基质金属蛋白酶-2，9在放射性肺损伤发生过程中的机制研究
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摘要
本课题受南方医科大学南方医院2005年度院长基金支助。
一、立项背景及国内外研究现状
胸部恶性肿瘤如肺癌、食管癌、乳腺癌等是我国最常见的恶性肿瘤，发病率呈逐年上升的趋势，是我国居民最主要的死亡原因之一。放射治疗作为无创或微创性治疗的手段，因其明显提高的局部控制率和生存率，越来越多地应用于胸部恶性肿瘤的治疗。放射性肺损伤作为胸部肿瘤放射治疗最显著的并发症，是影响肿瘤治愈患者生活质量的主要原因之一。研究放射性肺损伤的发生发展机制，并以此为基础寻找预防或治疗放射性肺损伤的途径，是目前国内外肿瘤放射治疗学者热切期待解决的问题。
到目前为止，放射性肺损伤的发病机制尚未清楚。关于放射性肺损伤发病机制的学说主要分为三种：①小血管及肺Ⅱ型细胞损伤学说；②自由基学说；③细胞因子学说。但是，任何一种学说都不能解释放射性肺损伤的整个病理生理过程。
然而幸运的是，放射性肺损伤的病理形态学却有一致的发现：放射性肺损伤的发生过程中，肺泡基底膜的损伤导致炎症因子和细胞在肺泡腔内及肺间质聚集，肺泡上皮细胞脱落、表面活性物质的丢失及胶原的沉积最终导致了肺泡塌陷及肺间质纤维化。显然，在这一演变过程中，以Ⅳ型胶原为主要成分的肺泡基底膜损伤对于肺间质纤维化的形成足十分必要的，是放射性肺泡炎和肺间质纤维化病理变化的关键事件。而包括肺泡基底膜在内的细胞外基质(extracellular matrix，ECM)的代谢失衡在放射性肺损伤的发生过程中可能发挥了关键作用，这是通过调节ECM合成与分解代谢实现的。
基质金属蛋白酶(matrix metalloproteinases，MMPs)是一类以降解ECM为主要功能的蛋白酶类，也是目前公认ECM降解最重要的蛋白酶类。包括胶原酶、间质胶原酶、弹性蛋白酶等，其作用底物是构成基底膜的纤维连接素、板层素、胶原、糖蛋白与弹性蛋白等。Ⅳ型胶原是构成基底膜的主要成分。明胶酶B(GelatinaseB，MMP9)和明胶酶A(GelatinaseA，MMP2)是体内Ⅳ型胶原的主要降解酶，是MMPs的亚类之一。它们在体内主要受三个水平的调节：①基因水平的调节；②酶原活化调节；③活化后调节。
显然，MMPS的代谢平衡是维持ECM代谢平衡的物质基础。根据放射性肺损伤发生发展的病理生理特点，结合MMPS的生物学特性，可以推断MMPS与放射性肺损伤之间可能存在一定的因果关系，这种因果关系可能会成为阻止放射性肺损伤发生发展的关键节点，成为临床预防或治疗放射性肺损伤的关键环节。
寻找简单易行的方法早期预测和防治放射性肺损伤的发生和程度，及早进行干预，减轻放射性肺损伤的程度，延缓放射性肺损伤的进程，是本课题研究的目的。
二、国内外研究现状
MMPs的研究目前是国内外各学科研究的热点之一，尤其在创伤修复、胚胎发育、恶性肿瘤的浸润与转移等方面均有大量的文献报道，其实验室技术趋于成熟。
上世纪90年代中期，国外学者开始将MMPs及其抑制物与肺组织的损伤与修复联系在一起。特发性肺纤维化、慢性阻塞性肺纤维化等疾病的研究已发现，ECM与MMPS之间的失衡，可能是肺纤维化类疾病发生发展的关键环节。同时，国内外研究者还发现，临床上能够观察到的肺纤维化改变往往比肺实质的分子生物学改变至少晚1-3个月。也就是说，在患者尚未开始出现肺纤维化症状的早期，由细胞因子介导的肺纤维化损伤已经悄悄开始了。这个结论被国内外文献大量的证实，如博来霉素、吸烟诱发的肺纤维化过程中MMPS表达均有改变。
放射治疗是一门新兴的边缘学科，放射性肺损伤是胸部肿瘤放射治疗最显著的并发症，，其最终发展成为不可逆的肺纤维化。放射性肺损伤的本质是一个渐进性的肺间质纤维化过程，它的发病机理很有可能与肺纤维化类疾病有共同特点，即与MMPs引起的代谢紊乱有关。国内外有学者猜测MMPs可能与放射性肺损伤的发生机制有关：Susskind等报道了肺癌、乳腺癌患者放疗后血浆中MMP-9的升高，猜测可能与放射性肺损伤有关；Araya等通过体外培养人支气管上皮细胞株，照射后发现MMP-2表达增高，由此得出MMP-2可能是放射性肺损伤的重要产物。杨坤禹等以12Gy照射C57BLP6J TGF-β基因敲除的雌性小鼠双侧肺，发现照射后小鼠肺组织中MMP-2和MMP-9的表达增加，预测MMP-2和MMP-9可能加剧了急性放射性肺损伤。
查阅文献可知，目前国内外尚无文献报道通过系统的动物模型实验，从组织学、蛋白水平、基因水平等多个层面证实MMPs在放射性肺损伤的过程中的重要作用；尚无报道通过动物实验前瞻性研究照射后引起的射野外淋巴细胞性肺泡炎的病理生理机制；尚无预测放射性肺损伤的发生及程度的血清学指标。
三、实验目的和内容
进一步证实MMP-2，9在放射性肺损伤的发生发展过程中动态改变及重要作用，寻找预测放射性肺损伤的血清学指标，初步研究射野外淋巴细胞性肺泡炎的病理生理机制，及早进行干预，减轻放射性肺损伤的程度，延缓放射性肺纤维化的进程。
我们建立了Wistar大鼠半胸照射的放射性肺损伤实验动物模型，主要实验内容如下：
1．观察照射后不同时间实验动物临床表现(包括毛发、体重、死亡：率等)，血清和肺泡灌洗液纤维化指标(HA，HPCⅢ，CⅣ，LN)的变化，肺泡灌洗液细胞总数量和成分比例的变化；
2．观察照射后肺组织病理变化(普通光镜HE染色，Ⅰ型、Ⅲ型和Ⅳ型胶原纤维特殊染色，MMP-2，9免疫组织化学)，活体组织内MMP-2，9mRNA的实时定量PCR(Real-time quantitative polymerase chain raction，RQ-PCR)变化，比较各观察指标随着放射性肺损伤不同时期动态变化的特点，并证实MMP-2，9在放射肺损伤发生机制中的作用；
3．比较正常对照组、照射一侧肺及未照射一侧肺组织的各项指标，分析射野外发生的淋巴细胞性肺泡炎的病理生理机制，研究散射线对射野外淋巴细胞性肺损伤发生几率的影响；
4．试探性应用能下调MMPS表达的干扰素γ，观察其是否能减轻放射性肺损伤，以及对MMPS的影响，为临床预防或治疗放射性肺损伤提供理论依据。
四、该项目的创新性
1．本项目将通过动物实验，从组织学、蛋白水平、基因水平多个层面证实MMPs在放射性肺损伤的过程中的重要作用；
2．通过比较各观察指标随时间动态变化的特点，解释放射性肺损伤早期、晚期发生发展的共性和特性，为预防和治疗放射性肺损伤提供临床干预可能的靶点；
3．比较血浆中纤维化指标与肺组织或支气管肺泡灌洗液的各项指标，寻找潜在的联系，为临床实践提供可行性强的预测和监测指标，这是本项目紧密联系临床需要的特色之一；
