【作者】 孙志超；

【导师】 肖湘生； 董伟华；

【作者基本信息】 第二军医大学 ， 影像医学与核医学， 2010， 博士

【摘要】 研究背景肺癌是最常见的恶性肿瘤之一,根据世卫组织(WHO)报道,每年有超过100万人死于肺癌(WHO,2002)。在我国城市人群中,肺癌是所有癌症死因中的头号杀手,而且,其发病率和死亡率在城乡仍在逐年递增。以手术、放疗及化疗为主体的治疗方式尚不令人满意,尤其是对中晚期肺癌。上世纪70年代发展起来的经支气管动脉化疗灌注术(Bronchial Artery Infusion Chemotherapy, BAI是针对中晚期患者疗效较好的方法之一。BAI是经导管支气管动脉内给予化学药物来治疗肿瘤的一种微创介入治疗方法,其目的是为了提高肿瘤内的药物浓度,并延长药物与病灶的接触时间,而同时并不增加外周血药浓度,从而提高疗效和减少毒副作用。但是由于目前对于动脉内给药的临床药物代谢动力学研究还不成熟,有关BAI的药动学的研究文献非常少,有代表性的几篇文献通过检测化疗药的血药浓度来计算药代动力学参数,存在较大局限性。究其原因之一在于稳定、可利用的、适合临床研究的大动物肺肿瘤模型难于建立；之二在于如何实时测定介入治疗过程中肿块内的化疗药物浓度。犬传染性性病肿瘤(Canine transmissible venereal tumor, CTVT)是一种犬类动物生殖系统自然发生的圆细胞类肿瘤,文献报道其具有可靠的稳定性、原位移植的多样性和良好的模拟性,为犬肺肿瘤模型的建立提供了良好的瘤株。微透析(Microdialysis)是上世纪70年代发展起来的一种在不破坏生物体内环境的前提下,对生物体细胞间液的内源性或外源性物质进行连续取样和分析的微量生物化学检测技术,微透析的原理与普通透析原理相同,即可透过膜的物质顺着浓度梯度通过半透膜进行扩散,只是取样装置小巧,可以置入各种组织中。微透析对组织损伤较小,可以最大程度的获取代表机体生理或病理生理情况下的样本,可应用于心肝、肺、肾、脑等几乎机体所有的器官和组织,并可实现多个位点同时取样不间断的实时监测。目前自然科学的众多基础领域应用广泛。本课题即为更好地指导临床BAI用药,探讨建立适合临床研究的大动物(犬)·CTVT同种移植性肺癌模型的最优方法,并在此基础上,借助微透析技术精确取样,联合质谱(MC)和高效液相(HPLC)测定CTVT肺肿瘤、正常肺组织内的游离药物浓度,并测定血药浓度,计算BAI的各项药代动力学参数,同时对比研究常规静脉化疗的变化趋势,分析药物-时间曲线,归纳房室模型,阐述可能存在的不同作用机制和原因,更好的指导临床BAI应用化疗药物,提供理论参考依据。同时拟建立一套经血管介入治疗药物代谢研究的方法,为今后科研工作提供参考。研究目的1、探讨建立犬传染性性病肿瘤(CTVT)移植性肺癌模型的理想方法。2、研究犬传染性性病肿瘤(CTVT)同种移植性肺癌模型的血供来源。3、基于微透析技术,在犬传染性性病肿瘤移植性肺癌模型上研究经支气管动脉灌注化疗的药代动力学。4、探索建立一套经血管介入治疗中活体内肿瘤药物代谢动力学的研究方法。研究方法1、Beagle犬18只分为2组,A组(细胞悬液接种组)12只,CT引导下经皮肺穿刺注射CTVT细胞悬液(活细胞浓度为108/ml)；B组(组织块接种组)6只,CT引导下经皮肺穿刺植入CTVT组织块(1.5mm至2.0mm)2～3枚。分别于接种后即刻、第4周、第6周、第8周、第10周行胸部CT平扫,观察有无肿瘤、肿瘤生长及转移情况；每日观察记录动物的一般情况,有肿瘤生长的动物待其自然死亡或于第10周后续研究后处死,行尸检和病理学检查,观察肿瘤有无坏死及微观病理学变化。重复测量数据分析方法分析各时间段肿瘤大小,研究两组间的差别,评判其优劣。2、犬传染性性病肿瘤同种移植性肺癌模型动物12只,分别行犬支气管动脉数字减影血管造影(Bronchial arterial digital subtraction angiography, BA-DSA)、肺动脉数字减影血管造影(Pulmonary artery DSA, PA-DSA)、经支气管动脉增强多排CT扫描(Trans-bronchial arterial contrast enhanced multi-slice compute tomography, BA-MSCT)、经肺动脉增强多排CT扫描(trans-pulmonary arterialcontrast enhanced multi-slice compute tomography, PA-MSCT)。分析所获图像,测量肺内结节的强化程度,并与肺动脉碘油栓塞CT扫描相结合,判断CTVT移植性肺癌模型的血供来源。3、CTVT同种移植性肺肿瘤模型犬6只,随机分为两组：BAI给药组(B组,n=3); Venous组(V组,n=3)。实验犬先在DSA下行经皮支气管动脉或体循环动脉造影,明确肺移植瘤血供,再于CT下以20G Chiba针行经皮肺肿瘤穿刺术,及肺实质穿刺术,分别于肿瘤内和肺实质内植入MD-2000 LM-5医用微透析探针(BASI公司,美国)各1枚。BAI组以卡铂按16.5mg/kg剂量溶于250ml 5%GS中,推注泵辅助下1小时内经动脉导管末端匀速滴完。静脉组经后肢外侧小隐静脉给药,给药剂量、给药时间同BAI组。自给药时起在微透析探针出口处收集透析液,并定时抽取静脉血。质谱及高效液相(HPLC)测定分析透析液内及血浆内的卡铂浓度。制作标准曲线,换算药物浓度,绘制化疗药在肿瘤组织内、正常肺组织及血浆内随时间变化的动态图谱,并以动力学程序处理数据,计算药代参数,以方差分析法行两组间及组内药代参数比较。研究结果1、细胞悬液及组织块两组肺内接种技术成功率均为100%,接种后并发症(气胸、血胸、肺出血、咯血等)发生数相当。无需特殊处理。组织块接种组成瘤率100%(12/12),明显高于细胞悬液接种组66.67%(9/15),两组差别有统计学意义(P=0.023<0.05)。细胞悬液组6、8、10周肿瘤的最大均径(最大层面垂径的平均值,cm)分别为1.059±0.113、1.827±0.084、2.189±0.153；组织块接种组6、8、10周肿瘤的最大均径(最大层面垂径的平均值,cm)分别为1.716±0.102、2.392±0.076、2.734±0.138；两组间6、8、10周肿瘤均径行重复测量分析得,总F=11.791,P<0.05,组织块组各时间段均径大于细胞悬液组之均径,差异有显著性意义。