【作者】 李丹叶；

【导师】 唐劲天； 钟美佐；

【作者基本信息】 中南大学 ， 肿瘤学， 2012， 博士

【摘要】 研究背景肝癌是最常见的恶性肿瘤之一,且70%-80%的肝癌在确诊时已失去手术机会。动脉栓塞治疗较目前其他介入治疗方法近期缓解率高,副反应较少,且容易开展,近年来成为不能手术的肝癌患者的首选治疗方式。但它存在着一定的缺点,表现为大多数肿瘤坏死不完全,远期疗效不佳,且需要多次治疗。动脉栓塞磁感应热疗是动脉栓塞与磁感应热疗相结合的治疗方式,通过经动脉导入含磁性介质的栓塞剂,在栓塞肿瘤供血动脉的基础上,利用磁感应介质在交变磁场下发热的特点,同时对肿瘤施行靶向热疗。目前其常用的栓塞介质为铁磁颗粒。磁感应栓塞热疗介质除需满足良好的生物相容性及磁致升温要求外,介质尺度也是影响疗效的关键因素。针对栓塞热疗介质的研究鲜有报道,目前的磁介质均为纳米级铁氧体或由高分子材料包裹纳米级铁氧体而成的微米级铁氧体。然而一些研究结果表明纳米级介质因尺度太小会渗入门静脉及肝静脉,因而可能影响动脉栓塞热疗的疗效及安全性,理想的热疗介质的颗粒粒径应在10微米左右。且经高分子材料包裹的纳米级铁氧体进入体内经降解后的安全性仍不明确。因此寻找微米尺度的、安全高效的热疗介质仍是磁感应栓塞热疗的关键问题。羰基铁粉是一种元素铁,美国International Specialty Products公司生产的微米级羰基铁粉(商品名Ferronyl(?))口服使用的安全性高,已经正式被美国药品管理监督局批准直接添加到食品中作为铁元素补给。本研究组对该羰基铁粉的物理表征的前期实验表明,羰基铁粉为表面光滑,且粒度比较均一的球形颗粒,粒径分布大约在2-10μm,平均粒径约5μm,符合肝动脉栓塞热疗磁介质最好在1-10μm粒径范围的要求。体内外实验证明羰基铁粉的安全性高。本研究中将羰基铁粉与常用栓塞剂碘油制备成栓塞剂,对其用于动脉栓塞磁感应热疗的可行性、安全性及疗效作出评价,并对其治疗机制进行初步分析,旨在寻找一种新的动脉栓塞磁感应热疗介质,为肝癌的治疗寻找新的思路。第一章羰基铁粉的体外细胞毒性及在交变磁场下升温情况的研究目的评估羰基铁粉的体外升温性能及细胞毒性,初步评价其用于动脉栓塞磁感应热疗的可行性。方法利用本研究室自主研发的500kHZ磁感应热疗仪,检测浓度为80、120、160、200、240mg/mL的羰基铁粉-碘化油混悬液在45Gs场强下的升温情况,并检测浓度为160mg/ml的羰基铁粉-碘化油混悬液在混悬液在25、35、45、55、65Gs场强下的升温情况。采用CCK-8法检测L929小鼠成纤维细胞与浓度分别为100%、75%、50%及25%的羰基铁粉浸提液共孵育24、48及96小时后的细胞增值率,根据细胞增值率结果判定浸提液的细胞毒级。结果磁场强度一定时,羰基铁粉-碘油混悬液的升温速率与其浓度成正相关；混悬液浓度一定时,其升温速率与磁场强度成正相关。160mg/L的混悬液在45Gs的场强下10min内升温可达60℃以上,满足热疗要求。L929小鼠成纤维细胞与羰基铁粉浸提液共孵育后,在各观察时间点100%、75%浸提液组细胞形态与对照组相比均表现为轻微毒性,而50%、25%两浸提液组细胞数量与形态均与阴性对照组相似。共孵育后第24、48、72小时,100%及75%浸提液的细胞毒级均为1,共孵育后第24、48小时,50%、25%浸提液的细胞毒级为1,第72小时50%、25%浸提液的细胞毒级均为O。结论羰基铁粉在500kHZ频率磁感应热疗仪下的体外升温性能良好,浓度为160mg/mL的羰基铁粉-碘油混悬液具有体内应用价值；羰基铁粉的细胞毒性表现为轻微毒性,符合国家关于材料体内应用的毒级标准。第二章羰基铁粉-碘油混悬液介导的肝动脉栓塞磁感应热疗对兔VX2肝癌模型治疗的可行性研究和对肿瘤生长的影响目的初步评价羰基铁粉-碘油混悬液用于肝动脉栓塞磁感应热疗的可行性、安全性及其对VX2原位肝癌模型生长的影响。方法将接种18天的荷瘤兔随机分为5组,即假治疗对照组(Ⅰ组)、碘油栓塞组(Ⅱ组)、羰基铁粉/碘油混悬液栓塞组(Ⅲ组)、羰基铁粉/碘油混悬液栓塞热疗组(Ⅳ组)。栓塞介入术后第3天将肿瘤暴露于交变磁场,43-45℃加热30分钟。栓塞热疗组3只实验兔于栓塞后立即和第3天及第14天行腹部常规CT扫描。所有荷瘤兔在栓塞术前、栓塞术后当天及栓塞术后3天(即热疗后当天)、栓塞术后10天采血行血常规、肝肾功能检查。栓塞术后14天处死所有实验兔,测量肿瘤大小,计算肿瘤体积抑制率,大体观察标本并进行病理学检查。结果(1)在场强为30-55Gs范围内,肿瘤内温度在10min内可达到43℃以上,瘤内温度可保持在43-45℃,瘤外1cm正常肝组织温度最高不超过40℃,肛温无明显变化。(2)CT可清晰地显示体内羰基铁粉-碘油混悬液的高密度影,混悬液分布于瘤周边多于中央。(3)栓塞术后当天Ⅱ、Ⅲ、Ⅳ组与Ⅰ组相比ALT、AST水平明显升高(P<0.01),Ⅱ、Ⅲ、Ⅳ组间无显著性差异(P>0.05)。术后第4天Ⅳ组与Ⅱ、Ⅲ组相比,ALT增高无显著性差异(P>0.05),AST增高有显著性差异(P<0.05)。第11天各组ALT、AST水平均接近正常值,组间无显著性差异(P>0.05)。(4)Ⅱ、Ⅲ、Ⅳ组较Ⅰ组肿瘤生长受到明显抑制,体积具有显著性差异(P<0.01),Ⅳ组较Ⅱ、Ⅲ组肿瘤生长受到明显抑制(P<0.05)。(5)肉眼及显微镜下观察见Ⅱ、Ⅲ组肿瘤组织较Ⅰ组明显减少,肺转移程度较Ⅰ组轻；Ⅳ组肿瘤组织更少,大片坏死,仅残留散在小片状癌巢。肺转移例数较Ⅱ、Ⅲ组少,转移结节更小。显微镜下Ⅲ、Ⅳ组微动脉内可见羰基铁粉及碘油沉积。Ⅱ、Ⅲ、Ⅳ组正常肝组织大体上未受影响,极少数被栓塞的微动脉周围肝组织出现萎缩、变性。各组肺组织无萎缩、变性及坏死。结论羰基铁粉-碘油混悬液介导的肝动脉栓塞热疗可显著抑制VX2肝癌模型的生长。肝动脉栓塞对肝功能有较大损伤,但羰基铁粉未加重对肝功能造成的负担,热疗可能加重对肝功能的损害,但肝功能损害具有可逆性。羰基铁粉-碘油混悬液在瘤内的分布依赖于瘤内血管的分布,对治疗血供丰富的肝癌具有优越性。羰基铁粉经肝动脉灌注后极少见回流入体循环,该尺度的羰基铁粉具有比较高的安全性,可适当增加羰基铁粉的粒径以进一步提高安全性。第三章羰基铁粉-碘油混悬液介导的肝动脉栓塞磁感应热疗与直接注射热疗治疗兔VX2肝癌的对比研究目的比较羰基铁粉-碘油混悬液介导的直接注射热疗与肝动脉栓塞热疗两种磁介质导入方式的优劣性,为肝癌磁感应热疗选择合适的磁介质植入途径提供实验依据。方法将接种18天的荷瘤兔随机分为动脉栓塞磁感应热疗组(AEH组)及直接注射热疗组(DIH组),AEH组实验兔于栓塞介入术后3天热疗,DIH组直接注射混悬液后立即进行热疗,两组均在43-45℃下加热30min。