【作者】 郑建伟；

【导师】 邹声泉；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 研究目的针对中晚期胆道恶性肿瘤的传统治疗方式,是以胆道支架为主的介入治疗和以氟尿嘧啶为主的区域化疗,邹声泉在国内外研究基础上,提出新的MTDDS模式:采用高新技术研制磁化纳米金属薄膜包覆的支架(磁化支架),在通畅胆道引流的同时,增强纳米磁小体5-FU靶向药囊(Targeted Magnetic 5-Fluorouracil Nanocapsules, TM5-FuNC)的靶向性,真正达到高选择性区域性化疗的目的,改变过去单纯安置支架的传统做法,更重要的是起到对胆道肿瘤的高靶向、低毒性和缓释性的综合治疗作用,实现胆管癌治疗观念中的创新和突破。该项目成功获得国家高技术研究发展计划(863计划)重大项目资助(编号2002AA214061)。本实验旨在通过在分子、细胞和动物整体水平,重点通过胆管癌移植瘤模型,检测TM5-FuNC和磁化支架作用胆管癌的效果,探讨TM5-FuNC的作用机制和在体分布特点,为TM5-FuNC联合磁化支架高靶向治疗胆管癌的理念实施提供实验依据。研究方法本课题对TM5-FuNC在内磁场作用下的靶向效应及其机制进行了初步研究和探索,着重探讨TM5-FuNC联合磁化胆道支架对荷人胆管癌裸鼠移植瘤生长的影响,以及磁共振成像技术评价TM5-FuNC靶向分布特点。此外,围绕TM5-FuNC的体外细胞生长抑制性实验、规模化肿瘤动物模型的稳定性评价、TM5-FuNC诱导胆管癌细胞凋亡与疗效的的相关性等进行初步研究,多角度评价TM5-FuNC给要系统,以期为TM5-FuNC进一步改进和临床实验提供前期研究。分别通过MTT观察胆管癌细胞生长抑制,DNAladder法和流式细胞仪观察细胞凋亡变化,半定量RT-PCR方法检测细胞Bcl-2和Bax的mRNA表达变,评价TM5-FuNC体外细胞生长抑制效应。构建生长行为及病理特征与人类胆管癌一致的胆管癌移植瘤裸鼠模型,并移植瘤体积一致性良好,在此基础上,进行实验分组,分别比较5-Fu、TM5-FuNC联合磁性胆道支架、TM5-FuNC与外磁场等疗效差异,评价TM5-FuNC动物水平抑瘤效应。采用高效液相色谱法(HPLC)检测离体组织中5-FU药物浓度,原子吸收光谱法(AAS)测定人胆管癌移植瘤裸鼠组织中的铁浓度,采用磁共振成像技术(MRI)在体检测TM5-FuNC在人胆管癌移植瘤裸鼠体内组织分布,探索TM5-FuNC组织分布特点。在裸鼠模型胆管癌移植瘤中运用RT-PCR和Western等方法检测各组肿瘤组织中caspse3、Bax/Bcl-2蛋白的表达变化,探索TM5-FuNC可能的作用机制和分子靶点。实验结果TM5-FUNC对胆管癌QBC939细胞株增殖的抑制作用呈剂量和时间依赖性,体外抑瘤率大于5-FU,并胆管癌QBC939细胞株DNA断裂成有规律的特征性梯状条带,调亡率为23.77%,大于5-FU组的13.18%(P<0.05)。实验建立的动物模型移植瘤为胆管癌,成瘤周期短,成功率达100%,带瘤生存期长,肿瘤体积一致好,符合正态分布,其生长行为及病理特征与人类胆管癌一致。在此模型基础上,TM5-FuNC联合磁化胆道支架组的移植瘤生长受到明显抑制,大于TM5-FuNC与传统的外磁场组的效应。在肿瘤组织内建立在磁场后,尾静脉注射TM5-FuNC,与对照组磁共振显示,在T1加权和T2加权肝肾信号都明显降低,在肿瘤信号在T2加权上降低明显,脑组织信号无变化。提示TM5-FuNC主要通过肝肾代谢,可以靶向性分布于肿瘤,TM5-FuNC不能通过血脑屏障。病理学检查也进一步证实;与无磁场的相同药物治疗组及单纯5-FU对照组比较,高效液相色谱法(HPLC)检测人胆管癌移植瘤裸鼠的肿瘤组织中5-FU浓度显著增加(P<0.01),原子吸收光谱法(AAS)检测肿瘤组织中铁浓度也显著增加。与对照组比,在TM5-FuNC联合磁化胆道支架组裸鼠移植瘤组织中, RT-PCR和Western-blot检测凋亡相关基因caspase-3和Bax表达增高,bcl-2表达无变化。实验结论:1. TM5-FUNC在体外细胞水平能显著抑制QBC939细胞株增殖,能导致QBC939细胞凋亡,细胞凋亡过程可能与上调Bax、下调Bcl-2 mRNA基因有关。2.建立了可重复表达人胆管癌生长及行为特性的异位移植瘤裸鼠模型,为评价TM5-FUNC在动物水平效应研究提供稳定模型。3. TM5-FuNC对磁化胆道支架提供的内磁场具有良好的磁响应性,通过TM5-FuNC加内磁场的方式给药,可靶向抑制荷人胆管癌裸鼠移植瘤生长;其基于内磁场的磁靶向治疗效果优于依靠外磁场的靶向治疗。4.在磁化胆道支架提供的内磁场引导下,TM5-FuNC中磁性载体和所载化疗药物氟尿嘧啶的体内分布可以通过内磁场的方式而改变,在人胆管癌移植瘤裸鼠体内具有肿瘤靶向性分布。磁共振成像(MRI)技术是检测TM5-FuNC在活体人胆管癌移植瘤裸鼠体内分布的有效方法。5. TM5-FuNC抑制管癌裸鼠移植瘤生长与诱导肿瘤细胞凋亡有关,并可能与上调凋亡相关基因caspase-3和Bax的表达有相关性, Caspase-3和Bax可能作为评价TM5-FuNC治疗胆管癌效果的的指标。通过以上研究,得出以下几个创新点:1.在靶向治疗为新内容的肿瘤综合治疗模式背景下,利用新材料新科技制备具有良好生物相容性的磁化胆道支架和TM5-FuNC,通过深入系统的实验研究,实现磁靶向治疗胆道肿瘤的新理念为中晚期胆管癌提供高靶向、低毒性、缓释性治疗途径。2.将内磁场概念应用到实验研究中。制备的磁化支架,既具有提供内磁场的功能,又保留记忆特点,解决了胆道记忆合金支架磁化的难题,使胆道支架不仅仅起通道的作用,更为TM5-FuNC提供内磁场,并通过研究,证实TM5-FuNC在磁化胆道支架提供的内磁场作用下,能高靶向在肿瘤组织中分布,克服了过去外磁场作用下的定位不准、磁场强度和作用时间不易控制、磁性颗粒聚集等难题,为进一步临床应用奠定基础。3.探索了应用磁共振成像技术,针对小动物,未偶联任何增强剂和核素示踪剂,在体观察TM5-FuNC的组织分布,为研究TM5-FuNC在体内靶向性治疗提供即时直观的信息。通过MRI研究证实,TM5-FuNC具有良好的组织相容性,排除率高,能在体内靶向性均匀分布。此外,我们也初步研究了TM5-FuNC靶向治疗胆管癌可能的分子机制,凋亡相关基因Caspase-3和Bax与TM5-FuNC抑制胆管癌移植瘤疗效具有相关性,为TM5-FuNC进一步分子水平深入研究提供基础。更多还原

