中文摘要：

背景：椎间盘退行性变的理想治疗方法是利用组织工程技术修复退行性变的椎间盘,而修复所需的髓核细胞来源十分有限。目的：观察在藻酸盐微球的介导下,髓核细胞与骨髓间充质干细胞非接触共培养时,髓核细胞对骨髓间充质干细胞的诱导作用。设计、时间及地点：细胞学体外观察,实验于2007-10/2008-08在华中科技大学同济医学院附属同济医院骨科实验室和中心实验室进行。材料：4月龄健康新西兰大耳白兔5只,取髓核细胞与骨髓间充质干细胞原代培养,利用传代法分离纯化。方法：①将纯化的骨髓间充质干细胞经胰蛋白酶消化、收集,并与适量的藻酸钠溶液混合,形成109L-1的单细胞悬液,将混合好的单细胞悬液经注射器滴加至35g/L的CaCl2溶液中,形成海藻酸钙凝胶珠。②将海藻酸钠凝胶微球与髓核细胞共培养。在7d和15d时,分组溶解海藻酸钙凝胶珠,收集骨髓间充质干细胞。主要观察指标：利用免疫组化技术、RT-PCR技术和Westernblot技术检测骨髓间充质干细胞中Ⅱ型胶原和聚集蛋白聚糖的表达,用以判断髓核细胞对骨髓间充质干细胞在非接触条件下的诱导作用。结果：在骨髓间充质干细胞的细胞爬片免疫组化染色中可见,Ⅱ型胶原和聚集蛋白聚糖染色阳性,RT-PCR和Westernblot结果显示经诱导后骨髓间充质干细胞中已有Ⅱ型胶原和聚集蛋白聚糖基因的表达,且诱导15d组的目的条带明显亮于诱导7d组的目的条带。结论：在藻酸盐微球的介导下,体外非接触共同培养时髓核细胞能够实现对骨髓间充质干细胞的诱导作用。

英文摘要：

BACKGROUND： The ideal therapy for degeneration of intervertebral disc is repairing the intervertebral disc by using tissue engineering technology. However, the resource of nucleus pulposus cells is limited. OBJECTIVE： To explore the effect of nucleus pulposus cells on bone mesenchymal stem cells （BMSCs）, which were encapsulated in alginate beads when they were non-contact co-cultured in vitro. DESIGN, TIME AND SETTING： The in vitro cytology observation was performed at the Orthopaedic Laboratory and Central Laboratory, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between October 2007 and August 2008. MATERIALS： The primary nucleus pulposus cells and BMSCs from 5 healthy, New Zealand rabbits, with 4-month-old, were separated and purified by passage cultural method. METHODS： After digested with trypsin, BMSCs were mixed with alginate solution to obtain cell suspension with 10^9/L, then adding dropwise of cell suspension to 35 g/L CaCl2 to harvest small droplets of cell-alginate beads. The nucleus pulposus cells and cell-alginate beads were co-cultured and dissolved at days 7th and 15 th respectively to collect BMSCs. MAIN OUTCOME MEASURES： The expression of type Ⅱ collagen and aggrecan were detected by immunohistochemistry, RT-PCR as well as Western blot, respectively to observe the induction of nucleus pulposus cells on BMSCs under non-contact condition. RESULTS： The immunohistochemistry staining showed that there were positive staining of collagen- Ⅱ and aggrecan in the BMSCs The results of RT-PCR and Western blot demonstrated that there was expression of collagen-Ⅱ and aggrecan, additionally, objective strap in the induced group of 15 days was brighter than that of 7 days. CONCLUSION： Co-cultured with BMSc in vitro, nucleus pulposus cells can play the induction effect mediated by alginate beads.