高渗环境结合氯化钴低氧预处理骨髓间充质干细胞移植治疗椎间盘退变  

Pretreatments with hypertonic solution and cobalt chloride in bone marrow mesenchymal stem cell transplantation for treatment of degenerative disc disease

作　　者：叶涵[1] 孟真[2] 林嘉琛[3] 李嘉伟[4] 张永兴[2] 林南河 赵庆华[2] 

机构地区：[1]复旦大学附属中学,上海市200433 [2]上海市第一人民医院,上海市201620 [3]上海交通大学医学院附属仁济医院,上海市200127 [4]解放军第四军医大学附属西京医院,陕西省西安市710032

出　　处：《中国组织工程研究》2016年第10期1452-1460,共9页Chinese Journal of Tissue Engineering Research

基　　金：上海市自然科学基金(14ZR1433100)~~

摘　　要：背景:有研究表明,干细胞可以用来预防和治疗椎间盘退变类疾病,但由于椎间盘的低氧高渗的特殊环境,使得体外培养的干细胞移植到椎间盘以后存活率及分化能力降低,治疗效果不佳。提高干细胞的移植效率及治疗效果成为干细胞移植治疗椎间盘退变研究的难点和热点。目的:观察氯化钴和高渗溶液预处理对骨髓间充质干细胞移植治疗椎间盘退变疾病效果的影响。方法:(1)细胞体外实验,将骨髓间充质干细胞分为3组,对照组正常培养,高渗组采用体积分数1%高渗母液干预,低氧组采用100μmol/L氯化钴干预,高渗低氧组采用体积分数1%高渗母液和100μmol/L氯化钴联合干预,培养1周后,使用体积分数2%高渗母液和200μmol/L氯化钴作为应激条件分别对预处理骨髓间充质干细胞和未处理骨髓间充质干细胞进行刺激24 h,以RT-PCR检测caspase-3的表达评估细胞凋亡情况;(2)动物体内实验:将SD大鼠分为3组,对照组建立椎间盘退变模型大鼠,细胞移植组造模后骨髓间充质干细胞移植治疗,高渗低氧治疗组造模后采用高渗低氧预处理后的骨髓间充质干细胞治疗,细胞分别移植至大鼠针刺椎间盘退变模型的椎间盘中,2周后取材,以免疫组织化学染色鉴定细胞分布情况、RT-PCR检测相关基因表达情况,评估预处理对于干细胞治疗效果的影响。结果与结论:(1)细胞体外实验:经预处理的骨髓间充质干细胞的caspase-3 mRNA的表达相比未经处理的对照组明显降低(P<0.05);(2)动物体内实验:相较于对照组,细胞移植组椎间盘caspase-3、白细胞介素1β以及多项椎间盘退行性指标的mRNA表达量均呈现下降趋势,高渗低氧治疗组的效果更佳(P<0.05);(3)结果证实:骨髓间充质干细胞具有治疗椎间盘退变的潜力,且经高渗低氧预处理后对高渗低氧应激的适应性增强,移植效果更佳。BACKGROUND： Stem cell therapy has been used for prevention and treatment of degenerative disc disease. Considering the special microenvironment in the intervertebral disc, the survival rate and differentiation ability of transplanted cells are decreased, which may lead to the poor efficacy of stem cell therapy. How to improve the survival ability and therapeutic effect of the transplanted cells is the focus of stem cell therapy for degenerative disc disease. OBJECTIVE： To investigate the effects of cobalt chloride combined with hypertonic solution pretreatment on bone marrow mesenchymal stem cells that will be transplanted for treatment of degenerative disc disease. METHODS：（1） In vitro cell experiment： bone marrow mesenchymal stem cells were divided into three groups and subjected to normal culture medium（normal control group）, 1% hypertonic mother solution（hypertonic group）, 100 μmol/L cobalt chloride（hypoxia group）, or 1% hypertonic mother solution plus 100 μmol/L cobalt chloride（combined group） for 1 week. Then, 2% hypertonic solution and 200 μmol/L cobalt chloride cobalt chloride were used to simulate the anaerobic and hypertonic environment intervenes in pretreated and untreated bone marrow mesenchymal stem cells for 24 hours. After that, RT-PCR was used to detect the expression of caspase-3 for apoptosis evaluation.（2） In vivo animal experiment： Sprague-Dawley rats were divided into model, cell transplantation and hypertonic plus hypoxic groups. Rat models of intervertebral disc degeneration were made in these three groups. After modeling, rats in these three groups were given no treatment, bone marrow mesenchymal stem cell transplantation or transplantation of bone marrow mesenchymal stem cells which were subjected to hypertonic and hypoxia pretreatments into the intervertebral disc. Two weeks later, immunohistochemistry and RT-PCR methods were used to detect cell distribution and related gene expression, respectively, thereby to evaluate the therapeutic effect of stem

关 键 词：干细胞 移植 椎间盘退变 干细胞治疗 骨髓间充质干细胞 高渗 低氧 增殖活性 凋亡 基质金属蛋白酶3 基质金属蛋白酶13 caspase-3 上海市自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]