【Abstract】 Objectives:Previous studies of Shengquan Zou’group long-term gone in for the study of pathogenesis and targeted medicine of cholangiocarcinoma, and gained systemic and penetrating achievement, especially in molecular biology of cholangiocarcinoma. Based on those performance record, Prof. Zou winned the financial assistance of National Hi-Tech Study and Progress Project (863 Project)“Experiment Study on targeted Magnetic 5-Fluorouracil Nanocapsules Treating Cholangiocarcinoma”(No: 2002AA214061). The key contents include: to aim directly at intermediate and advanced stage cholangiocarcinoma, employ magnetic stent and magnetic 5-Fluorouracil nanocapsules built by hi-tech to targeted chemotherapy cholangiocarcinoma at the same time of Supporting drainage the bile duct, witch alter the traditionary method and carry out innovate and breakthrough in treating cholangiocarcinoma.This study aims at detect the effect that TM5-FuNC associating with magnetic bile duct stent treat cholangiocarcinoma in vitro and in vivo, especially to implanted tumor of human cholangiocarcinoma in nude mice, explore bio-distribution characteristic of TM5-FuNC, provide experimental data to support the concept that TM5-FuNC Hi-targeted treat cholangiocarcinoma based on the internal magnetic fields.Methods:The targeted effection and its probalble mechanisms of TM5-FuNC in treating cholangiocarcinoma were studied, concentrating on the experimental study on TM5-FuNC to treat cholangiocarcinoma based on internal magnetic field, and the role of study on targeting distribution of TM5-FuNC on nude mouse bearing Cholangiocarcinom by MRI, trying to refer an experimental basis for further study.Mtt, DNA ladder and flow cytometry were employed respectively to detect the inhibition phenomenon or apoptosis expression. Bax and Bcl-2 genes were mesured by reverse transcription-polymerase chain reaction respectively (RT-PCR).Established allotopic transplantation model of human cholangiocarcinoma in nude mice, which provid steady and reproducibility animal mode.,and then, evaluate the effect of TM5-FuNCtargeting to treat cholangiocarcinoma in different groups.High Performance Liquid Chromatography (HPLC) method for the determination of 5-FU in tissues was established and applied to determine 5-FU in mouse tissue samples. A Flame atomic absorption spectrometer (AAS) was used to detect the iron concentration in mouse tissues; MRI was used to detecet the distribution of targeting magnetic nanoparticles in vivo.SP immunohistochemistry was used to detect the expression of caspase-3, bcl-2/bax protein in 38 cases cholangiocarcinoma; Caspase-3, Bcl-2/Bax protein were detected by RT-PCR and Western-Blot allotopic transplantation tumor.Results:The effect of TM5-FuNC inhibited proliferation in human Cholangiocarcinoma QBC939 Cell line was depended on relation of time and dosage, which was more powerful than 5-Fu group. Cholangiocarcinoma QBC939 cells presented some typical feasures of apoptosis: nucler shrinkage,chromatin condensation and DNA fragmentation. agarose gel electrophoresis appeared marked DNA ladder. The apoptosis rate of QBC939 cells treated by TM5-FuNC was 23.77%, which was bigger than 5-Fu group. The level of bax mRNA was up-regulated, the level of bcl-2 mRNA was down-regulated.Tumorigenic rate of 40 nude mice were 100%; average tumorigenic period was one week; average life span of nude mice bearing cholangiocarcinoma was fifteen weeks. Tumor volume shows that the difference among 40 nude mice have no significant. The study of pathology and electron microscope were in accordience with the biological