4．本项目首次通过建立动物实验模型，比较射野内和射野外肺组织的各项指标，研究射野外淋巴细胞性放射性肺损伤(“旁观者”效应)的发生机制；
4．通过建立动物实验模型，初步研究IFN-γ在放射性肺损伤发生发展过程的作用。
五、技术路线
本实验的主要技术路线如下：
六、主要结果
1．血清和BALF中HA水平在放射性肺纤维化早期显著升高，血清HA与肺间质纤维化水平呈正相关(r_s＝0.454，P＝0.010)，与Ⅰ型胶原含量呈负相关(r_s＝-0.427，P＝0.017)，与Ⅳ型胶原含量呈正相关(r_s＝0.450，P＝0.011)，说明了高水平的HA可能干扰了胶原代谢，从而参与了早期放射性肺纤维化的形成。血清或BALF中的显著升高，有可能预示着放射性肺纤维化已经开始；
2．血清LN水平在照射后几乎处于持续的低水平状态，而BALF LN在放射性肺泡炎、放射性肺纤维期持续处于高水平状态，可能与LN位于肺基底膜，照射后由于基底膜破坏导致间隙增宽，LN更有利于渗透至肺泡腔内，完成诱导炎症细胞的浸润、胶原纤维的生成等生物功能有关。因此，我们猜测LN参与了肺泡炎的全过程，尤其是在放射性肺纤维化期，BALF中LN持续处于高水平状态，可能在促进肺泡结构的消失，肺纤维化的形成有重要意义；
3．血清中PCⅢ在照射后1～2周就显著升高(P＝0.007)，至照射后4周降低，逐渐恢复至正常水平，其变化与肺间质炎症程度呈正相关(P＝0.022)。BALF中的PCⅢ在照射后2周达到峰值后持续维持在低于正常水平。提示Ⅲ型胶原可能参与了急性肺泡炎症的形成。即血清中PCⅢ的浓度有可能可以作为反映肺间质炎症的监测指标之一；
4．急性炎症期血清和BALF中IV型胶原一过性的升高，纤维化阶段维持在较低水平。而组织学却发现纤维化阶段Ⅳ型胶原在肺间质大量增生，占肺间质胶原的90％以上。可能与放射性肺纤维化阶段，由于基膜和间质的增生，毛细血管闭塞，肺泡结构消失，Ⅳ型胶原分子量大，不易穿透增生、闭塞的毛细血管壁释放入血液，故不能在BALF或者血清中显示出来；
5．肺巨噬细胞MMP-9、MMP-2的表达在放射性肺泡炎症最明显的时期表达最强烈，并持续至放射性肺纤维化早期。相关性分析表明肺巨噬细胞表达MMP-9、MMP-2与肺泡炎程度呈正相关(r_s＝0.808，P＝0.000)，与肺间质纤维化程度呈正相关(r_s＝0.415，P＝0.020)，这可能预示了巨噬细胞同时参与了放射性肺泡炎和早期放射性肺纤维化的形成；
6．肺血管旁肌成纤维细胞可能也是MMP-9、MMP-2的主要来源，照射后1天肌成纤维细胞MMPS呈强阳性，照射后1周呈持续强阳性表达，照射1周后开始进行性下降，至照射后4周，已几乎降低至正常水平，照射后3月，血管周围平滑肌几乎不表达MMP-9、MMP-2，显著低于正常对照组(χ~2＝23.624，P＝0.001)。实验结果提示肌成纤维细胞参与了早期放射性肺损伤的发生，是影响胶原代谢紊乱的主要效应细胞之一，从而诱导了放射性肺纤维化的形成，而且可能是纤维化进程中的关键步骤。因此，肌成纤维细胞可能成为控制放射性纤维化进展的重要靶细胞；
7．支气管粘膜上皮细胞在照射后一直处于低于正常水平的表达，猜测支气管粘膜上皮在正常情况下是维持ECM代谢平衡的主要来源之一，而在照射后粘膜上皮细胞高度水肿，大片脱落至支气管腔，细胞破裂崩解，可能成为MMP-9和MMP-2表达下降的原因之一；
8．照射后24小时内即可观察到肺组织分子水平的变化，说明放射损伤并不存在潜伏期。这一结果改变了我们以往对放射损伤的传统认识。从我们的实验可以看出，这个从分子水平到临床表现的所谓的潜伏期，实际上是一个肺组织内多种效应细胞如肺泡巨噬细胞、肌成纤维细胞、ECM等完成损伤信息传递，启动多种细胞因子瀑布效应的时期。因此，正常肺组织对放射损伤的反应是一个即时、动态的连续过程。正因如此，我们建议预防和治疗放射性肺损伤，应该早期、及时、多途径的阻断放射性肺损伤的“瀑布效应”；
9．本实验研究发现照射野内和野外肺组织在照射后12周内肺泡炎症程度有显著差异性(P＝0.000)。但其差异性主要表现在照射后2周前，照射野外比照射野内的肺组织炎症发生更晚且程度轻(P＝0.000)，而在照射后2周～12周，双侧肺组织肺泡炎程度无显著性差异(P＝0.170)。因此，我们认为在照射后放射性肺损伤发生的早期，放射性肺损伤的炎症因子处于动员及诱导阶段，并局限在受照射肺组织局部。随着炎症因子和可能产生的自身抗体逐渐累积并释放入血，放射损伤才开始逐渐诱导照射野外放射性肺损伤的发生，并使之与照射野内的放射性肺损伤达到同步；
10．在30Gy照射组的左侧肺，即照射野外发现MMP-2 mRNA和MMP-9mRNA表达增高，说明CD4＋T及其介导的细胞因子可能并不是唯一参与照射野外放射性肺损伤发生机制的生物介质。而临床上照射野外肺组织的微观变化，很有可能发生在每例胸部局部放射治疗的患者(其照射剂量或照射体积可能已经大于肺组织的耐受剂量或域体积)。而只有5％～10％的患者由于对血清中放射损伤产生的生物介质产生了“超敏”反应(类似“过敏性肺炎”或“自身免疫性肺炎”)，故产生了干咳、呼吸困难等症状。因为射野外的放射性肺损伤是免疫亢进诱导的肺炎，故用激素治疗显示了良好的效果，且不会遗留长期的后遗症，这些特点恰恰与我们临床看到的病例特征符合；
11．我们发现IFN-γ在放射性肺损伤的发生过程中降低了MMPS的表达，抑制了Ⅰ型和Ⅲ型胶原的合成，有效减少了照射后的放疗反应，提高了实验动物在照射后的存活质量，延迟放射性肺泡炎的发生时间并降低其发生的程度。而对放射性肺纤维化的作用有轻度减弱，但尚无显著性意义。其原因可能需要更长期的随访和进一步的实验研究证实。
Thoracic tumors such as lung cancer, breast cancer, lymphoma is the most widespread malignant tumor and is one of the supreme cause of death in China. Radiation is a common modality used for the treatment of thoracic malignancies, but injury to surrounding normal lung is a major limitation that dictates the ultimate dose, volume, and technique of radiation.
