鲑鱼皮明胶酶解产物对成骨细胞的作用机制

The dynamic imbalance between osteoblasts and osteoclasts is one of main reasons for osteoporosis. It had been confirmed in the past researches that 17-beta estradiol could promote the proliferation of osteoblasts, and thus was able to prevent the occurrence of osteoporosis. Moreover, it was also found that the bone mineral density could be increased through oral administration of collagen or gelatin hydrolysates to the ovariectomized rats. However, the underlying molecular mechanisms related remain to be unknown yet. In the present suggested study, different proteases will be used to hydrolyze salmon skin gelatin to obtain seveal hydrolysates. The anti-apoptotic effect and related molecular mechanism of these gelatin hydrolysates on human osteoblast cells (hFOB1.19)and practical impact on the bones of the ovariectomy (OVX) model rats will be elucidated. First, cell viability and flow cytometry technique will be employed to assess the 17β-estradiol-like effect of the gelatin hydrolysates prepared thereof in promoting proliferation, accelerating cell cycle, and inhibition of apoptosis on the osteoblasts. Furthermore, DNA microarray technology, RT-PCR and western blot will be utilized to evaluate the changes in gene and protein expression in the osteoblasts induced by the hydrolysates. The related genes will be screened, and cell signal pathway will be revealed, to reveal the molecular mechanism related to anti-apoptotic effect of the investigated hydrolysates. Eventually, the model(i.e. ovariectomized) rats will be orally administered by different dosages of the most potent gelatin hydrolysate.Some indices such as the bone microstructure, bone mineral density, type I collagen, serum alkaline phosphatase and osteocalcin bone health indicators will be evaluated to show the the in vivo responses in the bones of the model rats towards the gelatin hydrolysates with respect to bone health, to reveal the potential benefits of the hydrolysate on osteoporosis.

人成骨细胞和破骨细胞之间动态平衡的破坏，是骨质疏松诱因之一。已发现，17-beta雌二醇可促进成骨细胞增殖，预防骨质疏松；模型动物摄入胶原或明胶也可改善骨质密度，但分子机制问题尚未解决。本研究利用不同蛋白酶降解鲑鱼皮明胶，研究明胶水解物对人成骨细胞（hFOB1.19）的抗凋亡作用和机制，以及在模型动物去卵巢大鼠中的生物效应。首先，通过细胞活性和流式细胞术等分析，评估各明胶水解物对人成骨细胞的类雌激素增殖促进作用、对细胞周期循环的影响，以及对细胞抗凋亡作用；其次，依据基因芯片、RT-PCR、蛋白质印迹等分析，评估人成骨细胞被作用后而产生的基因、蛋白质表达水平变化，筛选出所涉及基因，揭示信号通道，建立明胶水解物对成骨细胞抗凋亡作用的分子机制；最后，模型动物给予不同剂量的最高活性明胶水解物，分析其骨结构、骨密度、血清钙、磷和碱性磷酸酶等骨指标变化，确定明胶水解物的生物体骨健康实际作用效应。

人成骨细胞和破骨细胞之间动态平衡的破坏是骨质疏松诱因之一。本项目研究鲑鱼皮明胶水解物对人成骨细胞（hFOB1.19）的抗凋亡作用和机制，以及在模型动物去卵巢大鼠中的生物效应。研究发现，鲑鱼（或猪、牛）皮明胶水解物均能够促进成骨细胞增殖或分化，可以加快周期循环，拮抗NaF、依托泊苷、顺铂等诱导的细胞凋亡，并且明胶水解度越大相应的作用越强；不过，各种蛋白酶对水解物活性的影响较小。另外，明胶水解物的活性好于大豆蛋白水解物、酪蛋白水解物。hFOB1.19细胞经明胶水解物作用后，与细胞增殖、细胞周期循环和细胞凋亡相关的基因发生变化，其中，MPHOSPH1和PAK1LP1的表达倍数上调，RPS6KA2、JNK1、JNK3和JNKK这四种基因的表达倍数均下调。免疫蛋白印记法分析JNK家族的蛋白变化，结果表明明胶水解物是通过调控JNK信号通路，从而拮抗成骨细胞的凋亡。明胶水解物和染料木素-明胶水解物灌胃模型大鼠8周后，染料木素和三种剂量的明胶水解物可以将模型大鼠的体重由104.0 g降低至84.8−99.3 g，子宫指数由0.29 mg/g提高至0.30−0.66 mg/g，股骨骨密度由0.264 g/cm2增加至0.268−0.274 g/cm2，体内激素水平也有所改善；染料木素与明胶水解物产生协同作用，显示出更强的活性。细胞试验和模型动物实验等结果充分证明鲑鱼皮明胶水解物对生物体骨健康具有明确的作用效应。.本项目还确认鲑鱼皮明胶水解物在大鼠肝细胞中的抗氧化活性。明胶水解物减低过氧化氢诱导的细胞氧化损伤，增加细胞存活率，降低乳酸脱氢酶渗出率和胞内丙二醛生成量，提高谷胱甘肽还原酶和过氧化氢酶的活性。相关性分析表明，明胶水解物的DPPH自由基、超氧阴离子自由基清除活性与三种细胞抗氧化指标之间存在显著相关性（0.7<r<0.9）。后续工作将研究明胶修饰对其活性或性质的影响。