SOST/sclerostin在骨细胞参与假体周围骨溶解过程中的作用和机制研究

Periprosthetic osteolysis mediated by wear debris is the principal cause of aseptic loosening which is the main complication of joint arthroplasty. However, there is still no effective way to prevention and treatment of prosthetic loosening. Recently, some studies showed that SOST gene and sclerostin, the protein product of SOST， were associated with sclerosteosis, fracture healing and osteoporosis. SOST/sclerostin inhibited bone formation and increased bone resorption through depression of osteoblast activity and promotion of osteoclast activity. Osteocyte which express SOST and sclerostin exclusively in the adult skeleton played an important role of regulation bone remodeling. Our latest research found osteocyte, SOST and sclerostin were concerned with periprosthetic osteolysis. But the mechanism has not been clearly clarified. Based on our previous study of osteolysis mechanism and the latest reports, we have a hypothesis that SOST gene and sclerostin play an important role in the effect of osteocyte on periprosthetic osteolysis. To test the hypothesis, we will plan to build the osteolysis model of SD rats and detect the expression of SOST and sclerostin. Then treating rats with sclerostin antibody, new bone formation and periprosthetic osteointergration will be assessed by using histomorphology, Micro-CT, mechanical pull-out test, and so on. Osteocytes will be cultivated with different concentration of particulates titanium alloy. cell proliferation, apoptosis, gene and protein expression of OPG/RANKL, SOST and sclerostin , the characteristic marker and inflammatory cytokine such as IL-1, TNF, MMP-2 will be evaluated or measured by Brdu assey, flow cytometry, real-time PCR, Western Blot and Elisa kit respectively. After downregulation SOST expression by RNA interference, the change of osteocyte will be detected again. Furthermore, osteocyte with siRNA-SOST gene will be co-cultured with osteoblast and osteoclast respectively. Then the characteristic marker will be detected to evaluate the anabolic state of osteoblast and the catabolic state of osteoclast. The effect of SOST and sclerostin on OPG/RANKL signaling pathways also will be examined. Through a series of experiments, we want to confirm our hypothesis and explict the role of SOST and sclerostin on osteocyte regulating osteoblast and osteoclast in the process of periprosthetic osteolysis. Therefore the research may provide a scientific basis for SOST/sclerostin as a target for prevention and cure of periprosthetic osteolysis and joint loosening.

假体周围骨溶解所致的人工关节无菌性松动尚缺乏有效防治手段。近年研究证明：SOST 基因及其蛋白sclerostin是骨细胞参与骨重建，调控成骨和破骨细胞的关键分子。预试验发现骨细胞、SOST/sclerostin 与假体周围骨溶解存在一定联系，但其机制不明确。在既往骨溶解机制研究基础上。拟通过：1）已构建的鼠骨溶解模型，检测SOST的表达，经sclerostin 抗体阻断，观察假体周围骨整合的变化。2）在磨损颗粒条件下，观察离体培养骨细胞功能的变化和SOST的表达，靶向干扰SOST后，再次观察其变化。并将SOST 干扰前后的骨细胞分别与成骨和破骨细胞共培养，观察其对成骨和破骨特征性标记物和OPG/RANKL信号通路的影响。以此探索骨细胞通过SOST/sclerostin调节成骨细胞和破骨细胞功能，参与假体周围骨溶解的机制，为开辟关节无菌性松动的靶向治疗提供理论依据。

SOST 基因及其蛋白sclerostin 是骨细胞参与骨重建，调控成骨和破骨细胞的关键分子。本课题研究了SOST/sclerostin在骨细胞参与假体周围骨溶解过程中的机制。1）通过构建的小鼠颅骨骨溶解模型，检测sclerostin的表达，继而颅骨表面局部注射病毒转染SOST-iRNA，观察颅骨骨溶解模型周围的的变化。研究中发现，SOST阻断后，形态学上观察到颅骨表面骨溶解小凹数量明显减少，免疫/病理学检测发现反应骨吸收的指标被抑制，骨形成的指标增加。证实了SOST阻断抑制骨溶解的有效性。2）在细胞离体培养中，首先检测了钛颗粒对骨细胞增殖、凋亡、Wnt信号通路蛋白和SOST的表达的影响。发现钛颗粒干预骨细胞后，细胞的增殖减少，凋亡增加，β-catenin表达减少，SOST表达明显增加。将受钛颗粒干预的骨细胞分别于成骨细胞和破骨细胞共培养，发现共培养条件下，随着钛颗粒浓度越高，成骨细胞的功能受到抑制（碱性磷酸酶活性降低，细胞外矿化减少等），而破骨细胞的功能变得活跃（TRAP染色阳性增多，吸收小凹陷增多，OPG/RANKL比率下降等）。病毒转染敲低SOST表达后，β-catenin表达恢复，但是细胞的增殖和凋亡没有改变。将SOST敲低后的骨细胞再次分别与成骨和破骨细胞共培养，成骨细胞的成骨功能有了部分恢复，破骨细胞的骨吸收功能下降，OPG/RANKL的比率被逆转。通过此研究，证实了假体周围骨溶解过程中，磨损颗粒可以影响骨细胞的功能，继而骨细胞可以通过Wnt信号通路中的SOST/sclerostin 调节成骨细胞和破骨细胞功能，参与假体周围骨溶解。该研究从另一视角探索了假体周围骨溶解机制，同时将sclerostin作为新型药物用于治疗关节无菌性松动提供了理论依据。