Radiation-induced pulmonary pneumonitis and fibrosis secondary is a major limiting factor in the successful treatment of thoracic tumors. The disease afflicts millions of individuals worldwide and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of the disease have been defined sufficiently to design appropriate targets for therapy. The radio-oncologists have focused on the molecular mechanisms through which growth factors could play a role in the development of radiation-induced pulmonary injury, and wish to get the key targets to prevention and cure the disease.
Radiation-induced pulmonary reactions have classically been viewed as distinct phases-acute pneumonitis and, later, fibrosis-occurring at different times after irradiation and attributed to different target cell populations.As yet our understanding of disease pathogenesis is still controversial. Shapiro presumed irradiation appears to trigger membrane receptor mediated surfactant release. The typeⅡpneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant. Cell cultures of typeⅡpneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 tad also in vitro. Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes. In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism. These studies establish that surfactant release in response to irradiation in vivo is a direct effect on typeⅡpneumocytes.
Bai discussed the role of the free radicals (FR) in the developing process of radiation interstitial pneumonitis. The morphologic changes were examined in the lung tissue of Wistar rats after chest irradiation and determined sequentially. The content of FR were examined in the lung tissue of Wistar rats after chest irradiation using electron spin resonance. The activity of superoxide dismutase (SOD) was assessed using the chemical glow method of xanthine oxidase. After chest irradiation, the content of FR in the lung progressively increased and the activity of SOD progressively decreased. De's study showed that mitochondria and microsomes in the lung of Wistar rats irradiated by gamma-rays from a Co source at the thoracic region had higher levels of lipid peroxidation. In the pneumonitic rat the activities of superoxide dismutase and catalase were markedly reduced in the cytoplasmic fraction but not significantly altered in lung mitochondria. On the contrary, lipid peroxidation and the two enzymes in lung tissue in the non-pneumonitic group of the irradiated rat were comparable with that of control animals. The results indicate that free radical-induced oxidative stress following thoracic irradiation may be one of the causative factors in the development of interstitial pneumonitis.
Rubin prefer to view these events as a continuum progression that is the result of an early activation of an inflammatory reaction, leading to the expression and maintenance of a cytokine cascade. The temporal sequence relationship between the elevation of specific cytokines and the histological and biochemical supports the concept of a perpetual cascade of cytokines produced immediately after irradiation, prompting collagen genes to turn on, and persisting until the expression of late effects becomes apparent pathologically and clinically.Although the mechanism of radiation-induced pulmonary pneumonitis is not known, it is believed that the histopathologic alterations get the conformity findings which include basal membrane injury after irradiation, macrophage infiltration in air spaces, edema in the alveolar wall and/or air spaces, desquamation of epithelial cells from the alveolar walls, thickening of the alveoli septa by infiltration of inflammatory cells, collagen deposition, progressive fibrosis of alveolar septa, and obliteration of the alveoli. Protein-rich edema and hyalin membranes were typical features of radiation-induced injury. In addition, focal fibrosis arose within inflammation between 5 and 7 months, especially in subpleural regions.
It has been demonstrated that radiation-induced pulmonary pneumonitis develops to be pulmonary interstitial fibrosis ultimately. The defects in the alveolar epithelium and basement membrane allow the migration of mesenchymal cells from the interstitium into the intraluminal compartment. Intraluminal fibrosis is one of the major characteristics of Radiation-induced pulmonary pneumonitis and is associated with recruitment of inflammatory cells, particularly, macrophages, neutrophils and lymphocytes in the airways leading to an imbalance between the synthesis and degradation of extracellular matrix molecules in the local lung environment. Matrix metalloproteinases (MMPs), or matrixins, are zinc and calcium-dependent enzymes being able to degrade virtually all extracellular matrix components including collagens, fibronectin, laminin, entactin, nidogen and heparan sulfate proteoglycans and modulate cell behaviour by creating influential cellular environment. MMPs can affect the adherence of cells to the extracellular matrix by their proteolytic activity as well as release both bioactive fragments of extracellular matrix molecules and "trapped" bioactive mediators, providing signals from the microenvironment to cells allowing them to react to stimulus. As for MMPs, TIMP expression in tissues is tightly regulated to maintain an equilibrium between proteolysis and proteolysis inhibition leading extracellular matrix as a stable medium maintaining plastic capacities. In pathological conditions, switching the expression and activity of MMPs leads to states of either exaggerated extracellular matrix turnover often leading to remodelling via impaired repair and scar formation or extracellular matrix accumulation leading to fibrosis.MMP-2 (gelatinase A) can degrade typeⅣcollagen, one of the major components of the basement membrane and is localized in structural cells like fibroblasts, bronchial epithelial smooth cells and muscle cells in healthy lung. MMP-9 (gelatinase B) is capable of degrading typeⅣcollagen, laminin, elastase, and fibronectin in the lung interstitial matrix. It is secreted by a wide range of cells, including neutrophils, macrophages, activated capillary endothelial cells, and trophoblasts, from both normal and malignant tissue. MMP-9 could be rather linked to inflammation-induced tissue remodeling, while MMP-2 may be associated with an impaired tissue remodeling leading to pathological collagen deposition and interstitial fibrosis.Yang explored that the expressions of MMP-2 were enhanced by 1.7 and 1.9 folds, and MMP-9 by 2.7 and 2.6 folds at 4 and 8 weeks after thoracic irradiation, respectively. The result presumed that enhanced expressions of MMP-2 and MMP-9 in the lung were involved in the development of acute lung injury after thoracic irradiation, leading to a disruption of the structure and fibrosis.Over-expressions of MMP-9 and MMP-2 after lung irradiation are involved in the inflammatory response associated with radiation-induced lung injury, and maybe further in radiation-induced lung fibrosis.Araya measured the activity of MMP-2 in normal human bronchial epithelial cells as well as in A549 cells with zymography and the MMP-2 mRNA level with RQ-PCR. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. These results indicate that MMP-2 expression by human lung epithelial cells may be involved in radiation-induced lung injury. However, Susskind's study presume the decrease in plasma MMP-9 after initiation of chest radiation therapy appears to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiation induced lung damage.
