人退变髓核细胞诱导脂肪间充质干细胞向类髓核细胞分化的实验研究  

Degenerative human nucleus pulposus cells induced differentiation of adipose-derived stem cells to nucleus pulposus like cells

作　　者：张永辉[1] 李玲慧[2] ZHANG Yong-hui;LI Ling-hui(Central Hospital of Luohe, Luohe 462300, China;Wangiing Hospital, Chi- na Academy of Chinese Medical Sciences, Beijing 100700, China)

机构地区：[1]漯河市中心医院关节外科,河南漯河462300 [2]中国中医科学院望京医院骨伤综合科,北京100700

出　　处：《中国矫形外科杂志》2018年第7期644-649,共6页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(编号:81674005);北京市自然科学基金资助项目(编号:7164313);中国博士后科学基金项目(编号:2017M611125;2016M591364)

摘　　要：[目的]探讨人脂肪间充质干细胞(adipose-derived stem cells,ADSCs)向类髓核细胞(nucleus pulposuslike cells,NPCs)诱导分化的可能性,为椎间盘退变的防治研究提供种子细胞。[方法]收集腰椎间盘退变患者手术中切除的脂肪组织及髓核组织,采用胶原酶消化法提取原代ADSCs和NPCs,倒置显微镜下观察共培养后ADSCs形态学变化。MTT法检测细胞增殖情况并绘制细胞生长曲线,采用孔径为0.4μm的Transwell小室建立细胞共培养体系,NPCs置于上层,ADSCs接种于下层,建立2个细胞培养组:ADSCs单独培养组和ADSCs/NPCs共培养组,甲苯胺蓝染色以检测蛋白聚糖表达情况,RT-PCR法检测细胞COL2A1、ACAN和SOX9基因表达水平。[结果]采用胶原酶消化法可较好的分离培养出人脂肪间充质干细胞和髓核细胞。与NPCs共培养后的ADSCs可被甲苯胺蓝染为天蓝色,且Ⅱ型胶原及SOX9基因表达水平上调(P<0.05)。[结论]采用胶原酶消化法分离培养的ADSCs活力较好、增殖旺盛,与退变NPCs共培养后,软骨特异性标记物的表达明显增加,细胞表现出向NPCs分化的趋势,可作为椎间盘组织工程研究的种子细胞。[Objective] To investigate the potential differentiation capacity of human adipose-derived stem cells （ADSCs） into nucleus pulposus-like cells （NPCs）, and to provide seed cells for the prevention and cure of intervertebral disc degeneration. [Methods] Adipose tissue and nucleus pulposus were collected from patients with lumbar intervertebral disc degeneration to extract ADSCs and NPCs by collagenase digestion methods. After generation and co-culturing, the morphological changes of ADSCs were observed under inverted microscopes. MTT assay was used to observe cell proliferation and growth curve was drawn. After isolation and proliferation, ADSCs and NPCs were co-cultured in a transwell plate with polycarbonate permeable membranes （pore 0.4μm）. Two experimental groups were designed as follows： ADSCs group and co-cultured group. Toluidine blue staining was adopted to assess the express of proteoglycan. The mRNA express of collagen II, aggrecan and SOX9 were detected by real time PCR. [Results] ADSCs and NPCs were successfully digested by collagenase. After one week of co-culture, the cells were positively stained by toluidine blue staining. The expression of collagen II and SOX9 mRNA were up-regulated （P〈0.05） . [Conclusion] Human ADSCs and NPCs with well growth and rapid proliferation were successfully isolated and cultured by collagenase digestion methods. During co-cuhure process, the interaction between ADSCs and NPCs could stimulate ADSCs differentiate into NPCs. The ceils could be used as seed cells for intervertebral disc tissue engineering.

关 键 词：髓核细胞 脂肪间充质干细胞 共培养 

分 类 号：R318[医药卫生—生物医学工程]