【作者】 聂春晖；

【导师】 曹喜才；

【作者基本信息】 天津医科大学 ， 影像医学与核医学， 2011， 硕士

【摘要】 目的探讨经气管插管由同轴微导管超选择精确定位,灌注不同剂量的甲基胆蒽(methylcholanthrene, MCA)、二乙基亚硝胺(diethylnitrosamine, DEN)及超液化碘油混悬液,诱导犬肺癌模型,为肺癌深入研究建立新的平台。通过影像学的胸部X线平片、计算机螺旋断层扫描摄像术(spiral computed tomography,螺旋CT)、数字减影血管造影(digital substraction angiography, DSA)检查、血.清肿瘤标志物(tumor maker, TM)检测及病理检查,探讨肺癌发生发展过程中早期的影像学表现,血清肿瘤标志物的改变及病理学表现,从分子生物影像学提出早期肺癌诊断的新标准,使肺癌综合治疗的临床效果产生一个新飞跃。材料与方法选用30只犬龄1-3岁,体重15-20Kg的杂种犬为研究对象,雌雄均有。实验前均行疫苗接种,观察数日确定犬仍处于健康状态后随机将30只犬分为A(8只)、B(8只)、C(7只)、D(7只)四组。饲养数日观察实验犬正常,经犬前腿取静脉血3m1,室温下保存0.5-1h,离心取上层血清,进行肿瘤标志物的检测。将犬进行全身麻醉,采用改良式气管插管出同轴微导管精确定位向右肺隔叶灌注致癌药物：A、C组灌注甲基胆葸(MCA)0.25g+二乙基亚硝胺(DEN)0.38g+超液化碘油混悬3m1；B、D组灌注甲基胆葸(MCA)0.35g+二乙基亚硝胺(DEN)0.48g+超液化碘油混悬3m1。分别在第6、12个月对四组实验犬再次进行血清TM检测。同时分别行X线胸片、CT、DSA检查。C、D组实验犬在饲养12月做完上述检查后处死,取注药部位肺组织,加10%中性福尔马林液固定,石蜡包埋(切片厚度5μm)HE染色,根据标本染色情况,选取结构完整标本进行后续染色观察及统计处理。检测四组实验犬不同时期CA125、Cyfra21-1、CEA、SCC-Ag、Cal 53、DR70、NSE 7种血清TM,对所得数据进行统计处理,观察TM指标是否随时间的推移增高。在不同时期对四组实验犬进行CT和DSA支气管动脉造影(Bronchial Arteriography, BAG)检查。实验犬全身麻醉后,先进行CT检查,观察实验犬注药部位是否有可见炎性改变、肿瘤样改变。DSA检查经右侧股动脉穿刺,采用改良式Seldinger技术,行选择性BAG。穿刺成功后,经导丝置入导管鞘和C3导管,在胸主动脉近气管隆突附近以后、右、前、左的顺序寻找支气管动脉开口。当导管头有嵌顿、落空感,不再随心脏搏动而上下移动时,经导管注入少量对比剂,确定导管进入支气管动脉后,行DSA支气管动脉造影。应用高压注射器以每秒2m1的速度注入4m1对比剂,观察支气管动脉造影的表现。是否左右支气管动脉共干,支气管动脉是否增粗、变形,有无肿瘤血管及染色,是否存在其它动脉向注药部位供血。通过仔细观察DSA支气管动脉造影表现,分析在动脉期、实质期(毛细血管期)和静脉期的BAG结果,确定肿瘤的供血动脉来源、形态及肿瘤染色情况。造影结束后,肌注抗菌素预防感染。病理学检查,所有标本行HE染色,通过光镜观察灌注致癌物部位在诱发癌变过程中肺组织病理变化。采用病理图像分析系统对标本HE染色切片进行形态计量与分别计数方法。结果1.胸部X线平片：6个月时沉积在犬右肺膈叶的致癌物质混悬液的密度较前变淡,范围缩小,12月时大小范围进一步缩小,未见确切软组织肿块影。2.螺旋CT平扫：6个月时所有实验犬致癌物混悬液沉积良好,注药部位支气管扩张,管壁增厚,周围可见磨玻璃密度影,邻近胸膜增厚粘连。B组2只实验犬混悬液沉积周边可见多发小叶中心结节。12月时致癌物混悬液大小范围较前缩小,其邻近胸膜仍可见增厚粘连。双肺纹理较前增多,肺野透过度不均,右肺可见多发磨玻璃密度结节影。支气管扩张更加明显,气管壁不规则增厚明显。D组1只实验犬增厚的气管周围可见不规则软组织块状密度影。B、D组各有1只实验犬可见片状肺不张。3.DSA支气管动脉造影：所有犬支气管动脉均与肋间动脉共干。注药后6个月的支气管动脉管壁光滑,走行自然。动脉早、中、晚三期均未见异常血管及肿瘤染色影。注药后12个月,B、D组各有1只实验犬可见支气管动脉增粗,走行迂曲,并可见肿瘤样血管向右肺致癌物质混悬液灌注部位的不规则肿块供血。肿瘤染色明显,从动脉早期持续到动脉晚期。其余实验犬支气管动脉走行自然,仍未见异常新生血管及肿瘤染色影。4.TM检测：实验犬A、C组和B、D组的CEA、Cyfra21-1、SCC、CA153、CA125、DR70、NSE7项TM浓度,灌注药物前和第6个月、第12个月,第6个月和第12个月比较,浓度差异显著,具有统计学意义(P<0.05)。A、C和B、D组灌注致癌物质混悬液的浓度不同,两种浓度对以上7种血清TM浓度影响,除Ca153,其余6项差异均无统计学意义(P>0.05)。5.病理学检查：15只实验犬右肺注药部位及周边可见各种改变。D组形成肿块的实验犬镜下可见大量的不典型增生的细胞巢或细胞团,似“癌巢”,其内可见炎性增生细胞及不典型增生的组织细胞,周边可见出血。部分支气管上皮假复层细胞鳞状化生为柱状上皮细胞。其他实验犬分别可见急慢性肺炎改变、肺气肿改变、纤维组织增生及大量炎症细胞(主要是淋巴细胞、浆细胞和中性粒细胞)浸润。结论1.首次联合影像学、血清肿瘤标志物及病理学检查探讨肺癌发生发展过程中的不同表现,提出肺癌早期诊断的新标准,为提高肺癌早期诊断率,提高综合治疗效果建立新的平台。2.提出犬早期肺癌诊断参考标准：犬CT显示肺脏有磨玻璃样改变及/或软组织肿块,DSA支气管动脉造影显示肿瘤样血管及染色,犬血清TM中Cal 25、CEA、SCC-Ag、Ca153、Cyfra21-1、NSE、DR70升高异常明显,病理学检查有不典型增生等改变时,既使犬胸部平片显示正常也高度提示为早期肺癌。3.联合检测犬血清Ca125、CEA、SCC-Ag、Ca153、Cyfra21-1、NSE、DR70浓度,测出其正常范围为：Ca125(19.253-19.465μt/m1)、CEA(7.707-27.543ng/1)、SCC-Ag(71.519-109.351pg/m1)、Cal 53(1.397-1.711μ/m1)、Cyfra21-1(0.738-1.7221μg/1)、NSE(175.095-195.425ng/1)、DR70(0.327-0.405μg/1),为进一步深入研究血清TM对犬肺癌早期诊断提供理论依据。4.提出诱导犬肺癌的最佳致癌物剂量为：0.35g MCA+0.48g DEN+3m1超液化碘油。更多还原