Just as we have mentioned, the key to future success in developing useful treatment strategies for the disease will be to focus on developing a more complete understanding of the fundamental molecular mechanisms. Extracellular matrix imbalance is one of the major features of the pathological process. It is our goal in this article to present the role of MMP-2 and MMP-9 in the radiation-induced pulmonary pneumonitis, so that we can propose some different approaches to reestablishing the protease/antiproteases homeostasis in the future. First, antiprotease therapy for instant will be considered to control the disease extension and maybe have a preventive effect simultaneously. Alternatively, an hypothetical therapeutic allowing the MMP activity to control the collagen accumulation by tight regulation of TIMP activity intervention could be a curative approach of the disease.
In this research a single-dose of 30Gy irradiation of right hemithorax and sham right lung irradiation in Wistar rats was delivered and then sacrificed respectively on d_1, d_7, d_(14), d_(28), d_(56) and d_84). The values will be measured at different stages, including clinical manifestation, HA, HPCⅢ, CⅣand LN in serum and bronchoalveolar lavage fluid (BALF), total cellular score and proportion of composition in BALF, pathological change (for instant hemotoxylin and eosin (H&E) stain, immunohistochemistry, collagen fibers specific stain), real-time quantitative polymerase chain faction (RQ-PCR). Firstly, it is expected to find out the characteristic of Radiation-induced pulmonary pneumonitis at different stages and to confirm the role of MMP-2 and MMP-9 in this disease. Secondly, we will analyze the pathophysiology of Radiation-induced pulmonary pneumonitis out of irradiation fields. Meanwhile interferon-γ(IFN-γ) will be admoved experimentally to explore the relationship between the MMP-2/9 and IFN-γand whether it will degrade the radiation injury
The results are as the following:
(1) Significant Increasing of HA was seen both in the serum and BALF in the early stage of radiation fibrosis of lung. There were positive correlations between HA in serum and degree of pulmonary fibrosis (r_s=0.454, P=0.010) or collogenⅠ(r_s=0.450, P=0.011), and negative correlation between the HA in serum and collogenⅠ(r_s=-0.427, P=0.017). We suspected that high-level of HA intervene in the metabolism of collogen, and participate the proceed of pulmonary fibrosis;
(2) LN in the serum was always almost low-level after irradiation, whereas LN in BALF was continusly high-level in the stage of Pulmonary radiation alveolitis and pulmonary fibrosis. This might be attributed to the location of LN in the basal membrane, and LN prefer to infiltrate into the alveolar space. We guess that LN participate the whole proceed of radiation induce lung injury, especially in the stage of pulmonary fibrosis;
(3) Significant increasing of collogenⅢin serum was seen on the 1st to 2nd week (P=0.007), and it decreased to normal level 4 weeks later, collogenⅢin BALF increased to the peak value on the 2nd week and then maintained to the normal level. It was proposed that collogenⅢparticipate the acute pulmonary alveolitis, and the level of collogenⅢin the serum might be one of the monitoring indicatror for the acute pulmonary alveolitis;
(4) CollogenⅣin serum and BALF increased templely on the 1st week whereas maintained in a very low level at the stage of pulmonary fibrosis. A great quantity hyperplasian of CollogenⅣwas seen in the interstitial substance of lung even to the proporation of 90%. That may due to the micrangium occlusion and high molecular weight of collogenⅣso that it was difficult to release into the blood;
(5) The express of MMPS mRNA increased to the peak value in the stage of the most severe pulmonary alveolitis and maintained until the early stage of pulmonary fibrosis. The spearman method analysed that there were positive correlations between MMP-9 in the pulmonary macrophage and degree of pulmonary alveolitis (r_s=0.808, P=0.000)or pulmonary fibrosis(r_s=0.415, P=0.020);
(6) The myofibroblasts beside the pneumoangiogram might be the main resourse of MMP-2 and MMP-9. The result was that myofibroblasts participate the early stage of radiation induced lung injury, and it might be the main effective cell which influence the metabolic disorder of collagen. We suspected that myofibroblasts could be the target cell to prevention and cure the radiation induced lung injury;
(7) The express of MMPS in tunica mucosa bronchiorum was constantly in a low-level. We suspected that MMPS in tunica mucosa bronchiorum was main sourse to main the metabolic equilibrium;
(8) The variance of molecular level occurred within 24 hours after irradiation. It was suggest that lung injury did not have stage of latency. It was evidence that the classic concept of "latency" was actually the time for multiple cytokines and cells to transfer information of irradiation injury. For this reason we suggest preventing and curing the radiation induced lung injury nonagely, promptly and multipathly;
(9) We found the "out-field" and "in-field" lung tissue got alveolitis differently significantly (p=0.000) within 2 weeks, but between 2 to 12 weeks they got alveolitis which appearance no significantly difference. The probalble explanation was that cytokines accumulated within 2weeks and released into blood so that the out-field lung injury was induced;
(10) We found increasing of MMPS in the out-field lung tissue and that at least suggest that CD4+T cell and with the induced cytokines were not the only biology mediums.Demonstration of involvement of pulmonary tissue outside the irradiated area is consistent with a hypersensitivity-type pneumonitis characterized by multiple cytokines that settles spontaneously without long-term sequelae or lethality;
(11) Early intervention with IFN-γcould inhibit the secretion of MMPS, collogenⅢand collogenⅠ, and prevent the development of alveolitis but not fibrosis of lungs in radiation induced pulmonary injury rats. The machanism was unknown as yet.