细胞悬液组6只犬可见胸壁及皮下种植,第7周起可出现胸腔积液,至第9周所有成瘤犬均发现中-大量胸腔积液及纵隔淋巴结肿大,犬呈恶病质状态,2只犬分别于第9、10周因呼吸衰竭死亡：组织块接种组胸壁种植2处,肺内转移2例,观察至第10周未见胸腔积液及纵隔淋巴结肿大,犬生长良好,未出现恶液质现象,两组犬均未见胸部外其它脏器转移发生。两组犬病理学检查肿瘤中心无坏死现象发生。2、14个直径大于2cm的结节中,12个(12/14)在BA-DSA上可见增粗的支气管动脉向肿瘤方向走行；5个直径在1～2cm的结节行支气管动脉造影,4个在DSA上染色明显；共8只犬行PA-DSA造影,未见结节内部或边缘肿瘤染色。14个大于2cm肿瘤中,12个BA-MSCTA上可见肿瘤内部杂乱无章的肿瘤血管影,以及造影剂渗入肿块内部所致的肿瘤染色。8只犬14个结节行碘油栓塞后CT扫描,2个小于1cm病灶内可见碘油沉积(2/5)。1个大于2cm结节,内部可见1油滴状碘油沉积；2个大于2cm结节内部可见散在点状碘油沉积；余病灶内部未见明显碘油沉积征象。3、BAI及静脉组滴注给药后,CBP在血浆中的浓度呈规律性变化,两组间的药代参数差异显著。BAI组及静脉给药组AUC值分别为58.66±4.768和76.992±8.873(mg/L*h),MRT值为5.093±0.064和2.141±0.928(h)；Cmax值为28.833±3.711和81.3±9.839(mg/L)；Vz值为10.613±3.225和2.09±2.688(L/kg)；t1/2z值为51.471±22.165和9.188±12.687(h),各组间t值分别为tAUC=-3.15216,tMRT=5.496557,tCmax=-8.64219,tVz=3.516387,差异均有统计学意义(p<0.05)。两种不同方式给药后肿瘤及正常肺组织内的药物浓度呈规律性变化。不同给药方式肿瘤内的药代参数：BAI组及静脉给药组AUC值分别为1660.76±339.77和459.49±49.99(mg/L*min),Cmax值分别为21500.39±4359.91和4627.27±722.676(mg/L)。MRT值分别为74.322±8.048和98.23±3.934(min),方差分析结果FAUC=17.336,P=0.001；FCmax=33.556,P=0;FMRT=9.091,P=0.006;差异均有统计学意义(p<0.05)。结论1、以经皮穿刺种植CTVT的方式建立犬同种肺移植瘤模型是可行的；组织块接种法较细胞悬液接种法简便、高效、成瘤率高、并发症少,是CTVT犬移植性肺癌模型制作的理想模式。2、CTVT同种移植性肺肿瘤的主要由支气管动脉供血,血供与肿瘤的大小相关,肿瘤越大,支气管动脉供血可能性越大；尚不能排除肺动脉参与该移植瘤的少量供血,特别在小于1cm的转移肿瘤。肺动脉可能参与供血。3、与静脉给药相比,BAI可明显提高肿块内的峰浓度,增加肿瘤局部药物的生物利用度,增强对肿瘤细胞杀伤；同时降低外周血药浓度,减轻全身毒副作用。4、卡铂经BAI给药可使药物的平均滞留时间有所延长,短时间内重复给药较静脉给药更容易发生药物蓄积,增加药物毒性。5、以经皮植入的方法建立犬CTVT肺肿瘤模型,结合CT引导下穿刺,微透析探针植入的方式,研究血管介入治疗的药物代谢动力学,是一套行之有效的方法,实践上切实可行。更多还原

【Abstract】 BackgroundLung cancer is one of the most common malignant tumors. According to WHO statistics, more than 1 million people die from lung cancer every year (WHO,2002). In China, lung cancer is the No.1 leading cause of death among all types of malignancies. in the cities, and the morbidity and mortality have increased significantly in both urban and rural areas. Developed in the late 1970(?), (?)al arterial infusion chemotherapy (BAI) one of the effactive treatment modalities for patients with advanced lung cancer. BAI is a minimally invasive interventional therapy which delivers chemical drugs directly to the tumor via bronchial, with the aim to increase intratumoral drug concentration and prolong drug stasis within lesions while maintain the blood drug concentration at low level. Therefore, the advantages of BAI are higher efficacy and less side effects. But the clinical pharmacokinetic studies of BAI are not mature, with few papers in the literature. Some representative studies calculate the pharmacokinetic parameters by detecting the blood concentration of several chemotherapeutic drugs. There are main reaseons for this limitation. Firstly, it is difficult to establish a suitable large animal model of lung cancer. Secondly, it is also difficult to measure the real-time concentration of chemical durgs inside mass in the course of interventional treatment.Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor, which has many characteristic such as stability, reliability and diversity in in-situ transplantation, is a good tumor strain for the canine lung cancer model.Microdialysis was developed in the 1970s. This technique provides a means of continuously sampling substances in the interstitial space fluid on the premise of not destroying environment in the organ. Principle of microdialysis is the same as conventional dialysis, with the exception that its sampling equipment (probe) is tiny which can be put into various kinds of organs and tissues. Since microdialysis has many advantages such as little damage, high authenticity and small deviation, it has been extensively applied in numerous fields of natural science.The aims of this study are:to explore the optimal method for the establishment of cTVT allograft lung cancer model, then try to discover the blood supply of the model, and finally to determine the intra-tumoral pharmacokinetics of BAI using microdialysis in order to better guide clinical BAI protocol and provide a theoretical frame of reference. By acompishing these work, we also hope to establish a set of mature methods to study drug metabolism after various vascular interventions in the futrue.Objective1. To explore the optimal method for the establishment of CTVT lung cancer model.2. To study the blood supply of CTVT lung cancer.3. To determine the intra-tumoral pharmacokinetics of BAI using microdialysis technique.4. To explore one series of pharmacokinetics study methods on vascular interventional treatment in vivo tumor.Methods1.18 beagle dogs were divided into 2 groups. Group A, cell suspension inoculation group, in which 12 dogs underwent percutaneous CT-guided puncture of lungs and innoculation of CTVT cell suspension (live cell concentration of 108/ml) into dog lungs. Group B, fresh tumor fragments inoculation group, in which 6 dogs underwent percutaneous CT-guided puncture of lungs and innoculation of 2～3 fresh tumor fragments (1.5～2.0mm). Chest CT was performed immediately after inoculation, and 4,6,,8 and 10 weeks after innoculation in order to observe the tumor growth and metastasis. Animals were observed daily for general state of health. Autopsy and pathological examinations were performed to observe whether tumor necrosis and micro-pathological changes until their natural death or at 10 weeks after furthur experiment. Repeated measure method was performed to analyse the tumor growth state, and determine the differences between the two groups.2. In 12 dogs with CTVT lung cancer, bronchial artery digital subtraction angiography (BA-DSA), pulmonary digital subtraction angiography (PA-DSA), trans-bronchial arterial contrast enhanced multi-slice CT angiography (BA-MSCTA) and trans-pulmonary