【作者】 张晓萌；

【导师】 蒋国梁； 杨焕军； 冯炎； 葛勇前；

【作者基本信息】 复旦大学 ， 肿瘤学， 2008， 博士

【摘要】 概述恶性肿瘤是当今严重威胁人类生命的疾病,目前肿瘤治疗的主要手段有手术、放疗、化疗和生物治疗,放疗是一种主要的非手术治疗方法,约有70%的患者在其病程的某一阶段需要接受放疗。因此,提高放疗疗效对提高肿瘤的临床疗效非常重要。提高放疗疗效主要可以从放射治疗学的两个大方面着手:放射物理和放射生物。提高肿瘤的放射敏感性是放射生物的一个主要研究方向,由此,放射增敏剂的研究应运而生。放射增敏剂定义为某种化学物质或生物制剂,当它们与放射线合并使用时,可以增强放射线对肿瘤的杀伤的作用。顺铂是临床常见的具有放射增敏作用的抗肿瘤药物,但由于其到体内迅速与血浆蛋白结合而限制了放射增敏作用的发挥。顺铂的缓释制剂有望延长药物的作用时间,增加疗效,并降低毒性,本研究自制纳米级脂质体包裹的顺铂(nanoliposomal cisplatin,NLDDP),观察其在小鼠体内的药物动力学过程,研究其体外和体内的抗肿瘤作用及与放射联合时的放射增敏作用,以为肿瘤的治疗找到一种更为有效的放射增敏剂提供实验基础。目的观察NLDDP在小鼠体内的药物动力学过程;研究NLDDP在体外对肿瘤细胞的活性抑制,观察其联合照射对肿瘤细胞体外生存的影响;在Lewis肺癌和B16黑色素瘤移植瘤小鼠模型中探讨NLDDP与放射治疗联合时的在体抗肿瘤作用及放射增效价值;在Lewis肺癌移植瘤小鼠模型中探讨NLDDP与放射联合时的放射生物学效应。材料与方法1.通过逆向蒸发法制备NLDDP,检测照射后不同时间药物的药物体外释放率。体内药物动力学实验选用C57BL/6N近交系Lewis肺癌荷瘤小鼠,分成两组,尾静脉分别注射普通顺铂(CDDP)和LDDP,剂量均为6 mg/kg。于注射后不同时间取血并处死动物,取肿瘤、肾、肝和肺组织。采用高效液相色谱法(HPLC)测定游离铂的含量,石墨炉原子吸收光谱法测定肿瘤及正常组织中总铂的含量。2.选用人肺癌A549细胞系,通过MTT分析法检测单纯脂质体、NLDDP和CDDP对细胞的半数抑制浓度(IC50);通过细胞集落形成观察NLDDP与放射联合对肿瘤细胞生长的影响。3.选用6～7周龄C57 BL/6N近交系小鼠,分别接种Lewis肺癌和B16黑色素瘤。小鼠尾静脉分别注射NLDDP和CDDP,6mg/kg,用药后1、24、72小时进行肿瘤局部不同剂量照射,观察肿瘤生长延迟情况;Lewis肺癌荷瘤小鼠尾静脉分别注入CDDP或NLDDP,在用药后不同时间点上处死小鼠。取肿瘤组织,进行流式细胞仪分析细胞周期分布和细胞凋亡率。4.选用Lewis肺癌荷瘤小鼠,分成单纯照射组、NLDDP联合照射组、CDDP联合照射组,每大组荷瘤小鼠又按照照射剂量不同分为亚组。观察肿瘤生长情况,根据Compertz模型得出剂量效应曲线,求出三组达到相同肿瘤生长延迟时间所需的照射剂量,单照组与药物联合照射组剂量之比即为放射增敏比。同时观察药物联合照射对小肠损伤的生物效应。C57 BL/6N小鼠按给与药物和照射剂量不同分组,进行全腹照射。照射结束后3.5天处死小鼠,取小肠进行切片,计数再生隐窝数。采用线性二次(LQ)数学模型进行细胞存活曲线拟合。放射增敏比为药物联合照射组和单纯照射组生存曲线LQ方程的β值之比。药物对肿瘤组织和对正常组织的放射增敏比之比为治疗增益因子。结果1.深部X射线对NLDDP体外药物释放没有明显影响。血浆药物动力学实验发现,CDDP组小鼠在注射后游离铂立即达到最高血药浓度3.24μg/ml,并迅速降低,2 h后在血液中已经无法检测出游离铂。NLDDP组小鼠注射后1 h,血浆游离铂达峰浓度13.79μg/ml,是CDDP组峰浓度的4倍多。72 h后血药浓度为1.04μg/ml。肿瘤和正常组织中总铂含量检测发现,NLDDP组荷瘤小鼠肿瘤组织中总铂含量峰浓度达8.96±1.01 mg/kg,明显高于CDDP组的1.99±0.77mg/kg;在用药后72小时内,NLDDP组中肿瘤组织中总铂曲线下面积(AUC)显著高于CDDP组,是它的7.48倍。在肝脏、肺及肾脏组织中,LDDP组AUC也高于CDDP组,分别是它的3.17倍、3.30倍和2.40倍。2.单纯脂质体外无细胞毒性;NLDDP对A549细胞的IC50为0.95μg/ml,毒性为CDDP(2.13μg/ml)的2.24倍;药物联合照射对肿瘤细胞增殖毒性大于单纯用药组和单纯照射组,其中NLDDP联合照射组肿瘤细胞的集落形成率低于CDDP联合照射组。3.在Lewis肺癌和黑色素瘤荷瘤小鼠中,NLDDP与CDDP比较均表现了更好的抗肿瘤作用。NLDDP与照射联合,最长可以使Lewis肺癌荷瘤鼠的TGD时间达15.29天,明显长于CDDP联合照射组的6.65(P=0.003)。用药后不同时间照射,小鼠的肿瘤生长差异显著,尤以用药后72小时照射肿瘤生长缓慢。NLDDP与CDDP联合照射可延长荷瘤小鼠的生存期,差异有统计学意义(P=0.0001)。荷瘤小鼠静脉注射NLDDP和CDDP后不同时间肿瘤组织流式细胞分析未发现差异。4.在Lewis肺癌荷瘤小鼠中,NLDDP与CDDP与对照组均表现了更好的抗肿瘤作用,SER分别为4.92和3.22。NLDDP与CDDP联合照射对小肠上皮的损伤增加,SER分别为1.154和1.192。NLDDP与CDDP这两种药物联合照射的的TGF分别为4.263和2.071。结论1.深部X射线对NLDDP体外释放的速度无明显影响;NLDDP静脉注射后在体内的循环时间长于CDDP,在肿瘤组织中的分布也高于CDDP组,说明NLDDP有长循环和被动靶向分布的作用。2.本研究制备NLDDP所采用的脂质成分体外无细胞毒性,NLDDP与CDDP相比,增加了对人肺癌A549细胞的毒性,与照射联合后对肿瘤细胞的体外增殖毒性增加。3.NLDDP可延长荷瘤鼠的局部肿瘤控制时间及生存时间,对细胞周期的影响和CDDP无差别。4.NLDDP和CDDP均有放射增敏作用,NLDDP的增敏作用大于CDDP,且可以获得更好的治疗增益。更多还原

【Abstract】 IntroductionMalignant tumor is a common disease threatening heath and life.The main treatment methods include surgery,radiotherapy,chemotherapy,biologic therapy and so on.Among them,Radiotherapy palys an important role,since more than 70 percent patients should receive radiation during their treatment. Thus,to improveme radiotherapy effect of cancer is very important.There are two directions through which we can improve radiaton effect,radiaton physilolgy and radiation bilogly.As to the radiation biology,the study of radiosensitizer,that can enhance the sensitivity of malignant tumor to radiation, has attracted a lot of attention during the past 50 years.Cisplatin is a kind chemothrepy drug with the potential of radiosensitization commonly used in clinic works,but its severe side effects limit its application.Therefore,if