一、立项背景及国内外研究现状
胸部恶性肿瘤如肺癌、食管癌、乳腺癌等是我国最常见的恶性肿瘤，发病率呈逐年上升的趋势，是我国居民最主要的死亡原因之一。放射治疗作为无创或微创性治疗的手段，因其明显提高的局部控制率和生存率，越来越多地应用于胸部恶性肿瘤的治疗。放射性肺损伤作为胸部肿瘤放射治疗最显著的并发症，是影响肿瘤治愈患者生活质量的主要原因之一。研究放射性肺损伤的发生发展机制，并以此为基础寻找预防或治疗放射性肺损伤的途径，是目前国内外肿瘤放射治疗学者热切期待解决的问题。
到目前为止，放射性肺损伤的发病机制尚未清楚。关于放射性肺损伤发病机制的学说主要分为三种：①小血管及肺Ⅱ型细胞损伤学说；②自由基学说；③细胞因子学说。但是，任何一种学说都不能解释放射性肺损伤的整个病理生理过程。
然而幸运的是，放射性肺损伤的病理形态学却有一致的发现：放射性肺损伤的发生过程中，肺泡基底膜的损伤导致炎症因子和细胞在肺泡腔内及肺间质聚集，肺泡上皮细胞脱落、表面活性物质的丢失及胶原的沉积最终导致了肺泡塌陷及肺间质纤维化。显然，在这一演变过程中，以Ⅳ型胶原为主要成分的肺泡基底膜损伤对于肺间质纤维化的形成足十分必要的，是放射性肺泡炎和肺间质纤维化病理变化的关键事件。而包括肺泡基底膜在内的细胞外基质(extracellular matrix，ECM)的代谢失衡在放射性肺损伤的发生过程中可能发挥了关键作用，这是通过调节ECM合成与分解代谢实现的。
基质金属蛋白酶(matrix metalloproteinases，MMPs)是一类以降解ECM为主要功能的蛋白酶类，也是目前公认ECM降解最重要的蛋白酶类。包括胶原酶、间质胶原酶、弹性蛋白酶等，其作用底物是构成基底膜的纤维连接素、板层素、胶原、糖蛋白与弹性蛋白等。Ⅳ型胶原是构成基底膜的主要成分。明胶酶B(GelatinaseB，MMP9)和明胶酶A(GelatinaseA，MMP2)是体内Ⅳ型胶原的主要降解酶，是MMPs的亚类之一。它们在体内主要受三个水平的调节：①基因水平的调节；②酶原活化调节；③活化后调节。
显然，MMPS的代谢平衡是维持ECM代谢平衡的物质基础。根据放射性肺损伤发生发展的病理生理特点，结合MMPS的生物学特性，可以推断MMPS与放射性肺损伤之间可能存在一定的因果关系，这种因果关系可能会成为阻止放射性肺损伤发生发展的关键节点，成为临床预防或治疗放射性肺损伤的关键环节。
寻找简单易行的方法早期预测和防治放射性肺损伤的发生和程度，及早进行干预，减轻放射性肺损伤的程度，延缓放射性肺损伤的进程，是本课题研究的目的。
二、国内外研究现状
MMPs的研究目前是国内外各学科研究的热点之一，尤其在创伤修复、胚胎发育、恶性肿瘤的浸润与转移等方面均有大量的文献报道，其实验室技术趋于成熟。
上世纪90年代中期，国外学者开始将MMPs及其抑制物与肺组织的损伤与修复联系在一起。特发性肺纤维化、慢性阻塞性肺纤维化等疾病的研究已发现，ECM与MMPS之间的失衡，可能是肺纤维化类疾病发生发展的关键环节。同时，国内外研究者还发现，临床上能够观察到的肺纤维化改变往往比肺实质的分子生物学改变至少晚1-3个月。也就是说，在患者尚未开始出现肺纤维化症状的早期，由细胞因子介导的肺纤维化损伤已经悄悄开始了。这个结论被国内外文献大量的证实，如博来霉素、吸烟诱发的肺纤维化过程中MMPS表达均有改变。
放射治疗是一门新兴的边缘学科，放射性肺损伤是胸部肿瘤放射治疗最显著的并发症，，其最终发展成为不可逆的肺纤维化。放射性肺损伤的本质是一个渐进性的肺间质纤维化过程，它的发病机理很有可能与肺纤维化类疾病有共同特点，即与MMPs引起的代谢紊乱有关。国内外有学者猜测MMPs可能与放射性肺损伤的发生机制有关：Susskind等报道了肺癌、乳腺癌患者放疗后血浆中MMP-9的升高，猜测可能与放射性肺损伤有关；Araya等通过体外培养人支气管上皮细胞株，照射后发现MMP-2表达增高，由此得出MMP-2可能是放射性肺损伤的重要产物。杨坤禹等以12Gy照射C57BLP6J TGF-β基因敲除的雌性小鼠双侧肺，发现照射后小鼠肺组织中MMP-2和MMP-9的表达增加，预测MMP-2和MMP-9可能加剧了急性放射性肺损伤。
查阅文献可知，目前国内外尚无文献报道通过系统的动物模型实验，从组织学、蛋白水平、基因水平等多个层面证实MMPs在放射性肺损伤的过程中的重要作用；尚无报道通过动物实验前瞻性研究照射后引起的射野外淋巴细胞性肺泡炎的病理生理机制；尚无预测放射性肺损伤的发生及程度的血清学指标。
三、实验目的和内容
进一步证实MMP-2，9在放射性肺损伤的发生发展过程中动态改变及重要作用，寻找预测放射性肺损伤的血清学指标，初步研究射野外淋巴细胞性肺泡炎的病理生理机制，及早进行干预，减轻放射性肺损伤的程度，延缓放射性肺纤维化的进程。
我们建立了Wistar大鼠半胸照射的放射性肺损伤实验动物模型，主要实验内容如下：
1．观察照射后不同时间实验动物临床表现(包括毛发、体重、死亡：率等)，血清和肺泡灌洗液纤维化指标(HA，HPCⅢ，CⅣ，LN)的变化，肺泡灌洗液细胞总数量和成分比例的变化；
2．观察照射后肺组织病理变化(普通光镜HE染色，Ⅰ型、Ⅲ型和Ⅳ型胶原纤维特殊染色，MMP-2，9免疫组织化学)，活体组织内MMP-2，9mRNA的实时定量PCR(Real-time quantitative polymerase chain raction，RQ-PCR)变化，比较各观察指标随着放射性肺损伤不同时期动态变化的特点，并证实MMP-2，9在放射肺损伤发生机制中的作用；
3．比较正常对照组、照射一侧肺及未照射一侧肺组织的各项指标，分析射野外发生的淋巴细胞性肺泡炎的病理生理机制，研究散射线对射野外淋巴细胞性肺损伤发生几率的影响；
4．试探性应用能下调MMPS表达的干扰素γ，观察其是否能减轻放射性肺损伤，以及对MMPS的影响，为临床预防或治疗放射性肺损伤提供理论依据。
四、该项目的创新性
1．本项目将通过动物实验，从组织学、蛋白水平、基因水平多个层面证实MMPs在放射性肺损伤的过程中的重要作用；
2．通过比较各观察指标随时间动态变化的特点，解释放射性肺损伤早期、晚期发生发展的共性和特性，为预防和治疗放射性肺损伤提供临床干预可能的靶点；
3．比较血浆中纤维化指标与肺组织或支气管肺泡灌洗液的各项指标，寻找潜在的联系，为临床实践提供可行性强的预测和监测指标，这是本项目紧密联系临床需要的特色之一；