arterial contrast enhaced multi-slice CT angiography (PA-MSCTA) were performed. Images were analyzed and enhancement of lung nodules were determined. CT after pulmonary arterial lipiodol injection was also performed. Finally, blood supply of CTVT lung cancer model was judged.3. Experimental dogs were divided into two groups:BAI treatment group (group B, n=3); Venous groups (group V, n=3). After the dogs were anaesthetized, percutaneous bronchial arterial angiography or other systemic arterial angiography was performed to clarify the blood supply to the lung tumors. Then chest CT scan was performed to decide puncture point, angle and route. Two 20G chiba needles were inserted to cross the semi-chest, one through the tumor and one through the adjacent lung parenchyma. A linear microdialysis probes were threaded through the canula, making sure the membrane (samping part) was within the tumor and the lung parenchyma of interest. For group B, carboplatin 16.5mg/kg dissolved in 250ml 5% GS was infused into the bronchial artery slowly within an hour using a micro-pomp. In groupⅤ, same dosage of carboplatin was administered through saphenous vein using the miaro-pump. Then dialysate samples were collected at the outlet. The drug concentration was determined by Mass Spectrometry Instrument. The time-curves of the drug concentration in extracellular fluid of tumor and lung tissue were drawn. Data were processed with Microsoft Excel and DAS2.0 software. Pharmacokinetic parameters were calculated.Results1. The technical success rates of both groups were 100%. Complications (pneumothorax, hemothorax, pulmonary hemorrhage, hemoptysis, etc.) after inoculation were almost equal. Tumor formation rate of group B (tumor fragment inoculation,100%,12/12) was significantly higher than group A(cell suspension inoculation,66.67%,9/15) (P=0.037<0.05). The mean largest diameter of Group A was 1.059±0.113,1.827±0.084 and 2.189±0.153cm at 6th,8th,10th week; while in Group B 1.716±0.102、2.392±0.076、2.734±0.138cm, respectively (p<0.05). Chest wall and subcutaneous plantings were found in 6 dogs in Group A, with pleural effusion appeared at 7th week and severe pleural effusion and mediastinal lymph node enlargement at 9th week in all dogs. All dogs in Group B showed cachexia status. In Group B, chest wall inoculatation occurred in 2 dogs, and lung metastasis was observed at 10th week. There were no pleural effusion and mediastinal lymph node enlargement. No metastases outside the chest were observed. Two dogs underwent pathological examination and no ecrosis was found.2. For 14 nodules larger than 2cm, hyperplastic bronchial arteries leading to the tumor were found in 12 nodules on BA-DSA. For 5 nodules 1～2cm in diameter, tumor staining were obvious in 2 nodules on