【作者】 唐滔；

【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】 研究目的胆管癌主要指源于肝外胆管的恶性肿瘤,预后不佳。流行病学少见,国外尸检发现率为0.01%～0.5%,占恶性肿瘤总数3%,我国尸检占0.07%～0.3%,其检出率逐年增高。胆管癌占所有胆道疾病总数的2.32%,男女之比为1.46:1。近年来其发病率有上升的趋势。胆管癌的治疗方法多采用以手术为主放化疗为辅的综合胆管癌的治疗方法多采用以手术为主放化疗为辅的综合治疗。但由于其位置深、隐蔽、早期发现困难,使手术治疗效果很差,生存率低。化疗药物通过作用在细胞繁殖的不同环节,抑制或杀灭肿瘤细胞,从而达到治疗的目的,已成为当今临床治疗肿瘤的重要手段。但是目前中晚期胆管癌的化疗效果很不令人满意。既往报道以5-氟尿嘧啶为主的单药或联合化疗的有效率为14%～18%。运用纳米技术和化疗药物相结合的方法,在磁导向的作用下靶向性治疗恶性肿瘤是近年来兴起的一种新的治疗手段。载药的磁性纳米微粒是纳米技术和化疗药物相结合的产物。在磁导向作用下聚集在肿瘤部位,这不仅提高了肿瘤局部的化疗药物浓度,而且纳米颗粒本身的可控性降解可使载体内的化疗药物持续性释放,使药物在血液中循环时间延长并维持有效的血药浓度,且可减少代谢产物,易于清除,副作用少。为恶性肿瘤的综合治疗带来了新的希望和途径。在邹声泉教授的领导下,我们课题组不仅成功获得了国家高新技术研究发展计划(863计划)资助的重大课题《纳米生物磁小体靶向药囊治疗肝胆胰恶性肿瘤的实验研究》(编号2002AA214061)。该项目的核心内容是:采用高新技术研制磁化纳米金属薄膜包覆的支架,在通畅胆道引流的同时,可增强5-Fu纳米磁小体靶向药囊的靶向性,真正达到高选择性区域性化疗的目的,以实现胆管癌治疗观念中的创新和突破。而且在前期的研究中我们对胆管癌肿瘤生物学特性有了深入的研究,认识到胆管癌的发生、发展过程中各种致癌因子和抑癌因子中存在着复杂的网络式的反馈机制,其中Rb的蛋白信号转导通路可能在这个网络中起到重要的作用。在肝外胆管癌的分子细胞遗传学研究取得了很大的进展。在此基础上,我们拟构建针针对Rb的RNAi真核表达载体,并将其转导人胆管癌细胞中,观察对Rb蛋白信号转导通路的影响及对胆管癌生长的影响。并与纳米磁小体靶向药囊联合作用人胆管癌细胞,观察在体内外实验中它们的联合作用对胆管癌细胞生长的影响。研究方法根据Rb基因cDNA序列,设计出针对目的基因的4个siRNA靶序列。克隆到真核表达载体pGenesil vector中,通过酶切鉴定和DNA测序鉴定正确与否。将构建好的4个siRNA表达载体分别转染到QBC939细胞中MTT法、FCM,检测Rb基因沉默后胆管癌细胞生长变化、细胞周期和细胞凋亡情况。RT-PCR和Western Blot检测Rb的mRNA和蛋白在胆管癌细胞系中的表达水平,同时检查与Rb相关的基因的表达情况。将QBC939细胞接种于裸鼠皮下,建立胆管癌裸鼠种植模型,观察体内种植瘤的发生率和生长情况。通过急性毒性试验找出TM5-FuNC对裸鼠的半数致死量(LD50)。根据急性毒性试验结果设定合适的药物剂量。MTT检测TM5-FuNC对转染psiRNARb-1前后QBC939细胞生长抑制的影响。将裸鼠模型再分成4组,分别为对照组,5-Fu组, TM5-FuNC组,psiRNARb-1和TM5-FuNC联合组。观察肿瘤体积变化,计算抑瘤率,绘制生长曲线。实验结果成功构建了针对Rb的真核表达载体,转染psiRNARb-1质粒48至72小时后QBC939细胞的生长相对于对照组细胞受到了一定程度的抑制,MTT实验结果显示当Rb基因在QBC939细胞中表达沉默时,对细胞生长有一定的抑制作用,在72小时内呈一定的时间依赖性。转染后QBC939细胞中Rb的mRNA表达相较于未转染细胞明显下调,Rb蛋白表达也明显受抑制。p16的mRNA和蛋白的表达变化情况与Rb相反。E2F1转录因子mRNA和蛋白的表达变化趋势与Rb相同。转染组中P27kip1蛋白的表达较未转染组有所增强。TM5-FuNC的急性动物实验中所有动物均未死亡,最大耐受剂量为1250 mg/kg。是临床给药剂量的5倍。TM5-FuNC 250和200mg/kg组的移植肿瘤体积在治疗后35天与对照组比较有显著差异(P<0.05)。电境结果显示,TM5-FuNC对荷瘤裸鼠的肿瘤细胞有诱导凋亡作用,且随着药物浓度的增大,细胞凋亡的形态改变越明显。转染psiRNARb-1的QBC939细胞可显著增强TM5-FuNC和5-Fu的生长抑制作用(P<0.05)。TM5-FuNC和TM5-FuNC+psiRNARb-1联合组与对照组和5-Fu组比较肿瘤体积的生长有明显差异(P<0.001)。TM5-FuNC和TM5-FuNC+psiRNARb-1联合组之间比较(P=0.44)有一定的差异显著性。实验结论1成功构建了4对针对Rb的siRNA真核表达载体,其中转染的psiRNARb-1质粒对胆管癌QBC939细胞有一定的抑制作用,并可诱导的胆管癌细胞G0/G1期阻滞和细胞凋亡。这种作用可能跟Rb与p16、E2F1、P27kip1之间的反馈调节机制相关。为我们将Rb作为基因治疗的靶目标提供了实验依据。2通过急性毒性实验,我们获得了TM5-FuNC最大耐受剂量为后续实验提供了用药依据。3 TM5-FuNC新剂型在内磁场的导向作用下能有效抑制人胆管癌裸鼠移植瘤的生长,使我们所研制的TM5-FuNC纳米药物制剂结合内磁场靶向治疗胆管癌的设计理念获得了实验的证实即可磁化的NiTi合金支架在保持胆道通畅引流的情况下,能高靶向的引导TM5-FuNC聚集在肿瘤部位实现对肿瘤高效地、持续地的治疗,并保证对机体的低毒性。4 TM5-FuNC联合psiRNARb-1作用组相比单用TM5-FuNC组能更有效的抑制胆管癌肿瘤的生长,为TM5-FuNC联合基因治疗提供了理论依据和实验平台,为治疗中晚期胆管癌指明了一条新的途径。更多还原