以AEH组行栓塞术当天为第1天,所有荷瘤兔在第0、4、11天采血行血常规、肝肾功能检查。第17天处死所有实验兔,测量肿瘤大小,计算肿瘤体积抑制率,大体观察标本并进行病理学检查。结果(1)热疗时两组动物瘤内温度均可在10min内上升至43℃以上,DIH组场强在25-35Gs范围内、AEH组场强在30-55Gs范围内时分别可使瘤内温度维持在43-45℃；DIH组肿瘤中心温度高于周边,而AEH组周边温度高于瘤中心温度。距肿瘤边缘1cm的正常肝组织温度最高均不超过40℃,肛温均无明显变化。(2)热疗后两组实验动物白细胞均升高,栓塞热疗组较直接注射热疗组白细胞计数及ALT、AST值均明显升高,具有统计学差异(分别为P<0.05,0.01)。至第11天,两组实验动物白细胞计数、ALT、AST值均恢复至接近正常水平,对比未见显著性差异(P>0.05)。(3)处理后14天观察AEH组肿瘤体积较DIH组体积小,差异具有统计学意义(P<0.05)。肉眼及镜下见DIH组实验兔肿瘤生长较AEH组旺盛,周边仍见大量残余肿瘤组织。DIH组全部发生肺转移,AEH组只有2只实验兔出现肺转移,且肺转移程度较前者轻微。(4)AEH组正常肝组织微动脉内均可见少量羰基铁粉及碘油,极少数被栓塞的微动脉周围肝组织出现萎缩、变性。肺微动脉内偶可见羰基铁粉沉积,肺组织结构保持正常。DIH组肿瘤所在肝叶、其余肝叶及肺组织中均未见羰基铁粉及碘油分布。结论羰基铁粉介导的DIH靶向性高于AEH,达到治疗温度所需的磁场强度较后者小,对正常组织的影响较后者小。43-45℃的治疗温度下,羰基铁粉-碘油混悬液介导的AEH对肿瘤的抑制作用优于DIH。AEH对肝功能的损害大于DIH。第四章羰基铁粉-碘油混悬液介导的动脉栓塞磁感应热疗对兔VX2肝癌模型血管作用的研究目的探讨羰基铁粉-碘油混悬液介导的肝动脉栓塞热疗对肝VX2肿瘤微血管的作用。方法荷瘤兔随机分为4组：假治疗对照组(Ⅰ组)、碘油栓塞组(Ⅱ组)、羰基铁粉/碘油混悬液栓塞组(Ⅲ组)、羰基铁粉/碘油混悬液栓塞热疗组(Ⅳ组)。栓塞后第14天处死动物,采用Super Vision二步法免疫组化法检测肿瘤组织内的VEGF和CD34的表达。根据CD34阳性血管数量计算MVD。结果两栓塞组间肿瘤组织MVD值、VEGF阳性表达率无显著性差异(P>0.05),两栓塞组MVD值、VEGF阳性表达率均明显低于对照组(P<0.01)。栓塞热疗组的肿瘤组织MVD值、VEGF阳性表达率较两栓塞组均低,具有统计学意义(P<0.01)。结论羰基铁粉-碘油混悬液介导的43-45℃肝动脉栓塞磁感应热疗可有效破坏兔VX2肝癌的微血管及抑制VEGF的表达,栓塞后2周内肿瘤组织的VEGF表达未出现上调。肿瘤的热疗对肿瘤营养血管的直接破坏可能是其治疗肿瘤的一项机制。更多还原

【Abstract】 Background Hepatocellular carcinoma(HCC) is one of the most common fatal malignant tumors world wide.70%-80%of tumors are inoperable because diagnosis is established in the late stages. Transcatheter arterial embolization(TAE) has been established as the preferred nonsurgical treatment for HCC, due to its higher tumor response rate and fewer adverse effects comparing with other interventional therapy. But some disadvantages still exists, for the performance of the majority of tumor necrosis is incomplete, and repeated treatments are needed. Magnetic arterial embolization hyperthermia (MAEH) is a combined therapy of arterial embolization and magnetic hyperthermia. It is based on selective arterial embolization of liver tumors with magnetic embolic agents. An external alternating magnetic field is then applied to produce heating in the embolized particles. The common mediators for MAEH are ferromagnetic particles.Magnetic mediators play critical role in the MAEH, especially on the size and size distribution concern. However, most of the works about mediators in MAEH are focused on the magnetic nanoparticles. It has been proposed that particles with smaller size (nano-scaled) may pass through the venous circulation, and the ideal hyperthermia medium particle size is about10microns. Also the safety of nanoscale ferrites remains unclear. However, seldom research was carried out on developing an ideal mediator of micro-size for MAEH.ICP (with trade name of Ferronyl(?), ISP pharmaceuticals) has been approved by American Food and Drug Administration (FDA) for dietary iron supplement by oral administration due to its low toxicity and excellent