共培养诱导人脐带间充质干细胞向纤维环样细胞分化

摘要

Purpose To induce the differentiation of hu-man umbilical cord mesenchymal stem cells ( HUCMSCs) into annulus fibrosus (AF) cells by in vitro co-culture technique and to investigate the morphological and histological changes of HUCMSCs after co-culture. Methods HUCMSCs and AF cells were isolated from the normal neonatal umbilical cord and New Zealand white rabbit. Transwell six-well plates were used for co-culture with the cells seeded at the ratio of 1 ∶ 1, HUCMSCs cultured alone served as controls. After two weeks of co-culture, morphological changes were observed by inverted microscope. Real-time PCR was used to detect the expression of typeⅠcolla-gen, aggrecan and SOX-9 gene in HUCMSCs. Immunocyto-chemical staining and toluidine blue staining were used to detect the synthesis of cell matrix such as type Ⅰ collagen and aggre- can. Results The morphology of HUCMSCs in control group was long-fusiform, the morphology of HUCMSCs in co-culture gradually became short-fusiform or polygonal, and began to ap-pear synapse, showing the morphological features of AF-like cells. Real-time PCR results showed that typeⅠcollagen, aggre-can and SOX-9 mRNA were significantly increased in the co-cul-ture group (P＜0. 05). Immunocytochemical staining and tolui-dine blue staining showed that type I collagen and aggrecan were positive, respectively. Conclusion In vitro co-culture technol-ogy can induce HUCMSCs to differentiate into AF-like cells, which is expected to provide a new kind of seed cells for the bio-logical treatment of degenerative disc disease.%目的 通过体外共培养技术诱导人脐带间充质干细胞(human umbilical cordmesenchymal stem cells, HUCMSCs)向纤维环(annulus fibrosus, AF)细胞分化,并探讨共培养后HUCMSCs形态学和组织学的变化.方法 取正常足月新生儿脐带及新西兰白兔,分别分离培养HUCMSCs和AF细胞.取第2代HUCMSCs和AF细胞,按1 ∶ 1比例利用Transwell 6孔培养板共培养.共培养2周后,用倒置显微镜观察细胞形态学变化;实时荧光定量PCR法检测共培养组HUCMSCsⅠ型胶原、蛋白聚糖及SOX-9基因的表达;免疫细胞化学染色和甲苯胺蓝染色法检测Ⅰ型胶原蛋白和蛋白聚糖等细胞基质的合成情况.结果 对照组HUCMSCs形态多呈长梭形,共培养后HUCMSCs形态逐渐变为短梭形或多角形,并开始出现细胞突触,出现类AF的形态特征;实时荧光定量结果显示,共培养HUCMSCs中Ⅰ型胶原、蛋白聚糖及SOX-9基因相对表达量显著上调( P＜0. 05);共培养HUCMSCs免疫细胞化学染色显示Ⅰ型胶原蛋白胞质阳性,甲苯胺蓝染色显示蛋白聚糖阳性,对照组均为阴性.结论 体外共培养技术可诱导HUCMSCs向类AF分化,有望为椎间盘退变性疾病的生物学治疗提供一种新的种子细胞.