全反式视黄酸抑制大鼠骨髓间充质干细胞成骨分化及其机制

All trans retinoic acid inhibits osteogenic differentiation of rat bone marrow mesenchymal stem cells

目的:探索全反式视黄酸(all-trans retinoic acid,at-RA)对大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSCs)成骨分化的影响及其机制。方法:成骨诱导培养基诱导rBMSCs,加入不同浓度(0.1、1.0、5.0μmol/L)at-RA,诱导第7天用组织化学染色法和检测试剂盒检测各组碱性磷酸酶(alkaline phosphatase,ALP)活性,第21天用茜素红染色后倒置显微镜观察并经提取法后检测钙盐沉积。于诱导第7、14、21天采用real-time PCR检测骨钙素(osteocalcin,OCN)、骨桥素(osteopontin,OPN),在上述时间点的基础上,再增加第1、3天2个时间点检测Runx2、Osterix以及视黄酸受体RARs(α、β)mRNA表达。结果:rBMSCs在成骨培养基诱导下经形态和茜素红染色观察证实能分化为成骨细胞,加入at-RA后培养7 d,ALP活性较对照组增加(F=107.177,P=0.000),与对照组比较,at-RA为1.0、5.0μmol/L 2组明显升高(P<0.05)。然而经茜素红染色测定,ALP升高组其钙盐沉积与对照组比较反而减少(F=57.554,P=0.000)。选at-RA浓度5.0μmol/L做real-time PCR检测,与对照组相比在上述3个时间点OCN(F=211.145,P=0.000)、OPN(F=30.835,P=0.005)表达均降低,Runx2(F=106.536,P=0.000)、Osterix(F=452.546,P=0.000)、RARα(F=253.159,P=0.000)的表达在第21天均下降,除Osterix表现为全程下调外,其他2个基因也在多个时间点有统计学差异;而RARβ表达则全程明显上升(F=2 294.377,P=0.000)。结论:at-RA抑制rBMSCs成骨分化,其机制可能与RARα的下调和RARβ的上调,进而影响Runx2、Osterix的表达有关。

Objective：To explore the effects of all trans retinoic acid（at-RA）on rat bone marrow mesenchymal stem cells（rBMSCs）osteogenic differentiation and mechanisms. Methods：The rBMSCs were isolated and induced to differentiate into osteoblast. At-RA at concentrations of 0.1,1.0,5.0 μmol/L was added to the osteoblast induced medium. Morphology of the cultured cells was observed using inverted microscope respectively. On the 7th d,histochemical staining and alkaline phosphatase detection kit were used to test the alkaline phosphatase（ALP）activity. On the 21 st d,alizarin red staining was employed to detect calcium salt deposition. During osteogenetic induction,mRNA expressions of both osteocalcin（OCN）and osteopontin（OPN）,biomarkers of osteoblast and related differentiation regulatory genes,i.e. Runx2,osterix of the osteogenesis regulation pathways and retinoic acid receptors（RARs,α,β）of the retinoic acid signaling pathways were measured using real-time PCR. Results：On the 7th d of differentiation,the activity of ALP of osteogenic induced rBMSCs with at-RA cells increased markedly compared with that without at-RA cells（F=107.177,P=0.000）. However,on the 21 st d,the calcium salt deposition of at-RA treated rBMSCs significantly reduced compared with that of non-treated rBMSCs（F=57.554,P=0.000）. During osteogenic induction process,OCN,OPN,Runx2,Osterix and RARα expressions of at-RA treated（5.0 μmol/L）rBMSCs decreased compared with those of non-treated rBMSCs（211.145,0.000;30.835,0.005;106.536,0.000;452.546,0.000;253.159,0.000,respectively for F and P values）on the 21 st d. Meanwhile,significantly increased RARβ expression was observed（F=2 294.377,P=0.000）during the whole differentiation process. Conclusion：at-RA can inhibit osteogenic differentiation of rBMSCs.The mechanism may be through up-regulating the expression of RARβ,down-regulating the expression of RARα,and inhibiting the expressions of Runx2 and Osterix.