bioavailability. It is elemental iron with high iron content and highly resistant to oxidation. The particle size ranges from2to10μm, and the average size is about5microns, which potentiates its further application as mediator for AEH. Our previous in vitro and in vivo experiments showed that carbonyl iron powder was safe. In the presented work, we develop ICP as magnetic mediator, to make an embolic agent by mixed with lipiodol. and the in vivo biocompatibility, inductive heating characteristics, as well as inhibitory effect of MAEH on VX2carcinoma-induced rabbit liver tumor model were studied. The deductions from the experiments may have clinical significance for application of MAEH mediated by CIP in liver cancer treatment.Part I In vitro evaluation of cytotoxicity of carbonyl iron powder and the inductive heating characteristics in alternating magnetic fieldObjective To assess the cytotoxicity of carbonyl iron powder and its potential by heated in alternating magnetic field, to evaluate the feasibility of its application to the MAEH.Methods By application of the magnetic induction hyperthermia instrument developed by our department, the elevated temperature properties of CIP-lipiodol suspension with concentrations of80,120,160,200,240mg/mL were measured by magetic field strength of45Gs, and the elevated temperature properties of CIP-lipiodol suspension with concentrations of160mg/mL were measured by magetic field strength of25,35,45,55,65Gs. Sterilized CIP samples were added to RPIM-1640cultural medium in a proportion of1cm2/mL and incubated for24h at37℃. Mouse fibroblast L-929cells were cultured in the medium of extraction of carbonyl iron powder with a concentration of100%,75%,50%and25%for24,48and72hours. Cell growth was tested by CCK-8assay and the cell relative growth rate (RGR) was calculated. The cytotoxicity was graded by generally accepted standard.Results The rate of the heating depend strongly on the CIP contents, higher concentration can ensure a higher temperature upon equilibrium finally, and higher field strength results in a greater increase in the temperature. Suspensions of160mg/mL had the ideal therapic temperature above60℃by heated within10min under45Gs of the alternative magnetic field. During the culture period, L929cells cultured in the extraction with a concentration of50%,25%showed normal morphology and proliferation compare with the control,50%and25%, extraction were demonstrated as non-toxic. While extraction with a concentration of100%、75%appeared mild cytotoxity, with a cytotoxicity level of1at24,48and72hours after incubation.Conclusions CIP has ideal inductive heating characteristics which can guarantee treatment temperature under the alternative magnetic field of500kHZ. Suspension of160mg/ml in concentration has the ideal therapic temperature in alternative magnetic field