BA-DSA. No tumor staining was observed in four dogs on PA-DSA. In 12 nodules more than 2cm, BA-MSCTA diaplayed chaotic tumor blood vessels and tumor staining within tumors. Chest CT scan was performed in 8 experimental dogs after pulmonary artery was embolized with lipiodol. Two lesions (less than 1cm) were shown to have lipiodol deposition (2/5). A droplet of lipiodol deposition was seen in one nodule larger than 2cm. Scattered dotted lipiodol deposits were found in two nodules larger than 2cm.3. Plasma carboplatin concentrations changed regularly after administration via both BAI or venous route. Some pharmacokinetic parameters between the two groups were significantly different. AUC values in BAI group and intravenous group were 58.66±4.768 and 76.992±8.873(mg/L*h), respectively. And Tmax value 1.053±0.046 and 1±0 (h), Cmax values 28.833±3.711 and 81.3±9.839 (mg/L), tl/2z values 51.471±22.165 and 9.188±12.687 (h), respectively, (p<0.05, for all). In both groups, drug concentrations in tumor and normal lung tissue changed regularly. The tumor pharmacokinetic parameters in two groups were:AUC values 1660.76±339.77 and 459.49±49.99 (mg/L* min), Cmax values 21500.39±4359.91 and 4627.27±722.676 (mg/L), MRT values 74.322±8.048 and 98.23±3.934 (min), in BAI groups andⅣgroup, respectively. Analysis of variance FAUC=17.336, P=0.001, FCmax=33.556, P=0, FMRT=9.091, P=0.006. (p<0.05).Conclusions1. It is feasible to establish a CTVT lung cancer model by percutaneous puncture. Tissue fragments inoculation method is simple, efficient, with few complications compared with cell suspension inoculation method. It is the ideal method to establish lung cancer model with CTVT.2. CTVT allograft lung tumor was mainly supplied by the bronchial artery.Tumor blood supply was related to tumor size, and the greater the tumor size was, the greater the likelihood of bronchial arterial supply was; Pulmonary artery may supply the tumor, especially in tumors smaller than 1cm. For larger tumors, blood supply was from the bronchial artery exclusively.3. Compared with intravenous administration, BAI could significantly increase the peak concentration within the tumor, prolong the local action time, increase drug bioavailability in local tumor and enhance tumor cell killing, while decreasing the blood concentration and reducing systemic side effects.4. The average residual time of carboplatin can be prolonged after dministered by BAI. But drug accumulation is prone to take place if administration is repeated in a short time compared with intravenous administration. 5. It is an effective way to study vascular interventional pharmacokinetics by establishing CTVT lung cancer model by percutaneous implantation, combined with microdialysis probe implanted by CT-guided puncture.更多还原