脊索细胞促进骨髓间充质干细胞增殖及定向诱导分化的研究  

Notochordala cells stimulate proliferation and differentiation of mesenchymal stem cells toward nu- cleus pulposus phenotype

作　　者：张彦男[1] 邵增务[2] 吴永超[2] 王佰川[2] 马凯歌[2] 杨述华[2] 

机构地区：[1]山西医学科学院山西大医院骨科,太原030032 [2]华中科技大学同济医学院附属协和医院骨科

出　　处：《中华实验外科杂志》2012年第7期1271-1274,F0004,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30872610）

摘　　要：目的分离兔髓核脊索细胞及骨髓间充质干细胞（MSCs），通过共培养观察脊索细胞对MSCs增殖能力及细胞表型的影响。方法4-6周龄新西兰兔4只，取胸腰段脊柱的髓核，用密度梯度离心法提取脊索细胞，同时取其股骨骨髓用Ficoll液分离得到MSCs，光镜观察脊索细胞和MSCs不同比例（1：2、1：1、2：1）共培养条件下细胞的生长，细胞计数试剂盒（CCK-8）法检测细胞增殖。脊索细胞和MSCs共培养（1：1）后行甲苯胺蓝染色及Ⅱ型胶原染色检测MSCs细胞表型的改变。对共培养后的MSCs进行相关基因表达的检测。结果光镜下观察原代脊索细胞呈圆形或椭圆形，胞体大，细胞增殖不明显。MSCs呈梭形贴壁生长，旋涡状排列。CCK-8检测发现脊索细胞／MSCs1：1组细胞增殖明显高于其余各组。甲苯胺蓝染色MSCs单独培养组呈阴性，共培养组呈阳性。Ⅱ型胶原染色MSCs单独培养组呈阴性，共培养组呈阳性。逆转录-聚合酶链反应（RT-PCR）检测发现共培养组蛋白聚糖及Ⅱ型胶原的表达分别为脊索细胞的2．00、1．55倍，而单独培养的MSCs则表达阴性。结论在共培养条件下脊索细胞可以促进MSCs增殖，且细胞比例为1：1时更为显著；同时可以诱导其产生Ⅱ型胶原及聚集蛋白聚糖，表现出类软骨细胞表型。Objective The notoehordal cells （NC） and bone mesenchymal stem ceils （MSCs） were isolated co-cultured from New Zealand rabbit immature nucleus pulposus （NP） , and the eontribution of NC to proliferation and differentiation of MSCs was assessed. Methods NC were got from immature NP of 4 New Zealand rabbits （4-6 week-old） and purified by discontinuous gradient density centrifugation, and MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. MSCs were cultured alone and co-cultured with NC （ 1 ： 1/1 ： 2/2：1 ）. Cell proliferation was evaluated by cell counting Kit-8 （CCK-8） -kit and expression levels of collagen 1I and proteoglycan detected by toluidine blue and immunocytochemistry staining. The expression of collagen II and proteoglycan mRNA was as- sayed. Results NCs and MSCs were isolated and purified. After co-culture, proliferation ability in the group of cell ratio at 1：1 （NC： MSC） was increased significantly as compared with other groups. In the co- culture group of MSCs and NC, the expression of collagen II and proteoglycan was 2.0 and 1.35 times higher than in NC cultured alone. Conclusion By co-culture of 1 ： 1, NC can stimulate proliferation of MSCs, and induce differentiation toward NP cells.

关 键 词：脊索细胞 骨髓间充质干细胞 髓核 椎间盘 共培养 

分 类 号：R681.53[医药卫生—骨科学]