【作者】 王宝中；

【导师】 王秀问；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2014， 博士

【摘要】 研究背景及意义放射性肺损伤(radiation-induced lung injury, RILI)是胸部肿瘤放疗后、骨髓移植预处理及战时核辐射常见的并发症。数据表明,临床上大约有30%的肺癌病人及10-15%其它胸部肿瘤病人放疗后可出现明显临床症状的放射性肺损伤。即便应用目前最为先进的适形调强放疗,也难以避免其发生。由于对于放射性肺损伤发生机制认识的局限性,数十年来有关放射性肺损伤的治疗一直没有突破性进展。临床上通过尽可能的缩小照射野或降低靶区剂量,来降低放射性肺损伤发生的可能。然而,肿瘤靶区剂量的限制,势必会影响肿瘤局控率。近年来,随着对放射性肺损伤的深入研究,人们逐渐认识到放射性肺损伤是由多种细胞因子介导的多细胞间的相互作用并维持着放射性肺损伤的全过程。已有研究证实,IL-1、IL-6、TGF-β、INF-α等多种细胞因子参与了放射性肺损伤的过程,并发挥重要作用,而以IL-6、TGF-β、TNF-α为靶点的治疗,可以减轻肺损伤的程度。然而,以单一细胞因子为靶点的实验性治疗,效果有限。这似乎说明可能还存在其它重要相关的细胞因子在放射性肺损伤过程中发挥重要作用。IL-17A是新近发现的一种前炎症因子,高表达于各组纤维化相关疾病的组织器官中。动物实验证实,IL-17A在博来霉素或二氧化硅诱发的小鼠肺损伤过程中发挥重要作用,并且通过IL-17A拮抗剂减轻了这种性质的肺损伤程度。Wilson等通过敲除小鼠IL-17A基因,发现IL-17A (-/-)小鼠化学性肺炎及纤维化程度明显弱于IL-17A野生型。基于以上研究,我们推测IL-17A在放射性肺损伤过程中,可能也发挥有重要作用。文献检索鲜见IL-17A在放射性肺损伤方面的研究。本课题研究IL-17A在放射性肺损伤不同阶段的表达,目的是为了证实IL-17A参与并在放射性肺损伤过程中发挥有重要作用,从而为防治放射性肺损伤提供一有效靶点。以IL-17A为靶点的放射性肺损伤治疗,可能是一个新的有效的临床途径。本研究分为三个部分进行阐述。第一部分研究目的：建立快捷、稳定的小鼠放射性肺损伤动物模型,并对其进行有效评价,为研究放射性肺损伤提供一可靠的平台。研究方法：近交系雄性C57BL/6小鼠90只,按照随机数目表法随机分为3组：(1)假照射组(n=20只),佯装照射,简称为Sham组；(2)单纯照射A组(n=35只),非麻醉状态,全胸一次性大剂量照射15Gy,简称为RT-A组；(3)单纯照射B组(n=35只),非麻醉状态,照射20Gy,简称为RT-B组。直线加速器照射小鼠后于第1、4、8、16周处死,分别取照射A、B组及假照射组小鼠肺组织,采用H&E、Masson染色法显微镜下观察肺组织病理学变化和胶原沉积,并按照Szapiel方法对放射性肺泡炎及肺纤维化的严重程度进行量化。通过碱水解法测定羟脯氨酸含量,用于评估肺组织胶原沉积情况。在生存期方面,将50只C57BL/6小鼠随机分为3组：(1)Sham组：n=10只；(2)RT-A组：n=20只；(3)RT-B组：n=20只。观察小鼠16周生存期,生存期分析应用Kaplan-Meier法及log-ranktest法。研究结果：小鼠全胸经照射后,肺组织经过一系列病理变化,包括：渗出期、增生期及纤维化期。HE染色切片显示照射8周内,肺组织主要是肺泡炎的表现：肺泡壁增厚、肺泡腔充血、肺间质水肿及毛细血管扩张、炎性细胞浸润等,且随时间延长肺泡炎程度逐渐加重。Masson染色显示8周开始出现灶状纤维组织增生,16周时纤维化病灶更为明显。按照Szapiel分级法,统计学分析显示肺泡炎程度：RT-A、B组均较Sham组严重,且RT-B组肺泡炎重于RT-A组。纤维化程度：RT-A、B组重于Sham组,且RT-B组纤维化程度较RT-A组严重。通过对各组小鼠肺组织中羟脯氨酸含量测定,发现RT-A、B组小鼠照射后,羟脯氨酸含量均随时间延长逐渐升高,且在病理学上出现灶状纤维化的第8周及第16周,羟脯氨酸升高趋势尤为明显。在该时间点上RT-B明显高于RT-A组,差异具有显著统计学意义(P<0.01)。小鼠生存时间方面,假照射组小鼠观察时间内无死亡。RT-A组小鼠16周存活率为80%。RT-B组小鼠16周存活率仅为40%。且照射B组小鼠死亡时间明显早于照射A组。两组小鼠存活率差异具有显著统计学意义(P=0.0241<0.05)。研究结论：直线加速器15Gy照射C57BL/6小鼠全胸,可以成功构建放射性肺损伤模型,为放射性肺损伤的分子机制的研究及防治靶点的筛选提供基础。第二部分研究目的：IL-17A作为新近发现的前炎症因子,在炎症或增殖性疾病中发挥重要作用。本部分研究旨在对IL-17A在放射性肺损伤不同阶段(肺炎及肺纤维化)的表达情况及可能发挥的作用进行探讨。研究方法：将61只C57BL/6小鼠随机分为两组：(1)假照射组,简称Sham组(n=25)；(2)照射组,简称RT组(n=36)；各组小鼠分别在观察时间点1周、4周、8周、16周处死,并进行性病理组织学及分子生物学检测。实验通过Real time-PCR方法,检测各组肺组织IL-17A mRNA的表达情况,用2△△Ct法进行相对定量分析。并通过Western bolt印记和免疫组化的方法,检测转录后各组肺组织中IL-17A蛋白表达的情况。研究结果：免疫组化结果显示：小鼠全肺经15Gy射线照射后,肺组织IL-17A在不同阶段,呈现出不同程度的高表达。IL-17A阳性颗粒显示在肺泡巨噬细胞、上皮细胞、淋巴细胞及支气管上皮细胞等细胞胞浆内。real-time PCR结果显示：照射后小鼠肺组织IL-17A mRNA均有不同程度的表达增强,4周时表达最为显著(观察时间内),8周开始下降,16周继续下降,但仍高于基线水平。结合组织学切片染色结果,8周内放射性肺泡炎程度随时间的延长逐渐加重,而IL-17表达量却在8周时开始下降,提示IL-17A可能在早期放射性肺损伤中作用更为重要。Western blot半定量检测显示IL-17A蛋白表达量在肺照射1周后便开始抬高,4周最高,此后表达量开始下降。IL-17A蛋白表达情况,与mRNA结果基本一致。研究结论：小鼠全胸照射后,肺组织IL-17A mRNA及蛋白表达均出现不同程度的升高,与肺泡炎和纤维化形成的时间基本一致,考虑IL-17A在放射性肺泡炎及肺纤维化过程中发挥一定作用。第三部分研究目的：阻断IL-17A信号途径可明显改善多种炎症性疾病的转归。本部分研究旨在探讨IL-17A中和抗体对放射性肺损伤小鼠的保护作用。研究方法：我们采用直线加速器一次性15Gy剂量照射C57BL/6小鼠全胸,构建小鼠肺损伤模型。将135只小鼠随机分为四组：①假照射组(Sham,n=30)；②照射对照组(RC group,n=35);③治疗组(Treatment group, n=35);④安慰剂组(placebo group, n=35)。后二组分别于照射后每月第1,3,7,14天,分别静脉给予IL-17A中和抗体或同型对照IgG抗体4μg/鼠。通过H&E和Masson染色来评估小鼠肺组织炎症改变及胶原沉积情况。按照Aschcroft评分标准对肺泡炎、肺纤维化程度进行定量分析。支气管肺泡灌洗液中IL-17A,TGF-β1和IL-6含量采用ELISA的方法测定。羟脯氨酸含量采用碱水解法测定。另外,我们采用Kaplan-Meier方法进行小鼠的生存期分析。研究结果：照射后16周,照射对照组(RC组)和安慰剂组(Placebo组)小鼠肺组织表现为明显的炎性细胞浸润和间质胶原沉积现象,而治疗组(Treatment组)小鼠这些病理学改变则相对较轻。Treatment组小鼠Ⅱ-Ⅲ度肺泡炎发生率(16%)明显低于RC组(72%)或Placebo组(64%)。纤维化程度方面,各组Aschcroft评分分别为Treatment组2.8、RC组5.2、Placebo组4.8。Treatment组小鼠的Aschcroft评分明显低于RC组(P<0.001)或Placebo组(P<0.001)。Treatment组小鼠支气管灌洗液中IL-17A,TGF-p和IL-6含量明显低于RC组及Placebo组(P<0.01)。小鼠180天死亡率方面,Treatment组小鼠明显低于RC组(16.7%vs75.0%)。研究结论：IL-17A中和抗体的应用,明显减轻了放射性肺炎及其后的肺纤维化程度,提高了照射小鼠的生存期,对照射小鼠产生了明显的保护作用。以IL-17A为靶点或许为治疗放射性肺损伤的另一有效途径。更多还原