of45Gs. The CIP particles showes mild cytotoxicity, excellent biocompatibility for further investigation. Part II Feasibility of arterial embolization hyperthermia mediated by CIP/lipiodol suspensions on rabbit VX2liver tumor model and the effect on tumor growthObjective To access the feasibility and security of CIP-lipiodol suspensions mediated arterial embolization hyperthermia on rabbit VX2liver tumor model and investigate the effect of it on growth of hepatic tumor.Methods Eigthteen days after tumor implantation, the rabbit bearing VX2liver tumor were randomly divided into five groups, including the pseudo-treatment control group(Group I), lipiodol embolization group(Group II), CIP/lipiodol suspensions embolization group(Group III) and CIP/lipiodol suspensions embolization hyperthermia group(Group IV). The rabbits were exposed to alternating magnetic field three days after embolization, and heated at43-45℃for30mins. Three rabbits in group IV were conducted abdominal CT scanning immediately, three days and fourteen days after the embolization procedure respectively. Blood routine and their function of liver and kidney were tested before the embolization procedure, and at0,3and10days after the embolization procedure respectively. All animals were sacrificed on the14th day after embolization. Tumor size was measured, volume and growth rates of tumors were calculated. The changes of tumors were observed macroscopically and microscopically.Results (1) The temperature of tumor core and rim both rose rapidly and reached the objective temperature above43℃in10min in magnetic field with a strength of30-55Gs, and was maintained within43-45℃by modulating the magnetic field strength.(2) The limited higher density imaging of intratumoral CIP/lipiodol suspension deposits compared to the surrounding tissue was observed clearly by CT scanning, the density was even higher in the tumor rim than the core.(3) ALT and AST values of rabbits in group Ⅱ, Ⅲ, Ⅳ were higher than group Ⅰ (P <0.05) on the day after transcatheter embolization, but without significant difference among those three treated groups. AST in group IV was elevated after hyperthermia, with significance difference from other groups (P<0.05). ALT and AST values in all groups went down close to the normal range10days after embolization, without significant difference(P>0.05).(4)14days after embolization, the tumor volume in group Ⅱ, Ⅲ, Ⅳ were significantly smaller than group Ⅰ (P<0.01), and those in group IV were even smaller than group Ⅱ and Ⅲ (P<0.05).