【作者】 张勇；

【导师】 吴兴裕； 陈苏民；

【作者基本信息】 第四军医大学 ， 航空航天医学， 2002， 博士

【摘要】 人类在借助现代科学技术进行太空探险的同时，也遇到了许多意想不到的困难。如何在太空维持生命并有效地发挥自己的各种功能，这是人类在航天探险过程中遇到的十分重要和急需解决的课题。其中，失重性骨质疏松已经成为目前航天医学界研究的重点，因为在太空失重环境下人体的骨骼系统会发生一系列结构和功能的变化，主要表现在骨量丢失、骨骼脱钙、负钙平衡和骨力学性能下降，而且是非自限性的病理变化，长期暴露在失重环境中可能导致骨折、电解质平衡紊乱、泌尿系结石、肌肉功能下降等表现，对人类征服太空的梦想提出了严峻的挑战。通过人们的不懈努力，在探讨失重性骨质疏松发病机制和寻找有效对抗措施方面已经取得了许多成果，但是有些难题尚未取得突破性进展。 现在比较一致的见解认为，失重时的骨丢失是一种局部机理起主导作用，并受多层次调节的复杂变化过程。也就是说，失重首先影响骨的再建系统，进而影响钙的内环境稳定调节系统，反过来再影响骨的再建系统。多数研究已经表明，失重和模拟失重导致的骨丢失是一个以骨形成抑制起主导作用的过程，因此成骨细胞和调节它的各种细胞因子，尤其是促进骨形成的因子之间的相互作用就成为目前研究的热点之一，这也是寻找有效对抗措施的重要突破口。骨形成蛋白(BMP)作为一种很强的促进骨质形成的细胞因子，在骨吸收方面也有重要的调节作用，近年来对它的研究非常广泛深入，但是在失重性骨质疏松中的相关研究却极少。因此，为了探讨失重环境下BMP在诱导成骨细胞发挥骨代谢作用方面 第四军医大学博士学位论文 中文钓耍ttase 的变化，以期进一步探索失重仕骨质丢失发病机末柳开辟新的对抗措施，我们设 计并进行了如下研究： l 利用回转器建立模拟失重环境下大鼠颅骨成骨细P培养模型。对成骨活 性标志物碱性磷酸酶（AlP）、骨钙素（OC）和体夕旧此能力u以确 定模型的成立。 二 采用一定齐临司叁诅BMPZ作为诱 咖入细 中，在主朗以失自摹付竟下 及正常重力下比较不同的时间段成骨细胞表达碱性磷酸酶和骨钙素量的变 化，以研究BMPZ fll＝rsx同重力环杉引勺方齿骨细胞成骨能力诱导作用的变化。 3 以破骨细胞活化因子（OPGL）和破骨细0因子（OPG）作为成骨细 胞调节骨吸收代谢的标志物，在模拟失重和正常重力环境下，比较不同的时 间段BMP－2诱导成骨细胞对这两种细胞因子表达的变化。 结果发现： l 模拟失重环境下，成骨细胞对BMP－2诱导产生WN敏感性陶B，对OC 的诱g敏感牲未见显著变化，提示成骨细胞对BMPZ的成骨诱导作用敏感性 降低。 2 模拟失重环摸和正常重力环境下，BMPZ诱导成骨细胞产生的Oo ITlrtrvA 较非诱导组均有显著增高，产生的 OPGL d较非诱导组均显著陶氏。 3 模拟失重环境下，BMP－2诱导成骨细胞产生的 Oo InRNA和 OPGL InRNA 与正常重力环境下培养细胞的表现相比没有显著差异，但是蛋白表达较正常 重力环境下细胞内的表达显著降低。提示模拟失重环境主要影响OPG和 OPGL的转录后调节过程。 4 模拟失重环境和正常重力环梭下，BMPZ诱导产生MwtA的表 达比值均较4［＠导组显著增高。 5．模拟失重环境下，成肴细胞诱导BMP－2产生的OWPGLANA表达t0直 与正常重力环杉n相比没有显著差异，提示BMI，J在促进骨吸收代谢的下调 方面有独特的作用，并有应用于治疗失重性骨质疏松的前景。 n （ 第o军医大学博士学位论文？RNZ’、 6．摸拟失重环境和正常重力环境下，BMP－2诱导产生OPG／OPG蛋白表达比值 均较非诱导组有显著性增高。 7．模拟失重环境下，成骨细胞诱导BMP－2产生的OIMpPGL蛋e表达t以彭咬 正常重力环境下的表达比值有显著降低。 总之，通过以上研究，我们认为：在模拟失重环境下，成骨细胞对BMPZ 成骨诱导作用敏感性阐氏，但是BMPZ对成骨细胞介导的骨吸 棺显著的下 调作用，一方面为失重性骨质疏松的发病机＄揽收了有力的证据，另一方面为失 重性骨质疏松的对抗措施提供了可能的新途径。更多还原

