动态压应力下软骨-软骨下骨间TGFβ分子信号交流的机制及其在骨关节炎进展中的作用

Osteoarthritis（OA） is a progressive disease, which has been characterized mainly by not only damaged articular cartilage, but also changes in subchondral bone. Currently no efficacious medical intervention is yet available to control the progression of this disease. Our previous research proved that there is abnormal biologic signaling crosstalk (or interaction) between osteoblasts of subchondral bone and articular chondrocytes from human knee OA. These crosstalks between subchondral bone and cartilage could be responsible for disease cycle between subchondral bone and cartilage. And it may be one of the central players in the initiation, long-term maintenance and progression of OA, and then result in the difficult therapy of OA. Sclerotic osteoblasts separated from subchondral bone of knee OA patients show an increased TGF-β expression, which has ability to promote the degeneration of chondrocytes and may play a role in regulating this signaling crosstalk between subchondral bone and cartilage..In future, when the abnormal expressed TGFβfactors was added and the abnormal expressed TGFβ signal way were obstructed between subchondral bone and cartilage respectively，we prepare to observe the whole disease progression of animal knee OA，and construct a co-culture model including subchondral bone and cartilage (osteoblast and chondrocyte) under compressure force in order to study the signaling crosstalk between subchondral bone and cartilage from different stages of disease and parts of lesions. .We would detect the pathology of cartilage, morphological structure changes of subchondral bone, bone remodeling, mineralization, the angiogenesis between the bottom of cartilage and the underlying subchondral bone plate, and articular function in various indicated time point by microCT, electron microscope, immunohistochemistry et al. It would also be tested that altered phenotype of subchondral bone and cartilage, as well as TGFβ- ERK，TGFβ-Wnt and TGFβ-NF-kB signaling pathway and the phosphorylation, acetylation and activities of the related proteins with molecular biological technique, such as Westernblotting、ELISA、RT－PCR. With this research, we expected to elucidate the mechanism of abnormal TGFβ crosstalk, and its role in osteoarthritis progression systematically.After that, the possibility would be further studied on drug efficacy testing by a co-culture model including subchondral bone and cartilage (osteoblast and chondrocyte) under dynamic compressure force. Finally, The patho-role and patho-mechanism of abnormal TGFβ crosstalk between subchondral bone and cartilage from OA patients under compressure force would be studied. With this research, it would provide both theory and method basis for guiding clinical intervention of OA progression, and new drug research and development.

骨关节炎（OA）以进行性的软骨下骨和软骨（BAC）共同病变为主要特征，目前的治疗尚不能完全延缓疾病进展。我们前期研究证实，压应力下人OA的BAC细胞间存在信息交流，TGFβ发挥潜在调节作用；该交流可能会使病变恶性循环，导致OA进展和难以治疗。本研究通过在BAC间添加TGFβ因子，以及用RNAi阻断TGFβ异常信号，对动物膝OA进行全程观察；并结合体外动态压力下细胞/组织的共培养，研究不同病变阶段和部位BAC间信号交流。采用microCT、电镜、免疫组化等方法，观察血管生成、软骨结构损伤、骨重塑、骨矿化和关节功能等改变；采用分子生物学技术，检测BAC表型及同TGFβ有关的ERK、Wnt和NF-kB通路中蛋白磷酸化和乙酰化，系统阐明该信号交流在OA进展中的作用及机制。以此为基础，应用BAC的共培养系统检测药物疗效，并验证人OA中该交流的机制与作用，为临床干预OA进展和新药研发提供理论和方法支持

本课题对原有的压力器进行升级改造，并构建了新的更稳定的细胞压力器和组织压力器。通过在共培养系统中，阻断骨-软骨细胞间TGFβ分子信号，采用microCT、电镜、免疫组化、染色、WESTERN-BLOT、RT-PCR等生物标记检测，证实在动态压应力作用下，兔子的骨关节炎模型细胞病变情况和骨关节炎患者病变的情况存在相似性。压应力作用下，骨关节炎中MAPKs信号通路参与着成骨细胞和软骨细胞之间的生物学交流，是软骨下骨成骨细胞促进软骨细胞退变的重要因素。OA 软骨下骨的成骨细胞在一定的压力下，高表达促进基质合成的因子TGFβ 1，能促进MAPKs 信号表达，且能进一步促进IL6 的表达。MAPKs信号通路可能是异常表达的细胞因子（TGFβ 1和IL6）之间级联通路，进而促进着骨关节炎病变的发生、发展过程。干扰TGFβ 1和其信号通路的表达，对于改善骨关节炎的病变，是一项有价值的潜在治疗靶点。收集关节炎术后患者标本，术前对患者行临床功能评分、和健康生活质量评估，同该患者压力下共培养模型中软骨下骨成骨细胞促进软骨细胞退变的程度有相关性。本实验显示，压应力下的骨和软骨的共培养系统，对于患者遗弃的组织标本进行检测，是一种有前景的检测方法，但由于对于骨组织标本特性认识不足和目前对检测标本的评判手段的限制，尚不能使其完全应用于临床检测。因此，我们构建了正常、骨质疏松、关节炎和骨质疏松性关节炎的动物模型，对其微结构等组织压应力特性进行进一步研究，研究显示，除了骨关节炎标本骨密度最高，四种组织微结构特性存在明显区别，后期实验拟对不同组织对压应力的反应进一步研究。应用去极化电流，交流阻抗等检测方法对骨的特性进行新的评判研究。研究显示，电化学方法可能对骨特性检测是一种有前景的检测方法，后期实验拟行深一步研究。