目的:探讨组蛋白去乙酰化酶8(histone deacetylase 8,HDAC8)对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的影响。方法:通过全骨髓贴壁法体外分离培养大鼠 BMSCs,转染 HDAC8过表达慢病毒载体,并于矿化诱导液中培养7 d后,实时荧光定量PCR、Western blot检测BMSCs成骨分化能力的变化;矿化诱导14 d后,茜素红钙结节染色检测BMSCs矿化能力的变化。组蛋白去乙酰化酶抑制剂---曲古抑菌素A(trichostatin A,TSA)刺激转染HDAC8过表达慢病毒载体的 BMSCs,于矿化诱导液中培养7 d后,实时荧光定量 PCR及 Western blot检测 TSA刺激前后过表达 HDAC8的BMSCs成骨分化能力的变化。结果:HDAC8过表达组经矿化诱导7 d 后,成骨分化相关基因 mRNA、蛋白表达水平均低于空白对照组;经14 d矿化诱导后,HDAC8过表达组茜素红钙结节的着色程度及范围低于空白对照组。TSA 刺激的 HDAC8过表达组矿化诱导7 d后,成骨分化相关基因 mRNA及蛋白的表达均高于未加刺激组(P<0.05)。结论:HDAC8对大鼠 BMSCs成骨分化具有抑制作用。
Objective:To evaluate the effect of histone deacetylase 8 (HDAC8)on osteoblast differentiation of rat bone marrow mes-enchymal stem cells (BMSCs).Methods:Rat BMSCs were isolated and cultured by the method of whole bone marrow adherent and transfected with HDAC8 overexpression lentiviral vector.Real-time PCR,Western blot were applied to estimate the change of osteogen-ic capacity after 7 days of osteogenic induction and Alizarin red stain was used to estimate the mineralization capacity after 1 4 days of osteogenic induction.Trichostatin A (TSA),one kind of histone deacetylase inhibitor,acted on BMSCs with HDAC8 overexpression and the change of osteogenic capacity of cells after 7 days of osteogenic induction was estimated by real-time PCR and Western blot. Results:For BMSCs with HDAC8 overexpression,the expression of osteogenesis-related genes at mRNA and protein levels were all low-er than those in control group after 7 days of ostogenic induction and the capacity to form calcified nodules was lower than that of the control group after 1 4 days of ostogenic induction.The osteogenesis-related genes expression of BMSCs with HDAC8 overexpression which was treated with TSA were all higher than those in untreated group after 7 days of ostogenic induction (P〈0.05).Conclusions:HDAC8 could suppress osteogenic differentiation of rat BMSCs.