【Abstract】 Obj iectiveTo evaluate the precise positioning intubation reperfusion methylcholanthrene (MCA),two diethylnitrosamine(DEN),and lipiodol suspension induced canine model of lung cancer,lung-depth studies provide new platform. Through the inspection X-ray, spiral computed tomograph digital substraction angiography, tumor maker and pathology. To disussion the relation between lung cancer and the method which is combining image science,tumor makers in serum with pathology for lung cancer, it is to find a new way for us to diagnose early lung cancer so that we can improve the clincic outcome of lung cancer.Material and methods30 dogs which are half-bred,1-3 years old and 15-20 kg weigh are studied, both male and female. Before we devide them into A(eight dogs),B(eight dogs),C(seven dogs),D(seven dogs) goups in random,we give them vaccination and look after some days so that the dogs are all health.1 month later,we get 3 ml boold from the forelegs of dogs. Then we take blood serum for detecting tumor makers(TM) after boold have been kept in outer at room temperature about 0.5-1 hour. After anaesthetization,tracheal intubation by Coaxial Microcatheter precise positioning leaves to the righr lung perfusion phrenic carcinogenic substances:for go up A,B,methylcholanthrene(MCA) 0.25g + diethylnitrosamine(DEN) 0.38g+ lipiodol suspension 3 ml; for goup C,D, MCA 0.35g+DEN 0.48g+ lipiodol suspension 3 ml. Then we detect the same TMs at 6,12,18 months again. At the same time, we give each goup the X-ray,CT, DSA dynamic observation. We kill the dogs of C,D groups after raised 12 month,get the injection sites tissue, plus 10% neutral formalin-fixed, Paraffin embedded (slice thickness of 5μm) HE staining. We Choose the complete tissue structure for the subsequent observation and statistical processing. CA125、cyfra21-1、CEA、SCC-Ag、Ca153、DR70、NSE 7 have been tested so as to we can find out if they are higher. For CT, we want to observe whether inflammation or tumor change can be found at the lung of dogs. Then we give dogs selective bronchial arteriography (BAG) testing with using modified Seldinger technique, punctured across the right femoral artery under general anesthesia. After the success of puncture, we put the guide wire,catheter sheath and C3 catheter into, then we find the open placement of the bronchial artery near by thoracic aorta. When we find out the bronachila artery, inject 4 ml contrast medium with the speed of 2 ml a second in order to know that if bronchial artery and arteriae intercostals are hemitruncus,or left and right bronchial artery are hemitruncus. At the same time,we also observe the shape of bronchial artery and its size, the tumor and its artery or other artery which take blood to tumor if they can be detected. Then we analyse the outcome which we have receive from digital substraction angiography(DSA). After angiography, we give each dog some antibiotic to guard against infection. At pathology, we stain each tissue with HE staining, so we can observe the pathological changes of the lung tissue which have been injected carcinogen and account the HE section with Pathologicalimage analysis systems.Results1. X-ray:6 month later, the carcingens suspension which is perfused at right lung of dogs is thiner and smaller. They become thiner and smaller than at 6month. There is no orther changes at the lung of dogs in study.2. CT scan:At 6 month, the carcingens suspension deposit very good and bronchus become dilatation,thickening there. Many glass-shadow can be seen surrounding them and adjacent pleura become thicker and adhesions. There are many centrilobular nodules can been seen at 2 dogs in group B. At 12 month,carcingens suspension is smaller than before and adjacen pleura are still thick and adhesioning. We can see markings at double lung of dogs