【Abstract】 Background & Objective: Cholangiocarcinoma is mainly named as the extrahepatic bile duct malignant tumor. The prognosis of it isn’t idealization. It’s scarce in epidemiology and is about 0.01%~0.5% in biopsy, about 3% in the total count of the malignant tumor. It’s about 0.07%~0.3% in domestic biopsy. The incidence rate of Cholangiocarcinoma is about 2.32% in all disease of biliary tract. The rate of female and male is 1.46:1. The incident rate is increasing progressively in recent years. The treatments of cholangiocarcinoma were given priority to operation or combine chemotherapy with radiation treatment. But the position of cholangiocarcinoma very deep and concealment in anatomy, it’s hard to detect cholangiocarcinoma in earlier period. As a result the effect of operation was discontentment, and the survival rate was very low. The chemiotherapy drug inhibit or kill tumor cells by act on different links of cell breeding, which can treat tumor. This is become a important method for clinical tumor treatment now. But the effect of chemiotherapy drug can not reach the aim of satisfactory. The report of the effect of 5-Fu is only reach 14%-18% regardless of simplex or combine medication.Targeting treatment to malignant tumor is a new therapeutic method which as a result of conjunct nanotechology with chemotherapeutic drug rising in recent years. The principle is that the carrier including various kinds species group affinity carry particles and magnetic targeting particles take drug which could targeted reach to the locus of medication in order to treat disease. This will afford experctation for cure malignant tumor.Our topic group lead by professor zoushengquan succeed get a great topic supported by the grant from The National Hi-Tech Research and Development Program of China (863 Program)(No. 2002AA214061). The core content of this project is that a magnetize cradle which be sacked by metal can reinforcement the TM5-FuNC targeting power while the cradle keeping bile duct drain smoothly. As a result that this will to achieve the real high selective and regional treatment. This will realizated break and new idea of Cholangiocarci- noma treatment, Furthermore, our group of the topic group has investigated deeply on the cholangiocarcinoma bionomics before. We concluded that there are complex network feed back mechanism between the oncogene and the anti-oncogene which is exist in the process of occurrence and develop of cholangiocarcinoma. pRb signal pathway were close importa- nt to the complex network feedback system of cholangiocarcinoma. It’s a great break in the research of the molecular cytogenetics in the cancer of extrahepatic biliary duct.We based on the prophase research, put our emphasis on the pRb signal transduction pathway, and make some investigate on the apoptosis of the cholangiocarcinoma and the Fuction of the sensitization of the chemotherapy. We association the siRNA eukaryotic expression vector Rb with TM5-FuNC act on human