基于PEPT1为载体的阿霉素靶向抗肝癌的减毒增效作用研究

发布时间：2018-05-12 15:22

本文选题：寡肽转运蛋白1 + 阿霉素　；参考：《天津医科大学》2016年博士论文

【摘要】：背景:原发性肝癌,以肝细胞癌(Hepatocellular carcinoma,HCC)常见。我国是乙肝高发国,故肝癌发病率尤为高[1]。世界范围平均每年有25-100万人死于HCC,我国竟占45%之多[2],在男女常见恶性肿瘤居于第五和第七位[3]。早期肝癌最佳治疗为肝切除术[4],由于该疾患隐匿,早期诊断困难,多数患者合并肝硬化,故临床上可获得手术机会的患者不多[5]。因此包括化疗的综合治疗是肝癌的重要治疗方式[6,7]。然而化疗对肿瘤细胞不具有特异性,其也作用于正常细胞,从而产生严重毒副作用,患者获益有限。靶向药物载体系统具有很好的应用前景[8]。其优点在于能够将药物特异性的运送到靶区,达到最高疗效,又能降低对正常细胞及组织的毒副作用,提高化疗总体有效性、安全性和患者依从性[9]。寻找安全有效的载体是目前的研究热点。寡肽转运蛋白(proton-independent oligopeptide transporter 1,PEPT1)对许多药物的吸收和分配起到重要作用[10]。研究报道PEPT1在正常细胞及组织中表达谱有限,而在很多恶性肿瘤中异常表达[11,12],提示PEPT1有可能在HCC中也特异性表达。因此,本研究目的为验证PEPT1在HCC中的表达,同时基于PEPT1为载体,将阿霉素改构,使其能被PEPT1识别并靶向转运入肝癌细胞,实现阿霉素靶向抗肝癌的减毒增效作用。本研究内容分为三部分:第一部分PEPT1在HCC的表达及靶向转运药物的功能研究目的:探讨PEPT1在HCC中的表达及其药物转运性能。方法:应用Western blot、免疫组化及Real-time RT-PCR验证PEPT1在人肝癌中的表达,同时检测PEPT1在肝癌细胞中药物转运的性能。结果:Western blot提示PEPT1在人肝癌细胞特异性表达;Real-time RT-PCR提示人肝癌细胞系中有PEPT1 m RNA的表达;PEPT1在正常组织、癌旁组织及HCC组织中表达阳性率分别为52.63%、53.85%、86.00%,差异具有显著性(P0.05);PEPT1在高、中、低分化肝癌组织中的表达阳性率分别为92.31%、90.00%、25.00%,差异具有统计学意义(P0.05)。人肝癌细胞系对PEPT1作用底物的摄取呈时间及浓度依赖性,存在竞争抑制现象。结论:PEPT1在HCC中高表达,与肿瘤分化程度相关。人肝癌细胞对PEPT1作用底物的摄取呈时间及浓度依赖性及竞争抑制现象。第二部分PEPT1靶向转运阿霉素抗肝癌的减毒增效作用的体外实验研究目的:合成阿霉素小肽偶联物,验证PEPT1转运该药物的效能。方法:合成阿霉素小肽偶联物并鉴定;应用荧光显微镜观察该药物在肝癌细胞内的分布,应用高效液相色谱法(High Performance Liquid Chromatography,HPLC)检测细胞中的药物浓度。通过动力学等实验验证PEPT1对该药物的转运性能。结果:阿霉素小肽偶联物成功合成;与阿霉素比较,阿霉素小肽偶联物更易进入肝癌细胞,其在肝癌细胞中具有较高的浓度(P0.05)。肝癌细胞对该药物的摄取亦呈时间浓度依赖性及竞争抑制现象。结论:阿霉素小肽偶联物能够被PEPT1特异性识别并靶向转运肝脏细胞,细胞内药物浓度稳定。第三部分以PEPT1为载体的阿霉素靶向抗肝癌的减毒增效作用的体内实验验证目的:验证以PEPT1为载体的阿霉素靶向抗肝癌的减毒增效作用。方法:构建Hep G2及Bel-7402肝癌模型,每一模型设对照组、阿霉素小肽偶联物组及阿霉素组,前者低剂量给药,后者高剂量给药,对比分析不同药物及同一药物不同剂量的疗效及安全性。结果:Hep G2实验发现:与对照组及阿霉素组比较,阿霉素小肽偶联物组裸鼠一般情况良好,生存期较对照组延长8.57%(3.0 d),较阿霉素组延长24.28%(8.5d)。阿霉素小肽偶联物组及阿霉素组肿瘤体积增长减慢,肿瘤重量减低,二者肿瘤抑瘤率分别为42.13%及25.28%,差异有显著性(P0.05)。与阿霉素比较,阿霉素小肽偶联物无明显心脏及骨髓损伤(P0.05)。Bel-7402成像可见该药物主要分布在皮下肿瘤及肝脏转移瘤组织中;增加阿霉素小肽偶联物剂量能提高其抗癌疗效,并不增加骨髓及心脏毒性,但增加阿霉素给药剂量虽可提高抑瘤率,同时毒副作用也增加。结论:PEPT1是阿霉素靶向抗肝癌的载体,阿霉素小肽偶联物能够实现阿霉素靶向抗肝癌减毒增效的作用。
[Abstract]:Background : Primary liver cancer is common in hepatocellular carcinoma ( HCC ) . Our country is the high incidence of hepatitis B , so the incidence of liver cancer is especially high . The world ' s average annual rate of 25 - 1 million people died of HCC , accounting for 45 per cent of the world ' s most common malignant tumors in the fifth and seventh positions . Early diagnosis of liver cancer is the best treatment for liver resection . Since the disease is hidden , early diagnosis is difficult , most patients complicated with liver cirrhosis , so the number of patients who can get the opportunity of surgery is not more than 5 million . Therefore , the comprehensive treatment of chemotherapy is an important treatment method for liver cancer , namely , 6 , 7 , but the chemotherapy has no specificity for tumor cells , and also acts on normal cells , thereby generating serious toxic and side effects , and the patient has limited benefit . The targeted drug carrier system has good application prospect . The invention has the advantages that the medicament - specific delivery to the target area can be achieved , the highest curative effect can be achieved , the toxic and side effects on normal cells and tissues can be reduced , the overall effectiveness of chemotherapy , safety and patient compliance can be improved . The search for safe and effective vectors is the focus of current research , and the absorption and distribution of many drugs plays an important role in the absorption and distribution of many drugs . Objective : To investigate the expression of PET1 in human hepatocellular carcinoma ( HCC ) and to evaluate its drug transport properties . Results : Compared with the control group and the adriamycin group , there was no significant difference in the tumor size and the tumor weight . The results showed that the adriamycin small peptide conjugate had no obvious heart and bone marrow damage ( P0.05 ) .

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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