【Abstract】 Radiation-induced lung injury (radiation-induced lung injury, RILI) is a common complication after thoracic radiotherapy, bone marrow transplantation pretreatment and wartime nuclear radiation. Clinically significant radiation lung injury occurs in up to30%of patients irradiated for lung cancer and in about10-15%of other thoracic tumor patients. Even the most advanced application of Conformal IMRT, it is still difficult to prevent its occurrence. Due to the limitation of understanding the mechanism, there have been no strategic advances in RILI treatment over the past decades. Currently, the only way available to decrease the risk of developing severe radiation pneumonitis is to minimize the dose of radiation or the areas of lungs that are exposed to the radiation.The disadvantage is that tumor target dose must be limited definitely, which will inevitably reduce the tumor control rate. In recent years, it is increasingly recognized that radiation-induced lung injury is associated with a variety of multi-cytokine mediated interactions between cells. Several studies showed that IL-6, TGF-β, IL-1, and TNF-α are the key cytokines involved in the pathogenesis of radiation-induced lung injury, and simultaneous inhibition of IL-6, TGF-β, as well as TNF-α attenuates the degree of radiation-induced lung injury. However, targeting a single cytokine has limited effect on lung injury prevention. It seems that there might be other important related cytokines playing important roles in radiation-induced lung injury. IL-17A is a newly discovered pro-inflammatory cytokines, which is expressed in fibrosis-related diseases. IL-17A antagonist reduced the degree of pneumonitis and pulmonary fibrosis in bleomycin-or silica-induced lung injury in mice.Based on these studies, we hypothesized that IL-17A may also play an important role in the process of radiation-induced lung injury. Literature search was rarely found research about the roles of IL-17A in radiation-induced lung injury.The purpose of the research is to confirm that IL-17A plays an important role in the pathogenesis of radiation-induced lung injury, thereby providing a reliable target for prevention and treatment of radiation-induced lung injury. Targeting at IL-17A may be a new and effective clinical approach for radiation-induced lung injury. The study is divided into three parts to elaborate. Part1Objective:The purpose of the study is to establish mice model of radiation-induced lung injury quickly and efficiently, evaluate for the method and provide a reliable platform for the field of radiation-induced lung injury.Methods:A total of90mice (C57BL/6, Inbred male) were randomly divided into three groups:(1) Sham irradiation group (n=20), abbreviated as Sham group;(2) Radiation group A (n=35), non-anesthesia, one-time high-dose chest irradiation15Gy, abbreviated as RT-A group.(3) Radiation group B (n=35), non-anesthesia, irradiation20Gy, abbreviated as RT-B group. Mice were sacrificed after1,4,8,16week post-irradiation by linear accelerator. Lung tissues were taken out from mice of RT-A, B and sham group. Pathological changes and collagen deposition in lung tissue were observed under the microscope by the methods of H&E and Masson. The severity of alveolitis and pulmonary fibrosis was quantified according to Szapiel scales. Hydroxyproline content was measured by alkaline hydrolysis to evaluate collagen deposition in the lung tissue. In terms of survival,50mice were randomly divided into three groups:(1) Sham group (n=10);(2) RT-A group (n=20).