大鼠椎间盘髓核来源间充质干细胞的体外分离及培养鉴定  

Isolation,culture and identification of nucleus pulposus-derived mesenchymal stem cells from adult rats in vitro

作　　者：芮云峰[1] 王善正[1] 谢鑫荟[1] 孙明辉[2] 林禹丞 李刚[3] 王宸[1] 

机构地区：[1]东南大学附属中大医院骨科,江苏省南京市210009 [2]南京大学附属鼓楼医院骨科,江苏省南京市210008 [3]香港中文大学干细胞与再生医学实验室

出　　处：《中国组织工程研究》2013年第49期8576-8582,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年基金(81201422);江苏省自然科学基金青年基金项目(BK2012334);东南大学"创新基金"项目(3290002401)~~

摘　　要：背景:目前椎间盘髓核组织的细胞组成和特性仍未阐明。目的:旨在建立大鼠椎间盘髓核来源间充质干细胞的体外培养体系,并对其体外多项分化潜能进行鉴定。方法:体外培养SD大鼠盘髓核来源间充质干细胞,取第3代细胞进行三系诱导分化,将成骨、成脂和成软骨分化作为实验组,基础细胞培养作为对照组。结果与结论:低密度培养获得的髓核来源细胞早期可形成葵花样细胞集落,克隆样生长。第3代后,细胞形态趋向均一,呈成纤维细胞样生长。成骨诱导28 d,实验组茜素红染色阳性,且RunX2、osteopontin及osteocalcin表达较对照组显著增高(P<0.05);成脂诱导21 d,实验组油红O染色阳性,C/EBPα及PPARγ2表达较对照组显著增高(P<0.05);成软骨分化诱导21 d,实验组番红O快绿染色和Ⅱ型胶原免疫组织化学染色阳性,aggrecan和Col2a1表达较对照组显著增高(P<0.05)。可见成年SD大鼠椎间盘髓核组织中可分离培养出体外呈克隆样生长的细胞群,且有向脂肪细胞、成骨细胞和软骨细胞发生定向分化的潜能。BACKGROUND：Currently, cellular composition and the features of the nucleus pulposus are stil not to be clarified. OBJECTIVE：To establish the in vitro culture system of rat nucleus pulposus-derived mesenchymal stem cells and to identify their multi-lineage differentiation potential. METHODS：Mesenchymal stem cells from the nucleus pulposus tissues of Sprague-Dawley rats were cultured in vitro. Then, cells at passage 3 were induced to differentiate into osteoblasts, adipocytes and chondrocytes as experimental group. cells cultured with basic culture medium served as controls. RESULTS AND CONCLUSION：cells isolated from rat nucleus pulposus could form the sunflower-like colonies and exhibit clone-like growth when they cultured at a low density. cells at passage 3 became homogeneous and exhibited fibroblast-like morphology. After 28 days of osteogenic induction, arizarin red positive signals were detected in the experimental group. The mRNA expressions of RunX2, osteopontin and osteocalcin were significantly increased in the experimental group, compared to the control group （P〈0.05）. After 21 days of adipogenic induction, oil red-O positive cells were detected in the experimental group. The mRNA expressions of C/EBPαand PPARγ2 were significantly increased in the experimental group, compared to the control group （P〈0.05）. After 21 days of chondrogenic induction, safranin O/fast green staining was positive in the experimental group. The mRNA expressions of aggrecan and Col2a1 were significantly increased in the experimental group, compared to the control group （P〈0.05）. Our findings in this study suggested that nucleus pulposus-derived mesenchymal stem cells could be isolated from the Sprague-Dawley rat nucleus pulposus and exhibited clonal-like growth when they were cultured in vitro. These cells were confirmed to have the potential to differentiate into adipocytes, osteoblasts and chondrocytes in vitro.

关 键 词：干细胞 干细胞培养与分化 椎间盘 髓核 间充质干细胞 成骨细胞 脂肪细胞 软骨细胞 II型胶原 国家自然科学基金 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]