characteristics of human cholangiocarcinoma. In following experiment, the group of TM5-FuNC and magnetic stent, shows significantly therapeutic efficacy to TM5-FuNC and external magnetic fields.The concentration of both 5-FU and Fe in tumor tissues increased significantly in the group of TM5-FuNC plus magnetic field detected by HPLC and AAS respectively, and the signal intensity of tumor tissue is lower than it in control group detected by MRI.The level of Caspase-3 and Bax mRNA were up-regulated in experimental groups detected by RT-PCR and Western-Blot (P<0.05), but the level of bcl-2 mRNA had no change.ConclusionAll the above study could be sumed up as follows:1. TM5-FuNC can inhibit proliferation human Cholangiocarcinoma QBC939 Cells, induce apoptosis of human Cholangiocarcinoma QBC939 Cells, which apoptosis could relate to the up-regulating of bax gene.2. The animal model is established simply and well ; There is no obvious difference in tumor volume and histology ; Biological characteristics of cholangiocarcinoma is kept .this kind of allotopic transplantation model of human cholangiocarcinoma can be used to provid steady and reproducibility animal model for animal experiment.3. Magnetic drug targeting based on internal magnetic field can inhibit the growth of tumor tissue, and its therapeutic efficacy is better than the traditional method based on external magnetic fields.4. TM5-FuNC possesses targeting distribution when treating nude mouse bearing cholangiocarcinoma based on the inducing of magnetic field. MRI is the effective method to detected biologic distribution of TM5-FuNC iv vivo.5. To some extent, caspase-3, bcl-2 and bax protein can predict the prognosis of cholangiocarcinoma. Molecular targets of caspase-3, bcl-2 and bax may also be important factors to the pathogenesis of cholangiocardinomaThis part of medical experimental studys was based on the prophase work, including preparing successedly magnetic bile duct stent and TM5-FuNC, and corresponding toxicology, pharmacology. After those systematical and in-depth studys, some innovations can be concluded as follows:1. On the background of the era of DTS, utilizing new materials and nanotechnique, we haved prepared magnetic bile duct stent and TM5-FuNC possessed well biocompatibility, and developing systemtical studys in depth, supplying a new road for cholangiocarcianoma in mid-advanced stage. 2. Makeing use of the concept of internal magnetic fields to experimental research. The magnetic stent can not only play a part in support bile duct, but also provide internal magnetic fields in the same time. Which overcome the defect of external magnetic fields, such as, locating error, hard controling the time of operation and the strength of magnetic fields, hard aggregating in targeted tissue, et al.3. Exploying the new method by utilizing MRI techquine to detect biodistribution of TM5-FuNC in vivo in little animal model, which can provide a realtime and visualized medium to study the characteristic of TM5-FuNC in pharmacokinetics.Whatmore, we study preliminarily the potential molecule target point of TM5-FuNC acting cholangiocarcianoma, which provide fundament for further study in molecular biology.更多还原