(3) RT-B group (n=20). Survival rates were observed in16weeks, and Kaplan-Meier method and log-rank test were used to perform survival analysis.Results:After the mice were irradiated, a series of pathological changes occurred in the lung tissue. General speaking, there are three stages in the process:exudative, proliferative and fibrosis stage. Within8weeks, lung tissue H&E stained showed mainly alveolitis performance:alveolar wall thickening, alveolar congestion, interstitial pulmonary edema and telangiectasia, inflammatory cell infiltration. And alveolitis gradually worsened with the time passed. In the8th week, lung sections Masson stained appeared spotty fibrosis and fibrotic lesions is more obvious at16weeks. According to Szapiel grading criterion and statistical analysis, alveolitis of group RT-A and B were more serious than group Sham, and group RT-B more severe than group RT-A. In terms of fibrosis, group RT-A and B were more severe than Sham group, and group RT-B more serious than group RT-A. The study found that hydroxyproline levels of lung tissues from irradiated mice were gradually increased with time, and hydroxyproline increasing trend was particularly evident in the8th and16th week which was consistent with the occurrence of spotty fibers in pathology. On both the time points, hydroxyproline content of group RT-B was significantly higher than that of group RT-A, and the difference was statistically significant (P<0.01). Terms of survival time, no sham irradiated mice died within the observation time.16weeks survival rate of mice of group RT-A was80%, and that of mice of group RT-B was40%. And mice of group RT-B died significantly earlier than group RT-A. There is a statistically significant difference between the two groups in survival (P=0.0241<0.05).Conclusion:The whole chest of C57BL/6mice were irradiated by15Gy dose of linear accelerator, which can successfully build radiation-induced lung injury model. The model provides the basis for research on the mechanisms and targeting drug to radiation-induced lung injury.Part2Objective:IL-17A, as a pro-inflammatory factor, play an important role in many diseases related to inflammation or proliferation. The objective is to investigate the expressions and roles of IL-17A in the process of radiation induced lung injury (puhnonitis or lung fibrosis).Method:A total of61C57BL/6mice were included in this study. They were radomly divided into two groups:1) Sham group, n=25;2) RT group, n=36. Mice were sacrificed for histological examination or molecular biology at the observed time (1st week,4th week,8th week, and16th week). Mice of RT group underwent15Gy total thoracic radiation, and Sham goup not irradiated. The expression of IL-17A mRNA was quantitatively detected by real-time RT-PCR, and2-△△Ct was used for Relative quantitative analysis. In addition, IL-17A protein levels in lung tissue were detected by Western blot and immunohistochemistry staining.Results:After receiving15Gy irradiation, IL-17A in lung tissue of mice shows varying degrees of over-expression at different stages by immunohistochemistry analysis. IL-17A positive particles located in the cytoplasm of alveolar