补肾方含药血清干预大鼠椎间盘软骨细胞miRNA的差异表达  

Study on the differentially expressed miRNA of drug-contained serum of “Bushen Decoction”on rat intervertebral disc chondrocytes

作　　者：潘欣[1] 梁兴伦[2] 曾思良[1] 嵇承栋[3] 周文锐[2] 李琛[2] 朱敏洁[2] 许畅[3] 付强强[3] 万悦竹[3] 马瑜[3] 于德华[3] 

机构地区：[1]同济大学附属杨浦医院中心实验室,上海200090 [2]同济大学附属杨浦医院老年科,上海200090 [3]同济大学附属杨浦医院科研部,上海200090

出　　处：《上海中医药杂志》2016年第1期65-69,共5页Shanghai Journal of Traditional Chinese Medicine

基　　金：科技部国家重点基础研究发展计划("973"计划)资助项目(2013CB531601);国家自然科学基金资助项目(81173312;30972633);同济大学附属杨浦医院学科带头人计划资助项目(YJ1201402)

摘　　要：目的筛选补肾方含药血清干预大鼠椎间盘软骨细胞后差异表达的miRNA,为补肾方治疗颈椎病提供可供选择的目标miRNA,以探讨其通过调控靶基因表达发挥治疗效应的机制。方法原代培养的大鼠椎间盘软骨细胞经Ⅱ型胶原酶免疫荧光染色和甲苯胺蓝染色鉴定后,分别用补肾方含药血清、空白血清干预椎间盘软骨细胞,在给定时间点收获细胞,抽提纯化总RNA,行miRNA芯片检测,通过芯片差异性分析,挑选差异表达4倍以上的miRNA行定量聚合酶链式反应(PCR)验证。结果二次酶消化法可简便高效培养大量原代椎间盘软骨细胞,所培养细胞具有典型的椎间盘软骨细胞形态和功能,Ⅱ型胶原酶免疫荧光染色和甲苯胺蓝染色均为阳性。补肾方含药血清干预组芯片数据分别与相应时间点的空白血清组芯片数据行组间比较后再行组内比较,与含药血清干预椎间盘软骨细胞1 d相比,干预3 d的共有121个2倍以上表达差异的miRNA,其中上调的53个,下调的68个。差异表达4倍以上的miRNA定量PCR结果与芯片数据一致。结论补肾方含药血清干预椎间盘软骨细胞获得了一套较为特异的miRNA,初步预测上调的miRNA可能通过靶向Opn、Col、Bcl212、Vdr、IGF-I、Mmp-13等基因在椎间盘营养代谢相关信号通路中发挥作用。Objective To Study on the differentially expressed miRNA of drug-contained serum of ＂Bushen Decoction＂on rat intervertebral disc chondrocytes,which aims to provide target miRNA for treating cervical spondylosis with＂Bushen Decoction＂,and to discuss the function mechanism by regulating the target of related genes. Methods The primary rat intervertebral disc chondrocytes were identified by collagen type Ⅱ immunofluorescent staining and tuluidine blue staining. The intervertebral disc chondrocytes were cultured in serum containing ＂Bushen Decoction ＂ or blank serum respectively. After a given time of intervention,the cultured cells were collected,and the total RNA were isolated and purified and used for follow-up miRNA microarray experiments. Real-time quantitative PCR was used to further validate the expression levels over 4-fold up-regulated. Results The twice-enzyme digestion method was practicable,efficient and ideal technique to obtain a large number of purified primary rat intervertebral disc chondrocytes in a short time and culture cells with typical intervertebral disc chondrocytes characteristics. Collagen type Ⅱ immunofluorescent staining and tuluidine blue staining were positive. The miRNA expression profiles in ＂Bushen Decoction＂containing serum groups compared inner the groups after comparing with that from blank serum control subjects. Compared with drug-contained serum of＂Bushen Decoction ＂on rat intervertebral disc chondrocytes cultrured 1d,121 over 2-fold different expression of miRNA（ 53 up-regulated and 68 downregulated） were identified in serum cultured 3d. The results of PCR were in accordance with the miRNA array results that the expression levels over 4-fold upregulated. Conclusion A set of specific miRNA were obtained after＂Bushen Decoction＂containing serum intervention on rat intervertebral disc chondrocytes for3 d. Target gene prediction software to predict the up-regulated miRNA mainly by improving the nutritional metabolism of intervertebral disc through th

关 键 词：补肾方 软骨细胞 大鼠miRNA芯片 差异表达 

分 类 号：R285.5[医药卫生—中药学]