(5) Either on macroscopically or microscopically observeion, the tumor growth were inhibited obviously in group Ⅱ and Ⅲ comparing with group Ⅰ, and the lung metastases in the formal were less and smallar than those in the later. Microscopically, deposition of carbonyl iron powders and iodized oil in arteriole could be seen in group Ⅲ and Ⅳ, large necrotic area for group Ⅱ, Ⅲ and almost complete necrosis for group Ⅳ were observed. There were a small amount of carbonyl iron powder and iodized oil remained in the normal liver tissue arterioles of rabbits in group Ⅲ and Ⅳ, litter liver tissue surrounding embolized arterioles occured atrophy and degeneration. There were tiny amount of remained carbonyl iron in pulmonary arterioles in1rabbit of group Ⅲ and Ⅳ respectively, but lung tissue structure kept being normal.Conclusions CIP/lipiodol suspension mediated hepatic arterial embolization hyperthermia is effective in suppresses the growth of VX2liver tumors. Arterial embolization induces a reversible liver function injury, while CIP makes no burden on the liver function, hyperthermia may increase injury on the liver function. Distribution of CIP-lipiodol suspension depends on the distribution of blood vessels within the tumors, which is advantageous for the treatment of hypervascular liver tumors. CIP rare flow into the body circulation, CIPs in this size have a relatively high security, a mild increase in the particle size of CIP may be appropriate in order to further improved security in clinical trials. Part Ⅲ Comparative study of magnetic arterial embolization hyperthermia and direct injection hyperthermia mediated by CIP/lipiodol suspension in treatment of hepatic rabbit VX2carcinomaObjective To compare the effect of direct injection hyperthermia (DIH)and hepatic artery embolization hyperthermia (AEH) mediated by CIP/lipiodol suspension, and offer an experimental basis for selecting a suitable implant method of magnetic medium in the magnetic induction hyperthermia for liver cancer.Methods18days tumor-bearing rabbits were divided into two groups as AEH group and DIH group. The rabbits in AEH group were treated by hyperthermia three days after the artery embolization operation, and the rabbits in DIH group were treated by hyperthermia just after the direct injection of suspension, the two groups were both heated in43-45℃for30min. Reguard the artery embolization surgery day as the first day, all tumor-bearing rabbits had blood routine tests and liver and kidney function tests in the zero,4th, and11th days. All rabbits were sacrificed in the14day, tumor size was measured, volume and growth rates of tumors were calculated. The changes of tumors were observed macroscopically and microscopically. Results (1) The intratumoral temperature of all the rabbits can be rised to above43℃in10min during the hyperthermia. Temperature of tumor could be maintained between43-45℃in magnetic field with a strength of30-55Gs in AEH group and25-35Gs in DIH group; The temperature was higher at the tumor core than the rim in the DIH group, in contrast to the AEH group. The temperature of normal liver tissues1cm away from the tumor marginin in both groups did not exceed40℃.