细胞死亡模式necroptosis在椎体成形术后再骨折的机制研究及新型载药骨水泥的研制

Osteoporotic vertebral compression fractures (OVCFs) has become a common disease in the elderly. Recently developed surgical treatment methods of Percutaneous vertebroplasty (PVP) and Percutaneous kyphoplasty (PKP) can fast relieve pain in patients by injecting PMMA bone cement into the compressed vertebra. But more and more attention has been paid to the postoperative vertebra refracture. The bone necrosis caused by PMMA implantation is an important risk factor for refractures occurrence. Necroptosis is a new mode of cell death discovered in recent years which is closely related to inflammation, although its mechanism in refractures is still unclear. Our previous studies demonstrated that the MMA monomer could promote the level of inflammatory mediators after it was injected into vertebral body. Through a combination of morphological and molecular biological methods, this research is aimed to firstly investigate the effect on osteoblasts apoptosis of TNF-α, a typical inflammatory mediator, which can lead to necroptosis; secondly, to detect the physical and chemical properties of improved PMMA bone cement containing Nec-1, the specific inhibitor of necroptosis；finally, to observe the difference of bone necrosis caused between the containing Nec-1 PMMA bone cement and traditional PMMA bone cement injecting in animal vertebrae， to clear the existence of necroptosis after PMMA implants and to clarify the feasibility of bone cement containing Nec-1. Ultimately, our research may provide an experimental support for the prevention of the postoperative vertebra refracture after PVP or PKP.

骨质疏松性椎体压缩骨折已成为危害中老年人健康的常见疾病。经皮椎体（后凸）成形术通过将PMMA骨水泥注入压缩椎体，可以迅速缓解患者疼痛症状，临床效果显著,但术后手术椎体的再骨折逐渐引起人们的重视。PMMA植入造成的骨坏死是其发生的重要危险因素之一。坏死性凋亡（Necroptosis）是近年来最新发现的与炎症关系密切的新型细胞死亡模式，但其在再骨折中的作用未知。我们的前期研究证实，MMA骨水泥单体植入椎体后可引起椎体内炎性介质TNF-α的升高。本研究拟联合组织形态学、分子生物学等多种方法，首先探究诱导坏死性凋亡的典型炎性介质TNF-α对成骨细胞凋亡的影响；第二检测载坏死性凋亡特异性阻断剂Nec-1改良PMMA骨水泥的理化性质；第三观察载Nec-1的骨水泥植入动物椎体内后骨坏死产生的情况，明确PMMA植入后坏死性凋亡的存在。最终为椎体（后凸）成形术后防止再骨折发生的干预治疗提供一定的实验依据。

主要研究内容：运用细胞培养建立成骨细胞MC3T3-E1分化模型，用炎性介质TNF-α，Nec-1（一种特异性坏死性凋亡抑制剂）和Z-IETD-FMK（一种特异性凋亡抑制剂）与成骨细胞MC3T3E1共同培养，利用分子生物学的多种方法明确典型炎性坏死性凋亡是否在TNF-α死亡中发挥作用。通过CPC与PMMA不同比例的混合制备混合型骨水泥，运用多种方法检测混合型骨水泥的理化性质，探究理想的混合骨水泥载体。构建动物模型，通过组织学、分子生物学等多种检测方法验证在植入PMMA后是否会引起骨组织的坏死，以及坏死性凋亡是否存在于骨组织的坏死中。.重要结果：TNF-α以剂量和时间依赖性的方式抑制细胞增殖；Nec-1可逆转流式细胞学中由TNF-a加Z-IETD-FMK引起的细胞群移位；TNF-α加Z-IETD-FMK增加RIPK3和p-MLKL；RIPK3，MLKL和caspase 3的mRNA水平的变化是与相应蛋白质表达水平的变化不一致。CPC与PMMA混合可以改良PMMA的理化性质，能够作为药物的理想载体系统，在CPC与PMMA比例为3：1时，载药释放最佳，但是考虑其生物力学较低，可以考虑1:1比例进行调整水泥比例。在动物实验中，HE染色及电镜扫描均表现出了植入水泥后，骨组织不同程度的骨组织坏死、骨细胞缺失及骨小梁的断裂，分子生物学检测发现坏死性凋亡特异性蛋白RIPK 3在载有Nec-1的混合型骨水泥组降低，说明坏死性凋亡在植入水泥引起的骨组织坏死中是存在的。.科学意义：首次阐明当使用Z-IETD-FMK阻断TNF-α诱导的细胞凋亡时，坏死性凋亡在成骨细胞死亡中扮演重要角色，而Nec-1可对成骨细胞起保护作用。完成了PMMA和CPC的混合型骨水泥的配制，并明确了释放Nec-1的最佳配比，为进一步的体内实验提供材料，也为临床应用提供一种新的选择。在动物实验中，首次验证了坏死性凋亡通路的存在，为临床中预防和治疗因骨水泥植入后引起的坏死性凋亡导致的椎体再骨折提供新的思路和方法。