4．本项目首次通过建立动物实验模型，比较射野内和射野外肺组织的各项指标，研究射野外淋巴细胞性放射性肺损伤(“旁观者”效应)的发生机制；
4．通过建立动物实验模型，初步研究IFN-γ在放射性肺损伤发生发展过程的作用。
五、技术路线
本实验的主要技术路线如下：
六、主要结果
1．血清和BALF中HA水平在放射性肺纤维化早期显著升高，血清HA与肺间质纤维化水平呈正相关(r_s＝0.454，P＝0.010)，与Ⅰ型胶原含量呈负相关(r_s＝-0.427，P＝0.017)，与Ⅳ型胶原含量呈正相关(r_s＝0.450，P＝0.011)，说明了高水平的HA可能干扰了胶原代谢，从而参与了早期放射性肺纤维化的形成。血清或BALF中的显著升高，有可能预示着放射性肺纤维化已经开始；
2．血清LN水平在照射后几乎处于持续的低水平状态，而BALF LN在放射性肺泡炎、放射性肺纤维期持续处于高水平状态，可能与LN位于肺基底膜，照射后由于基底膜破坏导致间隙增宽，LN更有利于渗透至肺泡腔内，完成诱导炎症细胞的浸润、胶原纤维的生成等生物功能有关。因此，我们猜测LN参与了肺泡炎的全过程，尤其是在放射性肺纤维化期，BALF中LN持续处于高水平状态，可能在促进肺泡结构的消失，肺纤维化的形成有重要意义；
3．血清中PCⅢ在照射后1～2周就显著升高(P＝0.007)，至照射后4周降低，逐渐恢复至正常水平，其变化与肺间质炎症程度呈正相关(P＝0.022)。BALF中的PCⅢ在照射后2周达到峰值后持续维持在低于正常水平。提示Ⅲ型胶原可能参与了急性肺泡炎症的形成。即血清中PCⅢ的浓度有可能可以作为反映肺间质炎症的监测指标之一；
4．急性炎症期血清和BALF中IV型胶原一过性的升高，纤维化阶段维持在较低水平。而组织学却发现纤维化阶段Ⅳ型胶原在肺间质大量增生，占肺间质胶原的90％以上。可能与放射性肺纤维化阶段，由于基膜和间质的增生，毛细血管闭塞，肺泡结构消失，Ⅳ型胶原分子量大，不易穿透增生、闭塞的毛细血管壁释放入血液，故不能在BALF或者血清中显示出来；
5．肺巨噬细胞MMP-9、MMP-2的表达在放射性肺泡炎症最明显的时期表达最强烈，并持续至放射性肺纤维化早期。相关性分析表明肺巨噬细胞表达MMP-9、MMP-2与肺泡炎程度呈正相关(r_s＝0.808，P＝0.000)，与肺间质纤维化程度呈正相关(r_s＝0.415，P＝0.020)，这可能预示了巨噬细胞同时参与了放射性肺泡炎和早期放射性肺纤维化的形成；
6．肺血管旁肌成纤维细胞可能也是MMP-9、MMP-2的主要来源，照射后1天肌成纤维细胞MMPS呈强阳性，照射后1周呈持续强阳性表达，照射1周后开始进行性下降，至照射后4周，已几乎降低至正常水平，照射后3月，血管周围平滑肌几乎不表达MMP-9、MMP-2，显著低于正常对照组(χ~2＝23.624，P＝0.001)。实验结果提示肌成纤维细胞参与了早期放射性肺损伤的发生，是影响胶原代谢紊乱的主要效应细胞之一，从而诱导了放射性肺纤维化的形成，而且可能是纤维化进程中的关键步骤。因此，肌成纤维细胞可能成为控制放射性纤维化进展的重要靶细胞；
7．支气管粘膜上皮细胞在照射后一直处于低于正常水平的表达，猜测支气管粘膜上皮在正常情况下是维持ECM代谢平衡的主要来源之一，而在照射后粘膜上皮细胞高度水肿，大片脱落至支气管腔，细胞破裂崩解，可能成为MMP-9和MMP-2表达下降的原因之一；
8．照射后24小时内即可观察到肺组织分子水平的变化，说明放射损伤并不存在潜伏期。这一结果改变了我们以往对放射损伤的传统认识。从我们的实验可以看出，这个从分子水平到临床表现的所谓的潜伏期，实际上是一个肺组织内多种效应细胞如肺泡巨噬细胞、肌成纤维细胞、ECM等完成损伤信息传递，启动多种细胞因子瀑布效应的时期。因此，正常肺组织对放射损伤的反应是一个即时、动态的连续过程。正因如此，我们建议预防和治疗放射性肺损伤，应该早期、及时、多途径的阻断放射性肺损伤的“瀑布效应”；
9．本实验研究发现照射野内和野外肺组织在照射后12周内肺泡炎症程度有显著差异性(P＝0.000)。但其差异性主要表现在照射后2周前，照射野外比照射野内的肺组织炎症发生更晚且程度轻(P＝0.000)，而在照射后2周～12周，双侧肺组织肺泡炎程度无显著性差异(P＝0.170)。因此，我们认为在照射后放射性肺损伤发生的早期，放射性肺损伤的炎症因子处于动员及诱导阶段，并局限在受照射肺组织局部。随着炎症因子和可能产生的自身抗体逐渐累积并释放入血，放射损伤才开始逐渐诱导照射野外放射性肺损伤的发生，并使之与照射野内的放射性肺损伤达到同步；
10．在30Gy照射组的左侧肺，即照射野外发现MMP-2 mRNA和MMP-9mRNA表达增高，说明CD4＋T及其介导的细胞因子可能并不是唯一参与照射野外放射性肺损伤发生机制的生物介质。而临床上照射野外肺组织的微观变化，很有可能发生在每例胸部局部放射治疗的患者(其照射剂量或照射体积可能已经大于肺组织的耐受剂量或域体积)。而只有5％～10％的患者由于对血清中放射损伤产生的生物介质产生了“超敏”反应(类似“过敏性肺炎”或“自身免疫性肺炎”)，故产生了干咳、呼吸困难等症状。因为射野外的放射性肺损伤是免疫亢进诱导的肺炎，故用激素治疗显示了良好的效果，且不会遗留长期的后遗症，这些特点恰恰与我们临床看到的病例特征符合；
11．我们发现IFN-γ在放射性肺损伤的发生过程中降低了MMPS的表达，抑制了Ⅰ型和Ⅲ型胶原的合成，有效减少了照射后的放疗反应，提高了实验动物在照射后的存活质量，延迟放射性肺泡炎的发生时间并降低其发生的程度。而对放射性肺纤维化的作用有轻度减弱，但尚无显著性意义。其原因可能需要更长期的随访和进一步的实验研究证实。
Thoracic tumors such as lung cancer, breast cancer, lymphoma is the most widespread malignant tumor and is one of the supreme cause of death in China. Radiation is a common modality used for the treatment of thoracic malignancies, but injury to surrounding normal lung is a major limitation that dictates the ultimate dose, volume, and technique of radiation.
