水率为(87.62±10.19)%、孔隙率为(92.56±1.12)%，细胞相容性好，无毒副作用，，适宜脐血干细胞培养用三维支架。 2、磁珠分选出脐血CD34+细胞，将细胞以5×104/ml的种植率种植在2D和3D体系中。扫描电镜观察细胞粘附于支架上，生长状况良好；3D培养第8d CD34+细胞可增殖(87.02±4.35)倍，2D培养到第5d达到峰值，细胞可增殖(20.78±5.03)倍，两者间具有统计学意义(P 0.05)；流式检测细胞膜表面标志物CD41+CD61+，培养到第7d、14d，3D体系测得的的结果为((28.70±1.75)、(82.40±2.91))%，2D体系中测得的结果为((27.18±2.63)、(80.33±3.56))%；3D培养系统所得类血小板颗粒较2D多，3D培养系统所得类血小板颗粒细胞数为(31.60±0.74)×106/ml，2D培养条件所得类血小板颗粒细胞数为(7.91±0.93)×106/ml，其黏附率为(43.05±1.93)%，2D为(41.37±1.06)%，两者无统计学意义；两种培养系统所收集的类血小板颗粒在镜下观察都具有聚集能力，流式细胞仪检测类血小板细胞膜有活化后标志物CD62p的表达，阳性率分别为((73.82±1.01)、(75.26±2.57))%，差异不显著，无统计学差异。 结论： 1、通过冷冻干燥法成功构建了适合脐血干细胞生长的壳聚糖-明胶三维支架。 2、壳聚糖-明胶支架和RCCS系统构建的三维培养体系利于脐血干细胞的增殖生长。
[Abstract]:Objective: to construct a three-dimensional scaffold of chitosan gelatin and culture CD34 cells isolated from umbilical cord blood by using three-dimensional scaffold and three-dimensional culture system (3D) formed by rotating culture system (RCCS). The differentiation of umbilical cord blood stem cells into platelets was observed. Methods: 1. Chitosan gelatin scaffolds with different concentrations were prepared by freeze-drying method. The surface characteristics of the scaffolds were observed by scanning electron microscope (SEM), the pore size of the scaffolds was measured, and the porosity, water absorption and compatibility were calculated. The scaffold suitable for cord blood stem cell growth was selected. 2. Human umbilical cord blood CD34 cells were isolated and identified by magnetic bead sorting. The CD34 cells were attached to chitosan gelatin scaffold and placed in RCCS system for 3D culture. The morphology of the cells was observed by inverted microscope. The adhesion and growth of cells on scaffolds were observed by scanning electron microscope (SEM), cell activity was detected by MTT assay, cell markers were detected by flow cytometry, and morphology and function of platelets were identified. And compared with 2 D culture system (2 D). Results: 1. Chitosan and gelatin scaffolds were successfully constructed. The scaffolds were prepared by using chitosan, gelatin 0.5 g and pre-freezing temperature -30 鈩

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