it can be made into a kind of delay-action and target-distribution preparation,the problem will probably disappear.In this study,Nanoliposomal cisplatin （NLDDP） was prepared in order to improve the circulating time and targeted distribution of the cispaltin in the body.The pharmokinetics of NLDDP was studied and the radiationsensitization effect was explored both in vitro and in vivo.This study will provide experiment references to the clinical study.ObjectiveTo observe the pharmacokinetics of nanoliposomal cisplatin（NLDDP） in mice bearing Lewis lung cancer.To explore the in vitro and in vivo anti-tumor effect of N/DDP when combined with radiation.To explore the radiation biological effect of nanoliposomal cisplatin（NLDDP） combined with radiotherapy in Lewis mice bearing lung carcinoma.Material and methods 1.NLDDP was irradiated by X ray,and then the drug release speed from the liposome was detected.As to the pharmacokinetics,Eighty C57 BL/6N mice bearing Lewis cancer were administrated with cisplatin solution（CDDP） and NLDDP respectively through their tail veins with dosage of 6 mg/kg.At different time points,five mice were killed after their blood was collected from eyepit, then their lungs,livers,kidneys and tumors were taken out.The content of free platinum in these plasmas was detected by HPLC,while the total platinum contents in tumor,liver,lung and kidney tissues were detected by Graphite Furnace Atomic Absorption Spectrometry.2.In vitro cytotoxicity was studied in A549 lung cancer cells.The inhibition to cell activity was detected by MTT test.Anti-tumor effect combined with radiation was evaluated through colony-forming experiment.3.C57BL/6N mice bearing Lewis lung carcinoma or melanoma were treated with single dose of radiation alone,NLDDP alone,CDDP alone,NLDDP followed by local single dose radiation or CDDP followed by single dose radiation after the tumor size reached 10mm in diameter.The drug was administrated through tail vein injection.Tumor size was measured three times a week.The tumor volume growth delay（TGD） time of each tumor was expressed as the difference between the tumor volume quintupling time（time of tumor volume to reach five times the original pretreatment volume,T5V₀） of treated tumors compared with the T5V₀ of untreated control tumors.The tumor cell cycle distribution in tumor tissue of the mice after NLDDP and CDDP injection was detected by flow cytometry.4.C57BL/6N mice bearing Lewis lung carcinoma were treated with single dose of radiation alone,NLDDP followed by local single dose radiation or CDDP followed by single dose radiation,drug was administrated through tail vein injection at a dosage of 6 mg/kg,and there were 6 levels of radiation dose for each group,including 0Gy、2Gy、6Gy、16Gy、28Gy and 40Gy.Tumor size were measured three times a week,The tumor volume growth delay（TGD） time of each groups was got,and the dose-effect curve was got through Compertz model,SER was the dose ratio of radiation alone group to the group combined with drug which was needed to reach same tumor growth delay. The damage enhancement of the drugs combined with radiation to intestinal epithelium was also observed.C57BL/6N mice were divided into 3 main groups,and then into 5 subgroups.The mice were irradiated at the whole abdomen with 0Gy、8Gy、10Gy、12Gy and 14Gy each subgroup 72 hours after NLDDP or CDDP injection through tail veins.All mice were sacrificed 