siRNA慢病毒载体介导DKK-1基因沉默对骨髓间充质干细胞分化影响的实验研究

Effect of DKK-DKK-1 gene scilence mediated by siRNA lentivirus vector on the differentiation of bone marrow mesenchymal stem cells

背景:激素性股骨头坏死(SONFH)发病机制尚不完全明确,可能与骨髓间充质干细胞(BMSC)的异常分化密切相关。目的:研究si RNA慢病毒载体介导的DKK-1基因沉默,通过调控Wnt/β-catenin信号通路对超生理剂量糖皮质激素作用下大鼠BMSC分化的影响,探索治疗SONFH的新途径。方法:提取、培养SD大鼠BMSC,并构建DKK-1基因慢病毒干扰载体。大鼠第3代BMSC分4组:对照组(A组)、激素组(B组)、激素+无序序列慢病毒载体组(C组)、激素+DKK-1基因慢病毒干扰载体组(D组)。A组用基础培养基培养,B组加入浓度为1×10^(-6)mol/L的地塞米松,C、D组除加入地塞米松外,各自加入相应的慢病毒载体转染BMSC。14 d后提取各组细胞RNA及蛋白,对DKK-1、成脂相关基因PPAR-γ2和成骨相关基因RUNX2进行Real-time PCR及Western-blot检测,并对各组进行油红O、茜素红染色,观察各组BMSC分化情况,对结果进行统计学分析。结果:Real-time PCR及Western-blot检测结果示B、C组与A、D组相比,成骨相关基因RUNX2的表达明显较低(P<0.05),而DKK-1及成脂相关基因PPAR-γ2的表达明显较高(P<0.05)。油红O染色结果显示B、C组呈阳性,A、D组呈阴性;茜素红染色结果显示A、D组呈阳性,B、C组呈阴性。结论:DKK-1基因沉默通过特异性抑制DKK-1过表达而重新激活Wnt/β-catenin信号通路,减少成脂基因PPAR-γ2的表达、增加成骨基因RUNX2的表达,抑制BMSC成脂分化,促进BMSC成骨分化。

Background： The abnormal differentiation of bone marrow mesenchymal stem cells （BMSC） plays an important role in the pathogenesis of steroid-induced osteonecrosis of femoral head （SONFH）. Studies showed that Wnt/βcatenin path- way could modulate the differentiation of BMSC. Objective： To investigate whether DKK-1 could regulate the differentiation of BMSC through Writ/β-catenin pathway in SONFH. Methods： BMSCs were isolated from rats. Lenfivirus siRNA interfer- ence vector ofDKK-1 gene was constructed. The third-generation of rat BMSCs were divided into 4 groups： control, glucocor- ticoid （GC, dexamethasone： 1 × 10^-6mol/L）, GC＋lentiviral vector empty sequence （ES） and GC＋lentiviral vector-DKK- 1-RNA interference （LDRi）, and were stimulated for 14 d. DKK- 1, PPAR-y, RUNX2 mRNA and protein expression were determined by RT-PCR and western blot, respectively. The differentiation of BMSCs was observed by oil red O stain and chinalizarin stain. Results： RUNX2 expression was suppressed and the expression of DKK-1 and PPAR-72 were up-regulated by dexameth- asone in mRNA and protein levels. RNAi targeting DKK- 1 significantly enhanced RUNX2 expression and inhibited the ex- pression of DKK-1 and PPAR-y2. Oil red O staining results showed that LDRi could decrease the adipogenic differentiation in BMSC induced by dexamethasone. In addition, we also observed that dexamethasone reduced osteogenic differentiation and LDRi could up-regulated the differentiation observed by chinalizarin staining. Conclusions： DKK- 1 gene-silence can reacti- vate Wnt/β-catenin signal pathway through specially inhibiting DKK- 1 overexpression, reduce the expression of PPAR-γ2, pro- mote the expression of RUNX2, inhibit the adipogenic differentiation of BMSC and accelerate the osteogenic differentiation.