cholangiocarcinoma cell in vivo and vitro, which will provid us a test base and realization pathway for the union opoerate of nanotechnology and gene therapy. Restricted to the time, we are limited and superficialis level, and we will make the Further step in the Future.1. Methods and results: Eukaryotic expression vector of siRNA Rb were constructed, and used to transiently transfect to QBC939 mediated with LipofectamineTM 2000. The growth of QBC939 cell has been inhibited in some degree compare to the control group form 48h to 72h after transfection. Rb slience caused cell cycle arrest at G0/G1. Apoptotic cell index also increased in some degree. It show certain time dependency in 72h.Rb mRNA and protein under expression significantly compare to the control after transfection.p16 mRNA and protein expression inversed to Rb. E2F1 have identical change tendency with Rb. No animal dead in Acute toxicity test. The most tolerance dosage of TM5-FuNC is 1250mg/kg, which is 5 times dosage of clinical administration. High and middle dosage groups of TM5-FuNC have difference significantly compare to the control group (P <0.05) at 35 after treatment.The high and middle dosage groups of TM5-FuNC have discovered cells apoptosis under electron microscope. The ratio of growth inhibiting of TM5-FuNC and 5-Fu increased significantly after transfection. The tumor volume of TM5-FuNC group and psiRNARb-1+TM5-FuNC group have greatly difference compare with those of the control group and 5-Fu group (P<0.001). The tumor volume of TM5-FuNC group compare to tumor volume of TM5-FuNC group also have some difference (P=0.44).Conclusions:1 We are succeed constructed the eukaryotic expression vector of siRNA Rb which can specifically inhibit Rb expression There are certain inhibited growth effects of QBC939 cell line after transfecting psiRNARb-1 into cell. And Rb slience caused cell cycle arrest at G0/G1. Apoptotic cell index also increased in some degree, which may correlation with the Feedback adjust mechanism of Rb、p16、E2F1、P27kip1 protein . It can be used for later test.2 We get TM5-FuNC the maximum tolerance dosage by acute toxicity experiment which is provided base for medication.3 The TM5-FuNC can inhibit the tumor growth of human cholangiocarcinoma xeno- graft on nude mice. It show that our TM5-FuNC and design ideal of infra-magnetic field targeting get confirmation by test. The Ni-Ti alloy cradle who can be magnetized can high targeting led TM5-FuNC collecting in tumor tissue for treatment effectually and lastly when it keeping bile duct drain smoothly.4 TM5-FuNC + psiRNARb-1 have better inhibit effect for cholangiocarcinoma compare to simplex TM5-FuNC. It would establish a test background for Further inosculatio TM5-FuNC with gene therapy.更多还原