Radiation-induced pulmonary pneumonitis and fibrosis secondary is a major limiting factor in the successful treatment of thoracic tumors. The disease afflicts millions of individuals worldwide and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of the disease have been defined sufficiently to design appropriate targets for therapy. The radio-oncologists have focused on the molecular mechanisms through which growth factors could play a role in the development of radiation-induced pulmonary injury, and wish to get the key targets to prevention and cure the disease.
Radiation-induced pulmonary reactions have classically been viewed as distinct phases-acute pneumonitis and, later, fibrosis-occurring at different times after irradiation and attributed to different target cell populations.As yet our understanding of disease pathogenesis is still controversial. Shapiro presumed irradiation appears to trigger membrane receptor mediated surfactant release. The typeⅡpneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant. Cell cultures of typeⅡpneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 tad also in vitro. Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes. In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism. These studies establish that surfactant release in response to irradiation in vivo is a direct effect on typeⅡpneumocytes.
Bai discussed the role of the free radicals (FR) in the developing process of radiation interstitial pneumonitis. The morphologic changes were examined in the lung tissue of Wistar rats after chest irradiation and determined sequentially. The content of FR were examined in the lung tissue of Wistar rats after chest irradiation using electron spin resonance. The activity of superoxide dismutase (SOD) was assessed using the chemical glow method of xanthine oxidase. After chest irradiation, the content of FR in the lung progressively increased and the activity of SOD progressively decreased. De's study showed that mitochondria and microsomes in the lung of Wistar rats irradiated by gamma-rays from a Co source at the thoracic region had higher levels of lipid peroxidation. In the pneumonitic rat the activities of superoxide dismutase and catalase were markedly reduced in the cytoplasmic fraction but not significantly altered in lung mitochondria. On the contrary, lipid peroxidation and the two enzymes in lung tissue in the non-pneumonitic group of the irradiated rat were comparable with that of control animals. The results indicate that free radical-induced oxidative stress following thoracic irradiation may be one of the causative factors in the development of interstitial pneumonitis.
Rubin prefer to view these events as a continuum progression that is the result of an early activation of an inflammatory reaction, leading to the expression and maintenance of a cytokine cascade. The temporal sequence relationship between the elevation of specific cytokines and the histological and biochemical supports the concept of a perpetual cascade of cytokines produced immediately after irradiation, prompting collagen genes to turn on, and persisting until the expression of late effects becomes apparent pathologically and clinically.Although the mechanism of radiation-induced pulmonary pneumonitis is not known, it is believed that the histopathologic alterations get the conformity findings which include basal membrane injury after irradiation, macrophage infiltration in air spaces, edema in the alveolar wall and/or air spaces, desquamation of epithelial cells from the alveolar walls, thickening of the alveoli septa by infiltration of inflammatory cells, collagen deposition, progressive fibrosis of alveolar septa, and obliteration of the alveoli. Protein-rich edema and hyalin membranes were typical features of radiation-induced injury. In addition, focal fibrosis arose within inflammation between 5 and 7 months, especially in subpleural regions.
It has been demonstrated that radiation-induced pulmonary pneumonitis develops to be pulmonary interstitial fibrosis ultimately. The defects in the alveolar epithelium and basement membrane allow the migration of mesenchymal cells from the interstitium into the intraluminal compartment. Intraluminal fibrosis is one of the major characteristics of Radiation-induced pulmonary pneumonitis and is associated with recruitment of inflammatory cells, particularly, macrophages, neutrophils and lymphocytes in the airways leading to an imbalance between the synthesis and degradation of extracellular matrix molecules in the local lung environment. Matrix metalloproteinases (MMPs), or matrixins, are zinc and calcium-dependent enzymes being able to degrade virtually all extracellular matrix components including collagens, fibronectin, laminin, entactin, nidogen and heparan sulfate proteoglycans and modulate cell behaviour by creating influential cellular environment. MMPs can affect the adherence of cells to the extracellular matrix by their proteolytic activity as well as release both bioactive fragments of extracellular matrix molecules and "trapped" bioactive mediators, providing signals from the microenvironment to cells allowing them to react to stimulus. As for MMPs, TIMP expression in tissues is tightly regulated to maintain an equilibrium between proteolysis and proteolysis inhibition leading extracellular matrix as a stable medium maintaining plastic capacities. In pathological conditions, switching the expression and activity of MMPs leads to states of either exaggerated extracellular matrix turnover often leading to remodelling via impaired repair and scar formation or extracellular matrix accumulation leading to fibrosis.MMP-2 (gelatinase A) can degrade typeⅣcollagen, one of the major components of the basement membrane and is localized in structural cells like fibroblasts, bronchial epithelial smooth cells and muscle cells in healthy lung. MMP-9 (gelatinase B) is capable of degrading typeⅣcollagen, laminin, elastase, and fibronectin in the lung interstitial matrix. It is secreted by a wide range of cells, including neutrophils, macrophages, activated capillary endothelial cells, and trophoblasts, from both normal and malignant tissue. MMP-9 could be rather linked to inflammation-induced tissue remodeling, while MMP-2 may be associated with an impaired tissue remodeling leading to pathological collagen deposition and interstitial fibrosis.Yang explored that the expressions of MMP-2 were enhanced by 1.7 and 1.9 folds, and MMP-9 by 2.7 and 2.6 folds at 4 and 8 weeks after thoracic irradiation, respectively. The result presumed that enhanced expressions of MMP-2 and MMP-9 in the lung were involved in the development of acute lung injury after thoracic irradiation, leading to a disruption of the structure and fibrosis.Over-expressions of MMP-9 and MMP-2 after lung irradiation are involved in the inflammatory response associated with radiation-induced lung injury, and maybe further in radiation-induced lung fibrosis.Araya measured the activity of MMP-2 in normal human bronchial epithelial cells as well as in A549 cells with zymography and the MMP-2 mRNA level with RQ-PCR. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. These results indicate that MMP-2 expression by human lung epithelial cells may be involved in radiation-induced lung injury. However, Susskind's study presume the decrease in plasma MMP-9 after initiation of chest radiation therapy appears to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiation induced lung damage.