3.5 days after irradiation and segments of jejunum were removed and transverse histological slice were cut and stained with haematoxylin and eosin,then the number of regenerating crypts per circumference was counted under microscope.Then,linear-quadratic model was got,and the SER was the ratio ofβvalue of combined group to radiation alone group.Results1.The drug release speed from liposome was not different with or without X ray irradiation in this study.In CDDP group,peak concentration of free platinum in plasma,3.24μg/ml,was achieved soon after drug injection,but it decreased rapidly,and no free platinum could be detected after two hours.In NLDDP group,the peak concentration reached 13.79μg/ml in 1 hour,4 times of which in CDDP group.In the tumor,kidney,liver and lung tissues,the contents and the AUC values of total platinum of NLDDP group were significantly higher than that of CDDP group.The biggest difference was in tumor tissue and the smallest was in kidney tissue.2.Lipid used in the preparation of NLDDP was proved no in vitro cytotoxicity. NLDDP got greater inhibition to A549 cells than CDDP,the IC50 of CDDP was almost 2.24 times of NLDDP.NLDDP combined with radiation provided the highest inhibition to colony forming.3.NLDDP produced better anti-tumor effect than CDDP both in Lewis lung cancer mice and B16 melanoma mice.Local radiation after NLDDP administration produced longer TGD time than radiation alone（p＜0.05）.When combined with local radiation of 6Gy,administration of NLDDP before radiation delivery produced an longest TGD time of 15.29 days,significantly longer than the 6.65 days owing to the combination of CDDP followed by radiation with the same intervals（p=0.003）.In the combination groups with NLDDP,radiation performed 72 hours after NLDDP injection caused greater inhibition of tumor growth delay than performed 1 hour after drug administration,the difference is statistically significant（P＜0.05）.Better survival were achived in NLDDP combined with radiation group than CDDP（P=0.0001）. Cell cycle distribution showed no difference in both groups after drug anmistration.4.Both NLDDP and CDDP produced radiosensitization effect in Lewis lung cancer mice,with the SER of 4.92 and 3.22 respectively.Also greater damage effects in jejunum tissue were got both in NLDDP and CDDP group than in radiation alone group,with the SER of 1.154 and 1.192 respectively。NLDDP proved a higher TGF（4.263） than CDDP（2.071） in the combination with radiation.Conclusions1.The drug release speed from liposome was not affected by X ray irradiation in this study.NLDDP prepared for radiosensitization in this study significantly extended the circulation time of free cisplatin in blood,brought more cisplatin into the tumor tissues,and markedly increased the effective bioavailability of cisplatin.2.NLDDP produces greater in vitro antitumor effect than CDDP,but the mechanism needs more investigation.3.NLDDP can provided a longer TGD time with or without combination with radiotherapy in Lewis lung carcinoma bearing mice and B16 melanoma mice,which is more effective than that of CDDP.The interaction between NLDDP and radiation maybe time dependent,which needs more investigation,as well as the mechanism.4.Both NLDDP and CDDP have radiosensitization effect,and NLDDP provided a better TGF than CDDP when combined with radiation for the anti-tumor effect without more damage to normal tissue.更多还原