are more than before and many centrilobular nodules, the degree of lung is not good. bronchus become un-regular and biger. A irregular soft tissue can been seen surround thicker bronchus at a dog in D group, two dogs (one at group B,the other at group D)have atelectasis sheet.3. DSA:bronchial artery and intercostal arteries of all dogs are from the same trunk. At 6 month,there doesn’t any abnormal at lung of dogs. At 12 month, we can find bronchial artery is thicking and running tortuos at 1 dog at each goup of B and D. there are also tumor-like blood vessels transported blood to the irregular tumor which are seen at the position of carcingens suspension at these two dogs. These tumor staining are clear and they are staining from early arterial phase lasted until late arterial. The orther bronchial artery are all normal.4. TM detection:When we compare the concentration of CEA、Cyfra21-1、SCC、CA153、CA125、DR70、NSE of these four group at the beginning,6month, 12month,they are significant difference and statistically signidicant (P<0.05). the concentration of carcingens suspension perfused to the dogs at group A and C are different with group B and D, but they don’t have different effect at the concentration of that seven TM, except Cal 53 (P>0.05)5. Pathological detection:There are almost no pathological changes in the left of lung of 15 dogs, but in the right lung there can be seen a variety of changes around the injection site. A large number of cell nests of atypical hyperplasia or cell groups can be seen in the right lung of one dog which has tumor in the right lung at group D. These cell nest or group are like "cancer nest", the cells are inflammatory cells and dysplasia cells,with blooding around. A part of pseudostratified bronchial epithelial cells become comlumnar epithelial with a change of squamous metaplasia. We can see some changes like emphysema, fibrosis, acute and chronic pneumonia in orther dogs. A large number of inflammatory cells also can be seen in these dogs, they are main of lymphocytes, plasma cells and neutrophils.Conclusion1. Imaging, serum tumor markers and pathological examinations were used to analyze the different manifestations of lung cancer, to find new standard for improving early diagnosis of lung cancer and improving outcome of comprehensive treatment in lung cancer..2. We first proposed a new reference standarad of early diagnosis of lung cancer:. When CT shows ground-glass-like changes and/or soft tissue mass in lung, DSA bronchial arteriography showed tumor-like blood vessels and staining, serum TM in Ca125, CEA, SCC-Ag, Ca153, Cyfra21-1, NSE, DR70 significantly higher than before, pathological changes had atypical hyperplasia, we can consider the early lung cancer is coming even when the chest film showed normal.3.7 serotypes TM(Cal25, Cyfra21-1, CEA, SCC-Ag, Ca153, DR70, NSE) had been detected one time and we find out there normal range:Ca125 (19.253-19.465μ/ml), CEA(7.707-27.543ng/1), SCC-Ag (71.519-109.351pg/ml), Ca153(1.397-1.711μ/ml), Cyfra21-1(0.738-1.722μg/l), NSE(175.095-195.425ng/l) DR70 (0.327-0.405u.g/l). It can provide some value in order to study the effect of serum TM in diagnosis of lung cancer further.4. The best dose of carcinogens for induceding lung cancer at dog is 0.35g MCA, 0.48g DEN and 3 milliliter of lipiodol.更多还原