组蛋白去乙酰化调控软骨细胞Ⅱ型胶原表达的机理研究

doi:10.13418/j.issn.1001-165x.2015.06.011

中国临床解剖学杂志 ›› 2015, Vol. 33 ›› Issue (6): 667-671.doi: 10.13418/j.issn.1001-165x.2015.06.011

组蛋白去乙酰化调控软骨细胞Ⅱ型胶原表达的机理研究

王光辉¹，　段莉²，　梁宇杰³，　费志强⁴，　王大平²

1.广州医科大学，广州 510182；　2.深圳市第二人民医院，深圳 518035；　3.北京大学深圳研究生院，深圳 518035
4. 香港大学深圳医院，深圳 518035

收稿日期:2015-04-12 出版日期:2015-11-25 发布日期:2015-12-18
通讯作者: 王大平，主任医师，医学博士，教授、博士生导师，E-mail: dapingwang1963@qq.com
作者简介:王光辉(1986-)，男，在读硕士，医师，研究方向：骨组织工程，创伤与运动医学，Tel： l3823207720， E-mail：873840741@qq.com
基金资助:
广东省自然科学基金（S2012010008129）; 广东省医学科研基金（b2013051）;深圳市科技创新委技术攻关项目（JSGG20140519 05550503）; 深圳市科技创新委国际科技合作项目（GJHZ201304121593 06739）;深圳市重点实验室提升项目(CXB201104220049A); 深圳市科技研发资金项目(CXZZ20120614160 234842, ZDSY20120614154551201); 中国博士后科学基金面上项目（2013M530385）

An in vitro study on the effect of histone deacetylation on type Ⅱ collage expression

WANG Guang-hui¹, DUAN Li², LIANG Yu-jie³, FEI Zhi-qiang⁴, WANG Da-ping²

1. Guangzhou Medical University，Guangzhou, 510l82, China；2. Department of Orthopedics，the Second People's Hospital，Shenzhen 518035，China；3. Peking University Shenzhen Graduate School，Shenzhen 518035，China; 4. The University of Hong Kong-Shenzhen Hospital, Shenzhen 518035, China

Received:2015-04-12 Online:2015-11-25 Published:2015-12-18

摘要/Abstract

摘要：

目的　探讨通过TSA抑制组蛋白去乙酰化水平，研究组蛋白去乙酰化对软骨细胞表型相关基因的影响及其机制，从表观遗传学角度为维持软骨细胞表型提供新的思路。方法体外培养人关节软骨细胞，建立软骨细胞去分化模型，采用不同浓度TSA刺激，在不同时间点收集细胞，提取总RNA，利用qRT-PCR检测Wnt-5a、SOX-9、COL-Ⅱ、COL-Ⅰ的mRNA表达情况。利用免疫荧光及Westerblot进行COL-Ⅱ蛋白表达水平的检测。结果　与对照组比较，TSA（0.25~1.0 μmol/L）显著降低COL-Ⅱ mRNA表达水平及蛋白表达水平，升高Wnt-5a 和SOX-9 mRNA表达水平；抑制Wnt-5a信号通路减弱TSA 对COL-Ⅱ的抑制效应。结论 TSA通过激活Wnt-5a和 SOX-9从而抑制COL-Ⅱ的表达，导致软骨细胞表型丧失。因此，组蛋白去乙酰化通过降低Wnt-5a和SOX-9，升高COL-Ⅱ的表达水平，发挥维持软骨细胞表型的作用。

关键词: 组蛋白去乙酰化, TSA, 软骨细胞

Abstract:

Objective　In the present study, we inhibited histone deacetylation of the chondrocytes by the treatment of TSA, aiming at investigating the effects of histone deacetylation on chondrocyte phenotype and related gene expression and the mechanisms，aiming at providing a new strategy to maintain the phenotype of chondrocytes from the perspective of epigenetics.　Methods　Human articular chondrocytes were cultured in vitro and a model of chondrocyte dedifferentiation was established first. Different concentrations of TSA were used for the stimulation and cells were collected at different time points for the extraction of total RNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for the detection of the expression of Wnt-5a, collagen typeⅠ(TypeⅠcollagen, COL-Ⅰ), collagen typeⅡ(TypeⅡcollagen, COL-Ⅱ) and SOX-9. Immunofluorescence and western blot were used to detect the protein expression of COL-Ⅱ.　 Results　Comparing with control group, a concentration of 0.25~1.0 μmol/L TSA was sufficient to block protein and mRNA levels of typeⅠcollagen and typeⅡcollagen expression in primary culture chondrocytes, while it could promote the expression of Wnt-5a and SOX-9. Conclusions Inhibition of COL-Ⅱ expression which leads to the dedifferentiation of chondrocytes and the phenotype change might be mediated by the up-regulation of Wnt-5a and SOX-9. Therefore, histone deacetylation may elevate the expression level of COL-Ⅱ through down-regulation of Wnt-5a and SOX-9, which might play an important role in maintaining chondrocyte phenotype in vitro.

Key words: HDAC inhibition, TSA, Chondrocyte

王光辉,段莉,梁宇杰,费志强,王大平. 组蛋白去乙酰化调控软骨细胞Ⅱ型胶原表达的机理研究[J]. 中国临床解剖学杂志, 2015, 33(6): 667-671.

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组蛋白去乙酰化调控软骨细胞Ⅱ型胶原表达的机理研究

An in vitro study on the effect of histone deacetylation on type Ⅱ collage expression

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