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

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（0.25~1.0μmol/L）显著降低COL-ⅡmRNA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。

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 0.25~1.0μmol/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-Ⅱexpression 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-Ⅱthrough down-regulation of Wnt-5a and SOX-9, which might play an important role in maintaining chondrocyte phenotype in vitro.