Lentiviral Delivery of PPARγ shRNA Alters the Balance of Osteogenesis and Adipogenesis, Improving Bone Microarchitecture
Lentiviral Delivery of PPARγ shRNA Alters the Balance of Osteogenesis and Adipogenesis, Improving Bone Microarchitecture
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DOI：
10.1089/ten.tea.2013.0736
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发表时间：
2014-10-01
影响因子：
4.1
通讯作者：
Soo, Chia
中科院分区：
文献类型：
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作者：
James, Aaron W.;Shen, Jia;Soo, Chia
骨骼老化不仅与基本代谢单位内成骨细胞（OB）和破骨细胞（OC）数量和活性的改变有关，还与骨髓脂肪增加有关。过氧化物酶体增殖物激活受体γ (PPAR γ)通常被认为是脂肪形成的主要转录调节因子，然而，它在成骨细胞和破骨细胞功能中也有已知的作用。在此，我们设计了PPAR γ shRNA的慢病毒递送系统，并在体外和小鼠髓内注射模型中检测了其对骨髓基质细胞（BMSC）的作用。方法：PPAR γ shRNA通过复制缺陷慢病毒载体传递，经过体外测试，以确定PPAR转录物的纯度、浓度和有效性。然后，将对照绿色荧光蛋白慢病毒或表达PPAR γ shRNA的慢病毒通过髓内注射注入雄性SCID小鼠股骨骨髓。分析包括每日监测动物健康，并在4周时进行死后分析。死后分析包括高分辨率微计算机断层扫描（microCT）重建和分析、常规组织学和组织形态学分析、定量实时聚合酶链反应分析Pparg转录物水平，以及脂肪细胞（PPAR γ、脂肪酸结合蛋白4 [FABP4]）、成骨细胞（碱性磷酸酶[ALP]、骨钙素[OCN]）和破骨细胞（抗酒石酸酸性磷酸酶[TRAP]、Cathepsin K）标志物的免疫组织化学分析。结果：在体外，PPAR γ shRNA递送显著降低小鼠BMSC中PPAR γ shRNA的表达，同时脂滴积累显著减少。在体内，注射后4周观察到成熟骨髓脂肪细胞几乎完全减少。这伴随着脂肪细胞特异性标记物的显著减少。显微ct和组织形态学分析均显示骨小梁的各项参数明显升高。通过免疫组化染色和半定量，观察到PPAR γ shRNA处理后，OCN+成骨细胞明显增加，TRAP+多核破骨细胞明显减少。讨论：这些发现表明骨髓间室中PPAR γ的急性缺失除了具有抗脂肪作用外还有重要作用。具体来说，我们发现PPAR γ shRNA治疗后具有促成骨、抗破骨作用，导致骨小梁结构改善。未来的研究将检验PPAR γ shRNA对OB和OC细胞类型的分离和直接影响，并可能有助于确定PPAR γ拮抗剂是否是骨质疏松性骨质流失的潜在治疗剂。
Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPAR gamma shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.Methods: PPAR gamma shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPAR gamma shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPAR gamma, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).Results: In vitro, PPAR gamma shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPAR gamma shRNA treatment.Discussion: These findings suggest that acute loss of PPAR gamma in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPAR gamma shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPAR gamma shRNA on OB and OC cell types, and it may help determine whether PPAR gamma antagonists are potential therapeutic agents for osteoporotic bone loss.