Just as we have mentioned, the key to future success in developing useful treatment strategies for the disease will be to focus on developing a more complete understanding of the fundamental molecular mechanisms. Extracellular matrix imbalance is one of the major features of the pathological process. It is our goal in this article to present the role of MMP-2 and MMP-9 in the radiation-induced pulmonary pneumonitis, so that we can propose some different approaches to reestablishing the protease/antiproteases homeostasis in the future. First, antiprotease therapy for instant will be considered to control the disease extension and maybe have a preventive effect simultaneously. Alternatively, an hypothetical therapeutic allowing the MMP activity to control the collagen accumulation by tight regulation of TIMP activity intervention could be a curative approach of the disease.
In this research a single-dose of 30Gy irradiation of right hemithorax and sham right lung irradiation in Wistar rats was delivered and then sacrificed respectively on d_1, d_7, d_(14), d_(28), d_(56) and d_84). The values will be measured at different stages, including clinical manifestation, HA, HPCⅢ, CⅣand LN in serum and bronchoalveolar lavage fluid (BALF), total cellular score and proportion of composition in BALF, pathological change (for instant hemotoxylin and eosin (H&E) stain, immunohistochemistry, collagen fibers specific stain), real-time quantitative polymerase chain faction (RQ-PCR). Firstly, it is expected to find out the characteristic of Radiation-induced pulmonary pneumonitis at different stages and to confirm the role of MMP-2 and MMP-9 in this disease. Secondly, we will analyze the pathophysiology of Radiation-induced pulmonary pneumonitis out of irradiation fields. Meanwhile interferon-γ(IFN-γ) will be admoved experimentally to explore the relationship between the MMP-2/9 and IFN-γand whether it will degrade the radiation injury
The results are as the following:
(1) Significant Increasing of HA was seen both in the serum and BALF in the early stage of radiation fibrosis of lung. There were positive correlations between HA in serum and degree of pulmonary fibrosis (r_s=0.454, P=0.010) or collogenⅠ(r_s=0.450, P=0.011), and negative correlation between the HA in serum and collogenⅠ(r_s=-0.427, P=0.017). We suspected that high-level of HA intervene in the metabolism of collogen, and participate the proceed of pulmonary fibrosis;
(2) LN in the serum was always almost low-level after irradiation, whereas LN in BALF was continusly high-level in the stage of Pulmonary radiation alveolitis and pulmonary fibrosis. This might be attributed to the location of LN in the basal membrane, and LN prefer to infiltrate into the alveolar space. We guess that LN participate the whole proceed of radiation induce lung injury, especially in the stage of pulmonary fibrosis;
(3) Significant increasing of collogenⅢin serum was seen on the 1st to 2nd week (P=0.007), and it decreased to normal level 4 weeks later, collogenⅢin BALF increased to the peak value on the 2nd week and then maintained to the normal level. It was proposed that collogenⅢparticipate the acute pulmonary alveolitis, and the level of collogenⅢin the serum might be one of the monitoring indicatror for the acute pulmonary alveolitis;
(4) CollogenⅣin serum and BALF increased templely on the 1st week whereas maintained in a very low level at the stage of pulmonary fibrosis. A great quantity hyperplasian of CollogenⅣwas seen in the interstitial substance of lung even to the proporation of 90%. That may due to the micrangium occlusion and high molecular weight of collogenⅣso that it was difficult to release into the blood;
(5) The express of MMPS mRNA increased to the peak value in the stage of the most severe pulmonary alveolitis and maintained until the early stage of pulmonary fibrosis. The spearman method analysed that there were positive correlations between MMP-9 in the pulmonary macrophage and degree of pulmonary alveolitis (r_s=0.808, P=0.000)or pulmonary fibrosis(r_s=0.415, P=0.020);
(6) The myofibroblasts beside the pneumoangiogram might be the main resourse of MMP-2 and MMP-9. The result was that myofibroblasts participate the early stage of radiation induced lung injury, and it might be the main effective cell which influence the metabolic disorder of collagen. We suspected that myofibroblasts could be the target cell to prevention and cure the radiation induced lung injury;
(7) The express of MMPS in tunica mucosa bronchiorum was constantly in a low-level. We suspected that MMPS in tunica mucosa bronchiorum was main sourse to main the metabolic equilibrium;
(8) The variance of molecular level occurred within 24 hours after irradiation. It was suggest that lung injury did not have stage of latency. It was evidence that the classic concept of "latency" was actually the time for multiple cytokines and cells to transfer information of irradiation injury. For this reason we suggest preventing and curing the radiation induced lung injury nonagely, promptly and multipathly;
(9) We found the "out-field" and "in-field" lung tissue got alveolitis differently significantly (p=0.000) within 2 weeks, but between 2 to 12 weeks they got alveolitis which appearance no significantly difference. The probalble explanation was that cytokines accumulated within 2weeks and released into blood so that the out-field lung injury was induced;
(10) We found increasing of MMPS in the out-field lung tissue and that at least suggest that CD4+T cell and with the induced cytokines were not the only biology mediums.Demonstration of involvement of pulmonary tissue outside the irradiated area is consistent with a hypersensitivity-type pneumonitis characterized by multiple cytokines that settles spontaneously without long-term sequelae or lethality;
(11) Early intervention with IFN-γcould inhibit the secretion of MMPS, collogenⅢand collogenⅠ, and prevent the development of alveolitis but not fibrosis of lungs in radiation induced pulmonary injury rats. The machanism was unknown as yet.
引文
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