梅花鹿鹿茸多肽调控骨关节炎软骨细胞TGF-β信号通路的物质基础和分子机制研究

Osteoarthritis is a chronic joint disease of pathological decompensation in articular cartilage, the increasing incidence of which is quite common in elderly people. Velvet antler is a renewable bone tissue, which is totally unique in mammalian. The total polypeptides extracted from fresh Cervus nippon antler could significantly accelerated fracture callus formation in rats, shorten the healing time, ameliorated the biomechanical properties of injured parts, and promoted the proliferation of chondrocytes. However, the exact molecular regulatory mechanism remains unclear. Our previous study demonstrated that total polypeptides had the repairable and protectable capability to rescue osteoarthritis chondrocytes injury. Further research revealed that the isolated nVAP monomer had effected on TGF-β signaling pathway . To identify the effect targets we present experiments including the yeast two-hybrid to elucidate the binding sites between nVAP and TGF-β1/TGF-β2; also nVAP and TβRI-ED/TβRII-ED. Observe the expression of downstream molecule Smad2/3 and the cell matrix degradation. Interpretate the dual regulation effects of nVAP in promoting the proliferation of cartilage cells and the inhibition the formation in osteophyte. Meanwhile, to provide new targets for gene therapy of osteoarthritis, we would detect the differential expression of TGF-β gene after effected by nVAP, and screen the changes of the microRNA who involved in the regulation. Preliminary exploration on the nVAP’s effect mechnism, could establish the theoretical and experimental basis of the modernization of traditional Chinese medicine resources.

骨性关节炎是一种以关节软骨代偿失调为病理特征的慢性关节病，常见于老年人且发病率逐年上升。鹿茸是哺乳动物中唯一可再生的骨类组织，从梅花鹿鹿茸中提取的多肽明显加速骨折大鼠骨痂形成，缩短愈合时间，促进软骨细胞增殖，但精确作用机制尚不清晰。本研究发现，鹿茸多肽主要成分nVAP单体对骨性关节炎软骨细胞的损伤有修复与保护作用，可能与影响TGF-β信号通路转导有关。为探讨nVAP单体调控机制，一方面采用酵母双杂交等方法，明确TGF-β及其受体胞外区与nVAP单体作用靶点，观察通路下游分子Smad2/3表达及细胞基质代谢情况，从而阐释nVAP促进软骨细胞增殖和抑制骨赘形成的双重调控效应，为鹿茸的老药新用提供理论支持。另一方面测定nVAP单体作用下MSC和软骨细胞TGF-β相关基因差异表达，筛选参与调控的microRNA，为骨性关节炎的基因治疗提供实验依据。本研究亦可为鹿茸的中药现代化应用提供新思路。

骨性关节炎是是一种严重影响患者生活质量的关节退行性疾病，以关节软骨代偿失调为病理特征，常见于老年人且发病率逐年上升。从梅花鹿鹿茸中提取的多肽明显加速骨折大鼠骨痂形成，缩短愈合时间，本研究通过western blot，qPCR，茜素红染色等方法表明，鹿茸多肽能够通过调控TGF-β信号通路，上调TGF-β1，Smad1，2，3等蛋白显著促进脂肪间充质干细胞的增殖，促进间充质干细胞产生矿化结节，诱导其成骨转化，促进骨损伤的愈合。骨肿瘤原发于骨骼及其附属组织，是一种高危性肿瘤疾病，具有较高的致残率、致死率和复发率，常见于青少年和老年。原发恶性骨肿瘤中骨肉瘤和软骨肉瘤占比过半，其余为纤维肉瘤、骨髓瘤、尤文肉瘤、恶性骨巨细胞瘤等。鹿茸是哺乳类动物唯一可再生的骨类组织，但其增殖迅速而不发生癌变的机制尚未明确。以鹿茸多肽干预骨肉瘤MG-63和U2OS细胞，发现从2.5μg/ml开始鹿茸多肽能够显著抑制细胞增殖，并且随着浓度升高和时间延长更加明显；细胞划痕实验发现鹿茸多肽能够抑制MG-63和U2OS细胞的迁移，也呈剂量依赖，即随着浓度的升高，抑制效果增强。通过western blot和qPCR验证发现鹿茸多肽能够下调MG-63细胞中TGF-β1表达，有趣的是上调U2OS细胞中TGF-β1的表达，同时还发现鹿茸多肽能够上调E-cadherin的表达，抑制MG-63细胞中EMT过程的发生，促进BCL-2的表达，抑制MG-63细胞的凋亡，下调U2OS细胞中E-cadherin的表达，促进EMT过程的发生，并调BAX的表达，促进U2OS细胞的凋亡过程。众所周知，TGF-β1在肿瘤细胞中作为主要调控因子能够促进肿瘤细胞的EMT过程，同时也能促进肿瘤细胞的凋亡，本项目的研究结果进一步验证了上述规律，表明鹿茸多肽对MG-63和U2OS细胞的干预主要是通过调控TGF-β1信号通路来实现的。