冻椎间盘复合表达hBMP7的髓核细胞体外构建生物椎间盘的可行性研究  

Cryopreserved intervertebral disc with injected nucleus pulposus cells expressing bone morphogenetic protein-7 : a feasibility study using organ culture in vitro

作　　者：王超锋[1] 阮狄克[1] 张超[1] 王德利[1] 

机构地区：[1]中国人民解放军海军总医院骨科,北京100048

出　　处：《骨科》2013年第2期62-65,79,共5页ORTHOPAEDICS

基　　金：国家自然科学基金资助(No.30730095)

摘　　要：目的探讨利用液氮储存的椎间盘与表达人BMP7的髓核细胞体外构建生物活性椎间盘器官的可行性。方法取液氮储存2个月的犬椎间盘48个,分为EGFP对照组、1×104、1×105和1×106共四组,每组12个椎间盘。对照组用22 G注射器自椎间盘后正中注入20μL含有1×105表达EGFP的髓核细胞,1×104组注入20μL含有1×104 PKH-26标记的表达hBMP7的髓核细胞,1×105组注入20μL含1×106 KH-26标记的表达hBMP7的髓核细胞,1×106组注入20μL含1×105 KH-26标记的表达hBMP7的髓核细胞。注射后立即置入50 mL离心管中,加入30 mL完全培养基,分别于培养第4、7、14 d,从外源细胞存活、存活细胞量、hBMP7的表达、蛋白多糖及总胶原含量变化对体外构建的生物活性椎间盘进行评价。结果在椎间盘培养的各时间点均可见PKH-26红色荧光和表达绿色荧光蛋白的髓核细胞存在。对不同组、不同时间点的椎间盘中荧光强度定量后发现:培养第7天、14天时1×105组荧光强度明显高于1×106组和1×104组(P<0.05)。1×105组在第7、14天时hBMP7 mRNA表达量明显高于1×106组和1×104组(P<0.05),EGFP组未见表达;且1×105组在培养的第7天、14天时蛋白多糖及总胶原含量均较另外三组明显增高(P<0.05)。结论应用液氮冻存的椎间盘与1×105个表达hBMP7的髓核细胞复合,外源性髓核细胞可较长时间存活,并能增加蛋白多糖及胶原含量,是一种体外构建生物活性椎间盘的可行性方法。Objective To investigate the feasibility of constructing the biological intervertebral disc using cryopreserved intervertebral disc with injected nucleus pulposus cells （NPCs） expressing bone morphogenetic protein-7 （hBMP7） in vitro. Methods The 48 intervertebral discs stored in liquid nitrogen for 2 months were divided into EGFP control, 1× 10^4 NPCs, 1× 10^5 NPCs and 1× 106 NPCs groups. In EGFP group, 20 vL cell suspension including 1× l0s NPCs expressing EGFP was injec- ted into the intervertebral disc. In I x 10^4, 1 ~ l0s and 1× 106 NPCs groups, 20 t,L cell suspension including 1× 10^4 , 1× l0s and 1× 106 NPCs expressing hBMP7 by PKH-26 dying were injected into the intervertebral discs respectively. Intervertebral discs injected were immerged into 30 mL culture medium. The cell survival, cell fluorescence intensity, hBMP7 mRNA expres- sion, PG and collagen contents of the intervertebral discs in each group were assessed at culture days 4, 7 and 14. Results= En- dogeneous NPCs remained alive at 4th, 7th and 14th day of culture. Results showed that cell fluorescence intensity in 1× 10^5 NPCs group was higher than that in I x 106 and i x 10^4 NPCs groups. The hBMP7 mRNA expression levels in 1× 10^5 NPCs group were higher than those in i x 106 and 1× 10^4 NPCs groups at 7th and 14th day of culture. The hBMP7 mRNA expression in EGFP group was not detectable at any time points. Moreover, the PG and collagen contents in 1× 10^5 NPCs group were higher than those in other grou^os at 7th and 14th day of culture. Conclusion It is feasible that the biological intervertebral disc is constructed to use eryopreserved intervertebral disc with injected NPCs expressing hBMP7, and then cultured for the suitable time in vitro.

关 键 词：椎间盘 骨生成 骨髓细胞 骨骼蛋白 注射 关节内 

分 类 号：R681.5[医药卫生—骨科学]