(2) Compare with the DIH group, white blood cell count, ALT and AST values of rabbits were higher in AEH group after hyperthermia, with significant difference (P<0.05). Till the11th day, the white blood cell count, ALT and AST values were restored to near normal levels in both groups of experimental animals, without significant difference (P>0.05).(3) The tumor volumes of rabbits in AEH group were smaller than the DIH group in the14th day, and the difference was statistically significant (P<0.05). Tumor growth of rabbits in DIH group was more active than the rabbits in AEH group both in macroscopically and microscopically observeion. All the rabbits occurred lung metastases in the DIH Group, while only two experimental rabbits in the AEH group occurred lung metastases, and the metastasis was not as large as the DIH group.(4) There were a small amount of carbonyl iron powder and iodized oil remained in the normal liver tissue arterioles of rabbits in the AEH group, and a very small number of liver tissue surrounding embolized arterioles occured atrophy and degeneration. There were tiny amount of deposited carbonyl iron powders in pulmonary arterioles, but lung tissue structure kept being normal. No carbonyl iron powder was observed in the normal liver tissues outside the tumors or in lung tissues in the DIH group.Conclusions More excellent target effect of CIP is conformed in DIH than AEH, the required magnetic field strength for the objective treatment temperature is also smaller. While the tumor inhibition effect of AEH is superior to DIH in the same treatment temperature as43-45℃. Part IV Effect of CIP-lipiodol suspensions mediated arterial embolization hyperthermia on rabbit VX2liver tumor vessels.Objective To investigate the effect of CIP-lipiodol suspensions mediated arterial embolization hyperthermia on rabbit VX2liver tumor vessels.Methods The rabbits bearing VX2liver tumor were randomly divided into five groups, including control group(Group Ⅰ), liPiodol embolization group(Group Ⅱ), CIP/lipiodol suspensions embolization group(Group Ⅲ) and CIP/lipiodol suspensions embolization hyperthermia group(Group Ⅳ). Two weeks after the embolization, expression of CD34and VEGF of tumors were detected by Super Vision immunohistochemical methods. MVD calculation was based on the number of CD34positive blood vessels.Results The MVD in tumor tissues and the positive rate of VEGF in two embolization groups were significantly lower than that in the control (P<0.01), and those in the AEH group was even more lower than in two embolization groups (P<0.01)Conclusion CIP-lipiodol suspensions mediated arterial embolizati-on hyperthermia at43-45℃can effectively injury tumor vessels and inhibit the positive expression of VEGF, which may play an important role in treatment of tumor.更多还原