【Abstract】 While human exploit the space taking advantage of modern technology, we also have encountered many unexpected problems. Among them the most important and urging one is how to sustain man’s life and function effectively in space. Osteoporosis induced by microgravity, one of the serious diseases acquired in space, is now becoming a more and more absorbing target in the space medicine field. Exposure to microgravity or weightlessness will cause a series of malignant alterations to human’s skeleton system, including loss of bone mass and bone mineral, declines in bone mechanical function and negative calcium balance which all cannot ameliorate automatically at all. A long period of time staying in space may lead to bone fracture, unstable in inner environment balance, stone formingin urine system and decline in muscle function as a result, which make great challenges toward realizing our dreams of conquering space. Although scientists have achieved fruitful success during investigation of pathogenesis of osteoporosis under microgravity and finding effective countermeasures, several difficult problems are remained unsolved yet.Until now it’s well recognized that the bone loss under microgravity is caused by a malfunction in focal area concerning many complex modulation systems. Studies have confirmed that the decline of bone formation is mainly responsible for the bone loss induced by microgravity or weightlessness. So the mutual reaction between osteoblasts and their regulators-relative growth factors has now become a focus in this field, which will also be one of the break points in finding effective countermeasures. Bone morphogenetic proteins, as a proteins family which have remarkable stimulation both on bone formation and on bone absorption, are studied profoundly and exclusively in recent years. But they are seldom concerned in studies of osteoporosis induced by microgravity. In terms of this context we designed and carried out the experiment below in order to uncover the alterations on the induction of osteoblasts by BMP under simulated weightlessness and to explore the further pathogenesis in osteoporosis induced by simulated weightlessnes, expecting to find any possibilities of new countermeasures.1. Study on culture of osteoblasts from neonatal rat’s calvaria in simulated weightlessness. Constructed cell model under simulated weightlessness by clinostat and confirmed the credibility by assessing the expression of biomarkers-alkaline phosphatase and osteocalcin.2. Addition of recombinant human BMP-2 into the culture media andobserved the alteration on the exact expression of ALP and osteocalcin under simulated weightlessness in different period of time.3. Addition of recombined human BMP-2 into the culture media and observed the alteration on the mRNA and protein expression of osteoprotegerin and osteoprotegerin ligand under simulated weightless -ness in different period of time.Results as follows.1. Under simulated weightlessness, the ALP expression of osteoblasts induced by BMP-2 was remarkably lower than that under normal gravity. No difference was found in expression of osteocalcin between groups under simulated weightlessness and the ones under normal gravity. The data indicated that the osteoblasts’ osteogenic sensitivity against BMP-2 was lower than normal.2. Under simulated weightlessness or normal gravity conditions, BMP-2 induced osteoblasts to produce more OPG mRNA and inhibited the OPGL mRNA production.3. There’s no significant difference in the expression of OPG mRNA and OPGL mRNA under simulated weightlessness or normal gravity. But simulated weightlessness inhibited both two factors’ protein expression. The data indicated that there may be some posftranscriptional factors downregulating the protein expression of OPG and OPGL.4. The proportion of OPG mRNA compared with OPGL mRNA was increased remarkably in BMP-2 stimulating groups.5. There was no significant difference in the proportion between BMP-2 stimulating groups under simulat更多还原