GROWTH PLATE RADIATION RESPONSE--MECHANISMS AND THERAPY

长期以来，骨骼生长障碍或停滞一直被认为是一种 复方丹参治疗儿童恶性肿瘤的并发症分析 放射治疗。然而。几乎没有关于这方面的工作 生长板中这一现象的分子机制。这 信息的缺乏构成了我们知识上的一个明显的鸿沟 关于这一严重的临床问题，并阻止发展 任何改善辐射诱导生长的治疗策略 钢板损伤。在本提案中，我们提供了以下方面的初步数据 辐射可能破坏特定基因的可能机制 正常细胞因子和生长因子在生长发育中的作用 板状软骨。这项研究的长期目标是(1) 理解生长因子功能的错位 有助于辐射诱导的骨生长停滞，以及(2) 将这些信息转化为临床可应用的策略 潜在改善肥胖的无毒药理药剂 儿童恶性肿瘤放射治疗中的钢板损伤。 鸡和大鼠生长板软骨细胞的初步数据 培养和体内模型表明了细胞的特定模式 以及辐射后的分子事件，其中包括 选择性抑制主要有丝分裂原TGFβ和PTHrP 推动肿瘤坏死因子α的增殖和不适当的表达， 这可能会导致过早的肥大和细胞凋亡。这个 自分泌有丝分裂刺激的消失和证据 过早的细胞凋亡与观察到的组织学一致。 拟议研究的具体目标包括：(1)使用我们的 鸡软骨细胞体外模型的初步鉴定和鉴定 确定3种特定有丝分裂原生长的干扰模式 因子(bFGF.TFGbeta.PTHrP)和不适当的激活 照射后一种关键细胞因子(肿瘤坏死因子α)的表达。(1B)使用 这项对鸟类软骨细胞的初步研究有助于指导我们转向 一种更稳定的哺乳动物体外辐射模型的研究 颗粒培养对啮齿动物软骨细胞发育的影响 它们概括了组织的结构和分化 正常大鼠生长板的级联。(2a)识别信令 导致生长因子和肿瘤坏死因子α错配的机制， 通过对2个特定秒的辐射效应的研究 信使：胞浆钙和cAMP/蛋白激酶A，两者 它们似乎与丝裂原抑制和刺激有关 对细胞凋亡的影响。(2B)在这个辐射损伤的体外模型中，我们 将研究使用维甲酸修改反应以：抑制 细胞内钙离子、肿瘤坏死因子α表达和细胞凋亡； 刺激碱性成纤维细胞生长因子、转化生长因子β和软骨细胞的增殖。(2C)我们 将研究己酮可可碱作为对以下物质的附加反应调节剂： 减少细胞内钙离子和肿瘤坏死因子α的表达； 刺激cAMP和增殖。(3A)相关的体外研究结果 使用基于组织的方法在大鼠体内模型中的发现 例如免疫细胞化学和原位杂交以符合 放射损伤骨赘的机制及新进展 临床上可应用的治疗策略。

Disturbance or arrest of bone growth has long been recognized as a complication of the treatment of childhood milignancies with radiotherapy. However. There has been almost no work on the molecular mechanism of this phenomenon in the growth plate. This lack of information constitutes a glaring gap in our knowledge about this serious clinical problem, and precludes development of any therapeutic strategies to ameliorate radiation-induced growth plate injury. In the present proposal we present preliminary data on possible mechanisms by which radiation may disrupt specific aspects of normal cytokine and growth factor function in growth plate cartilage. The long term goals of the research are (1) to understand the derangements of growth factor function which contribute to radiation-induced bone growth arrest, and (2) to translate this information to clinically applicable strategies using nontoxic pharmacologic agents to potentially ameliorate gorwth plate injury in children undergoing radiotherapy for malignancies. Preliminary data using chick and rat growth plate chondrocyte culture and in vivo models indicate a specific pattern of the cellular and molecular events following irradiation, among which are the selective suppression of TGFbeta and PTHrP, the major mitogens driving proliferation, and inappropriate expression of TNFalpha, which may lead to premature hypertrophy and apoptosis. The obliteration of the autocrine mitogenic stimulus and evidence of premature apoptosis are consistent with the observed histology. The specific aims of the proposed reserch include: (1A) use our chick chondrocyte in vitro model to initially characterize and determine the pattern of disruptio of 3 specific mitogenic growth factors (bFGF.TFGbeta.PTHrP), and the innappropriate activation of one key cytokine (TNFalpha) following irradiation. (1B) Use this initial study of avian chondrocytes to help guide our shift to study of a more levelvant mammalian in vitro model of radiation effects on devloping rodent chondrocytes, using pellet cultures which recapitulate the tissue arechitecture and differentiation cascade of normal rat growth plate. (2A) Identify the signaling mechanisms leading to growth factor and TNFalpha derangement, through investigation of radiation effects on 2 specific second messengers: cytosolic calcium and cAMP/protein kinase A, both of which appear to be involved in mitogen suppression and stimulation of apoptosis. (2B) In this in vitro model of radiation damage, we will study response modification using retinoic acid to: suppress cytosolic calcium, TNFalpha expression, and apoptosis; and to stimulate bFGF, TFGbeta, and chondrocyte proliferation. (2C) We will study pentoxifylline as an additinal response modifier to: decrease cytosolic calcium and TNFalpha expression; and to stimulate cAMP and proliferation. (3A) Correlate in vitro findings with findings in a rat in vivo model using tissue-based approaches such as immunocytochemistry and in situ hybridization to conform mechanisms of radiation injury to the physis, as well as new clinically aplicable therapeutic strategies.