macrophages, alveolar epithelial cells, lymphocytes and bronchial epithelial cells of lung tissue. Stained histological sections showed alveolitis gradually aggravated within8weeks, while the expression of IL-17A peak at4weeks, which suggested that IL-17A may participate in the process of the early radiation-induced lung injury. According to real-time PCR results, IL-17A mRNA expression were increased in varying degrees within the observation time, started at1week, peaked at4weeks, and began to decline at8weeks. IL-17A mRNA continued to decline at16weeks, but still higher than the baseline level. Semi-quantitative Western blot analysis showed that IL-17A protein expression in lung began to rise post-irradiation after one week, four weeks peaked, then the expression level began to decline. These results show that the expression of IL-17A protein is consistent with the mRNA results.Conclusion:IL-17A expression significantly elevated after irradiation to the whole chest of mice, which was consistent with the occurrence time of alveolitis and fibrosis. Thus, IL-17Amay participate in radiation induced lung injury.Part3Objective:Blocking IL-17A signaling pathway improved outcomes variety of inflammatory diseases significantly. The study is to investigate the effect of interleukin-17A (IL-17A) antibodies on radiation-induced lung injuries in mice.Method:The thoraces of C57BL/6mice were irradiated with15Gy dose only once, which established mice model of RILL The total of135mice were divided into Sham (n=30), radiation control (n=35), treatment (n=35, IL-17A-neutralizing antibody,4μg/mouse, IV,4days per month for4months) and placebo group (n=35) before a single dose irradiation (15Gy) to the thorax. Inflammation and collagen contents in the lung tissues were examined by H&E and Massion staining method, and the concentration of IL-17A, TGF-β1, and IL-6in broncho alveolar lavage fluid (BALF) were measured by ELISA method. The degree of alveolitis and fibrosis was judged according to an established grading scale. Hydroxyproline content was measured using the alkaline solution. In another50animals,180-day survival rate following the irradiation and treatment was calculated by Kaplan-Meier method.Results:Sixteen weeks after the irradiation and treatment, there was significant inflammatory cell infiltration and interstitial collagen depositions in the radiation control and placebo groups, whereas these changes were relatively mild in the treatment group. The percentage of grade II and III alveolitis in the treatment group (16%, P<0.05) was lower than in the radiation control (72%) or placebo group (64%). The mean Aschcroft fibrosis scores were2.8(treatment group),5.2(radiation control) and4.8(placebo group) respectively. The scores of treatment group was lower than that of radiation control (P＜0.001) or placebo group (P＜0.001). The IL-17A, TGF-β and IL-6concentrations in the treatment group were lower than in the radiation control and placebo group (P＜0.01) following the irradiation. The180-day mortality rate in the treatment group was lower than in the radiation control group16.7%vs75.0%(P<0.05). Conclusion:IL-17A antibody treatment alleviates radiation-induced pneumonitis and subsequent fibrosis, and improvise post-irradiation survival. IL-17A neutraulizing antibody play an protective effect on mice undergoing thoracic radiation. Targeting at IL-17A maybe is an effective way for the treatment of radiation-induced lung injury.更多还原