应用Edu标记技术对髓核样细胞移植治疗椎间盘退变进行示踪标记的实验研究  

Experimental Research on the Application of Edu to Track the Labeled Nucleus Pulposus Cells Transplantation in Treatment of Intervertebral Disc Degeneration

作　　者：陈杰[1] 薛超[1] 陈欣[1] 田谦[1] 李浩鹏[1] 

机构地区：[1]西安交通大学第二附属医院骨二科,西安710004

出　　处：《中国中医骨伤科杂志》2015年第11期9-14,共6页Chinese Journal of Traditional Medical Traumatology & Orthopedics

基　　金：陕西省社发攻关项目(2011K14-08-06)

摘　　要：目的:建立一种可行的骨髓基质干细胞(BMSCs)成软骨细胞诱导分化方法,并用Edu标记明确移植入椎间盘髓核内的髓核样细胞的分布及分化情况。方法:密度梯度离心和贴壁培养兔BMSCs,观察、鉴定后加入TGF-β1+ITS-A为主的成软骨诱导液中进行分化,2周后免疫组化检测Ⅱ型胶原的表达。将标记后的髓核样细胞微创注射入采用髓核抽吸法建立的椎间盘退变(IDD)模型内,荧光镜下检测移植细胞的分布及分化情况,同时可进行组织学检测,判定移植效果。结果:密度梯度离心结合贴壁法能分离培养出纯度较高的BMSCs,1μg/mL TGF-β1+1%ITS-A成软骨诱导液将BMSCs诱导2周后,Ⅱ型胶原免疫组化检测示表达阳性。10μmol/L的L5-乙炔基-2’脱氧尿嘧啶核苷(Edu L5-ethynyl-2'-deoxyuridine)孵育72h可获得较高的标记率。移植入椎间盘髓核内后4周、8周取出椎间盘髓核检测,标记细胞有荧光表达,红色荧光主要分布在纤维环中,髓核中较少,并且随着时间的推移,阳性细胞由中央区域向外周迁移。组织学检查显示,退变模型组髓核细胞的数量及Ⅱ型胶原含量进行性减少(P<0.05),细胞凋亡率明显增加,干细胞及诱导组髓核细胞数量及Ⅱ型胶原含量较退变模型组均有增多(P<0.05),细胞凋亡率下降,但以诱导组尤为明显。结论:BMSCs在TGF-β1+ITS-A作用下可定向分化为软骨样细胞,通过微创注射方法移植入体内后可修复椎间盘髓核退变;Edu可用于BMSCs的体内外标记,移植的细胞在椎间盘髓核进行迁徙,其规律是逐渐向纤维环趋化。Objective： To establish a feasible method for inducing the differentiation of bone marrow stromal cells （BM- SCs） into chondrocytes, and to make clearly defined the distribution and differentiation of nucleus pulposus cells which transplanted into intervertebral disc degeneration （IDD） by Edu method. Methods： Purified BMSCs were isolated with density gradient centrifugation method and adherence culture method. Then the chondrogenically inductive medium containing TGF-β1 and ITS-A were used to induce the BMSCs differentiation into nucleus-like cells. The expression of collagen type Ⅱ was detected by immunohistochemistry two weeks after intervention. After establishment of the IDD model by the nucleus aspiration methods, the labeled nucleus pulposus-like cells were injected into the degeneration disc. The distribution and differentiation of transplanted cells were detected by fluorescence microscope, and the effect of transplantation was determined by histological examination. Results： The density gradient centrifugation method combined with adherent method was effective for isolating and purifying the BMSCs. The immunohistochemical assay used to detect expression of type Ⅱ collagen showed positive 2 weeks after inducing by 1 μg/mL TGF-β1＋1%ITS-A. High labeling rate can obtain 72 hours after incubation by L5 ethynyl - 2 ＇deoxyuridine （10 μmol/L）. The nucleus pulposus of intervertebral disc was detected 4 weeks and 8 weeks after transplantation. The cells were labeled with fluorescence, and the red fluorescence was mainly distributed in the annulus, but less in the nucleus. The positive cells migrated from the central area to the periphery as time went by. Histological examination showed that the nucleus pulposus cell number and type Ⅱ collagen content were decreased in degeneration model group （P〈0.05）, cell apoptosis rate increased significantly. The nucleus pulposus cell number and type If collagen content in stem cells group and induced group were significantly increased tha

关 键 词：骨髓间充质干细胞 椎间盘髓核退变 Edu标记 

分 类 号：R-33[医药卫生]