人脐血基质细胞的体外培养及向成骨细胞诱导分化

In vitro culture and differentiation of human cord blood stromal cells into osteoblasts

背景:研究证实婴儿脐带血里含有类似骨髓间充质细胞的细胞,但在培养过程中其成功率很低且难度高。目的:拟在体外分离培养人脐血基质细胞,并观察其分化潜能。设计、时间及地点:细胞学体外观察,于2007-03/2008-06在辽宁医学院中心实验室完成。材料:健康足月新生儿脐带血12份,每份30～60mL,由锦州市妇婴医院提供。方法:取采集的脐带血,使用淋巴细胞分离液离心后取界面上的白色混浊状细胞层,加入含体积分数为10%胎牛血清、20μg/L碱性成纤维细胞生长因子、0.3g/LL-谷胺酰胺、100U/mL青霉素和100g/L链霉素的L-DMEM培养液悬浮细胞,按2×109L-1密度接种于25cm2培养瓶中,待细胞达70%汇合时消化传代。取处于对数生长期的脐血基质细胞,将其培养基更换为含有地塞米松、β-甘油磷酸钠、维生素C、体积分数为10%胎牛血清的L-DMEM培养基成骨诱导21～30d。主要观察指标:倒置显微镜观察细胞形态学特征,描绘生长曲线,流式细胞仪检测细胞周期,碱性磷酸酶染色和Von-Kossa染色检测成骨能力。结果:原代培养的人脐血基质细胞呈长梭形,需21～30d长满瓶底,传代后细胞增殖缓慢并逐渐死亡,细胞平均倍增时间为42h,处于G0/G1期的细胞达(86.54±2.31)%。在成骨诱导培养基里,脐血基质细胞增殖速度减慢,细胞形态无明显变化,成骨诱导14d后碱性磷酸酶阳性率为(46.0±2.1)%,成骨诱导21d形成钙化结节。结论:应用体积分数为10%胎牛血清、20μg/L碱性成纤维细胞生长因子的L-DMEM培养液可在体外成功分离培养出人脐血基质细胞,但细胞增殖能力有限,诱导后可向成骨细胞方向分化。

BACKGROUND： Infant umbilical cord blood contains cells similar to bone marrow mesenchymal cells, but success rate during culture is low. OBJECTIVE： To culture stromal cells from human cord blood in vitro, and to observe the differentiation. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Central Laboratory, Liaoning Medical University from March 2007 to June 2008. MATERIALS： A total of 12 samples of healthy full-term newborn cord blood （30-60 mL each） were obtained from Jinzhou Maternal and Child Hospital. METHODS： Collected cord blood was used and centrifuged by lymphocyte isolation. White unclear cell layer was obtained, and cultured in L-DMEM containing 10% fetal bovine serum, 20 μg/L basic fibroblast growth factor, 0.3 g/L L- glutamine, 100 U/mL penicillin and 100 g/L streptomycin. Cells at a density of 2×10^9/L were incubated in 25 cm^2 flask, and digested when 70% cells were confluent. Cord blood stromal cells at logarithmic phase were obtained. The medium was changed into L-DMEM containing dexamethasone, β-sodium glycerophosphate, vitamin C, and 10% fetal bovine serum for 21 30 days induction. MAIN OUTCOME MEASURES： Cell morphology was observed under an inverted microscope. Growth curve was described. Flow cytometry was used to measure cell cycle. Alkaline phosphatase staining and Von-Kossa staining were utilized to measure osteogenic differentiation. RESULTS： The cord blood stromal stem cells presented spindle shape, and it needed 21-30 days to reach confluence. The proliferation ability of cord blood stromal cells was lower and cells gradually died. Mean doubling time was 42 hours, and about （86.54±2.31）% cells in the G0/G1 phase. In osteogenic medium, the cord blood stromal cells grew slowly, and there was no obvious change in morphology. Alkaline phosphatase staining showed the positive rate of cord blood stromal cells was （46.0±2.1）% at 14 days following induction. At 21 days, hydroxyapatite nodules were formed in cord blood stromal cells. CONCLUSION： L-DMEM containing 10% fetal bovine serum, 20 μg/L basic fibroblast growth factor can successfully isolate human cord blood stromal cells in vitro, but the proliferative ability of cord blood stromal cells is limited. It could differentiate into osteoblasts following induction.