shRNA干扰PPARγ基因表达预防兔激素性股骨头坏死的研究

摘要

Objective To investigate the pathogenesis of steroid-induced avascular necrosis of the femoral head (SANFH), and explore the preventive effect of shRNA targeting PPARγ gene on SANFH in rabbit models.Methods Forty-eight healthy New Zealand rabbits were randomly into the four groups including Group M treated with dexamethasone only, Group S with dexamethasone and a recombinant adenovirus vector carrying shRNA targeting PPARγ gene and infecting the rabbits' bone marrow stromal cells (BMSCs), Group E with dexamethasone and a vector carrying irrelative sequence of shRNA adenovirus vector infecting BMSCs, and Group N with Saline as the control group.The levels of serum triglycerides (TG) and cholesterol (CHO) in the four groups were detected and the pathological changes of femoral head were observed.RT-PCR was used to assay the expression levels of PPARγ mRNA and Runx2 mRNA, and the immunohistochemistry was performed to detect the expression of PPARγ and Runx2.Results The contents of serum TG and CHO in Groups M, E and S increased, and they were significantly different from those in Group N (P<0.05).Both Group M and Group E showed obvious osteonecrosis and changes such as adipocyte proliferation and hypertrophy, thinner and sparse trabeculae, and plenty of empty osteocyte lacunae, while the osteonecrosis and changes were not found in Group S and Group N.The expression levels of PPARγ mRNA and protein were low while the expression levels of Runx2 mRNA and protein were high in Group S.The expression levels of Group S were significantly different from those of Group M and Group E respectively (P<0.05), while they were not significantly different from those of Group N (P>0.05).Conclusion The adenovirus vectors shRNA targeting PPARγ gene can effectively inhibit the expression of PPARγ mRNA in BMSCs and the adipogenic differentiation, promote osteogenic differentiation of BMSCs, and finally prevent steroid-induced osteonecrosis in rabbits, which may provide a novel approach to the treatment of steroid-induced osteonecrosis by means of gene targeting therapy.%目的 研究激素性股骨头坏死(steroid-induced avascular necrosis of the femoral head,SANFH)的发病机制,观察靶向过氧化物酶体增殖激活受体γ(PPARγ)基因shRNA腺病毒载体预防兔SANFH的作用效果.方法 健康新西兰兔48只,随机分为4组,每组12只.单纯激素组(M组)仅肌注地塞米松;实验组(S组)在肌注地塞米松同时,加用靶向PPARγ基因shRNA腺病毒载体感染兔活体股骨头内骨髓基质干细胞(bone marrow stromal cells,BMSCs);无关序列组(E组)在肌注地塞米松同时,加用搭载无关序列的shRNA重组腺病毒感染兔活体股骨头内BMSCs;对照组(N组)肌注等量生理盐水.测定4组兔血清甘油三酯(TG)、胆固醇(CHO)含量.观察股骨头组织病理学改变.应用RT-PCR 技术检测PPARγ mRNA和Runx2 mRNA表达;应用免疫组化技术检测PPARγ和Runx2蛋白表达情况.结果 M、E、S组血清中TG和CHO含量增高,与N组比较差异均有统计学意义(P<0.05).M组和E组可见明显骨坏死,脂肪细胞增生肥大,骨小梁变细和稀疏,大量空骨陷凹,S组和N组未见骨坏死及上述改变.S组中PPARγ mRNA和蛋白均呈低表达,Runx2 mRNA及蛋白呈高表达,与M组和E组比较,差异有统计学意义(P<0.05);与N组比较,差异无统计学意义(P>0.05).结论 靶向PPARγ基因shRNA腺病毒载体可有效阻断BMSCs内PPARγ mRNA表达,抑制其成脂分化;增强Runx2 mRNA表达,促进其成骨分化,预防SANFH的发生,为通过基因靶向治疗SANFH提供新思路.