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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 To explore the effect of histone deacetylation on the chondrocyte phenotype-related genes through TSA inhibition of histone deacetylation and to provide a new idea for the maintenance of chondrocyte phenotype from epigenetic perspective. Methods Human chondrocytes were cultured in vitro. The chondrocyte decellularization model was established. The cells were collected at different time points and stimulated with different concentrations of TSA. Total RNA was extracted and the expression of Wnt-5a, SOX-9, COL-Ⅱ, COL -I mRNA expression. The expression of COL-Ⅱprotein was detected by immunofluorescence and Westerblot. Results Compared with the control group, TSA (0.25 ~ 1.0μmol / L) significantly decreased the expression of COL-ⅡmRNA and protein, and increased the expression of Wnt-5a and SOX-9 mRNA. -II inhibitory effect. Conclusion TSA inhibits the expression of COL-Ⅱ by activating Wnt-5a and SOX-9, leading to the loss of chondrocyte phenotype. Thus, histone deacetylation enhances the expression of COL-II by decreasing Wnt-5a and SOX-9, thereby maintaining the chondrocyte phenotype.