【作者】 李征；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 【IONP-PLL的特性及前期研究结果】氧化铁纳米颗粒是一种在生物研究领域得到广泛应用的纳米材料。我室向娟娟博士制备了一种自组装的多聚赖氨酸修饰的氧化铁磁性纳米颗粒（poly-L-Lysine modified iron oxide nanoparticles,IONP-PLL）。IONP-PLL为应用二价和三价铁在高浓度的葡聚糖体系中共沉淀制成分散状态良好的氧化铁磁性纳米颗粒后经多聚赖氨酸修饰而成。前期研究表明:IONP-PLL颗粒表面能大量吸附生物大分子,如DNA、RNA,被该纳米颗粒吸附的物质能抵御各种生物酶的降解作用。且IONP-PLL颗粒对于细胞几乎没有毒性,可被体外培养细胞吞噬,在细胞浆和细胞核均有分布。表面修饰有多聚赖氨酸的氧化铁纳米颗粒已成功地将报告基因GFP,Beta-galactosidase转运至细胞内,并获得高效表达。同时IONP-PLL也可以将FITC标记的反义寡核苷酸转运进入细胞。体内试验研究结果表明IONP-PLL纳米颗粒大部分不被肝,脾的巨噬细胞吞噬,可以经肾脏排泄,且能通过血脑屏障在脑胶质细胞分布。转运报告基因GFP至体内后,用流式细胞仪观察,在肺部的分布最高,有34.9%。综上所述,IONP-PLL纳米颗粒具有将外源基因转运至体内、外细胞,且基因获得表达的能力。那么这种能力的具体机制是什么?我们能否利用这种能力进行基因治疗的应用呢?本论文将集中于这两方面的研究。【IONP-PLL入胞机制研究】电镜和普鲁士兰染色结果显示IONP-PLL进入细胞的表现类似于铁转运的过程。考虑到IONP-PLL的主要成分就是不同价态铁离子的混合物,那么IONP-PLL进入细胞的机制是否和铁离子进入细胞的机制存在相同或类似的原理呢?本文利用数种转铁蛋白—转铁蛋白受体途径的阻断剂进行了分析。结果发现在转铁蛋白存在状态下,IONP-PLL进入细胞表现出温度和时间的依赖性,在1h内随着时间的增加进入细胞的量增多。使用NH₄C1、CH₃NH₂和Trypsin对转铁蛋白—转铁蛋白受体途径进行干扰后均减少了IONP-PLL进入细胞的量,但并不能完全抑制IONP-PLL进入细胞。这说明IONP-PLL确实能通过转铁蛋白—转铁蛋白受体途径进入细胞,同时还存在非转铁蛋白—转铁蛋白受体途径,且该途径同样存在温度和时间的依赖性。因此,IONP-PLL进入细胞的机制同时依赖于转铁蛋白—转铁蛋白受体途径和非转铁蛋白途径。【IONP-PLL/DNA在体内器官和肿瘤组织中的分布】为了验证IONP-PLL能携带外源基因在体内器官分布,将IONP-PLL/EGFP-C2复合物从C57/BL6小鼠尾静脉注射入小鼠体内。在30min、1h、2h取血液进行涂片,48小时后,处死小鼠,取各组织器官观察荧光信号。结果显示:在肺,脾,肾均可见荧光信号,而在肝,胃,心基本未见荧光信号,该结果与向娟娟报道的实验结果一致。除此以外,本研究发现血液涂片中明显看到IONP-PLL/DNA复合物颗粒粘附于血细胞表面,部分分散在血液当中。淋巴结组织有明亮的绿色荧光信号,提示IONP-PLL可携带外源基因在血液系统中表达。将B16F10细胞通过尾静脉注射进入C57BL/6小鼠体内,成功建立了小鼠黑色素瘤肺转移动物模型。将IONP-PLL/pNM23-GFP复合物从尾静脉注射入肺转移瘤建模小鼠体内,NM23-H1抗体和GFP抗体进行免疫组化分析发现肿瘤细胞中有明显的NM23-H1-GFP融合蛋白的阳性表达。以上结果验证了IONP-PLL携带外源基因进入体内各器官和淋巴结组织以及肿瘤组织,并使基因在细胞获得表达的能力。【IONP-PLL携带pNM23-GFP质粒对小鼠黑色素瘤肺转移模型的实验性基因治疗及与化疗的联合治疗观察】肺整体外观观察和HE染色实验证明成功建立了小鼠黑色素瘤肺转移动物模型。pNM23-GFP质粒所表达的NM23-H1基因是在黑色素瘤高转移与低转移株之间差异表达分析发现的抑制肿瘤转移的基因,能明显的抑制黑色素瘤的转移。实验建模小鼠共分为6组:生理盐水注射对照组,IONP-PLL组,DNA组（空pNM23-GFP质粒）,环磷酰胺（CTX）组,IONP-PLL/pNM23-GFP组,IONP-PLL/pNM23-GFP/CTX组。生理盐水注射空白组于肿瘤接种前1天,接种后6天,13天注射生理盐水各一次;IONP-PLL/pNM23-GFP混合物为将15μg的IONP-PLL滴加入30gg的DNA混匀合成,亦同样注射三次;IONP-PLL组仅注射15μg IONP-PLL;DNA组仅注射30μg pNM23-GFP质粒。环磷酰胺溶于生理盐水中,以100mg/Kg剂量按同样规律给药三次;IONP-PLL/pNM23-GFP/CTX组以相同剂量混合使用IONP-PLL/pNM23-GFP和CTX以同样规律注射C57BL/6建模小鼠三次。通过肺的整体外观观察,转移瘤灶数目统计以及HE染色实验,检测了基因治疗及与化疗联合治疗抑制肿瘤转移、生长的效果。注射生理盐水的空白组、注射裸DNA和单独IONP-PLL的处理组均可见大量的黑色素瘤转移灶,转移灶布满整肺,形成巨大癌巢,有坏死、出血。单用DNA组转移灶发生率为214±32,单用IONP-PLL组转移灶发生率为210±19,两组与空白组相比没有太大差别（P＞0.05）,其中,较大转移灶（直径大于0.7mm）的比例分别为16.9%和13.2%。而在IONP-PLL/pNM23-GFP复合物治疗组可见散在癌巢分布,未见明显巨大癌巢及坏死,转移灶形成率54±22,与前三组区别明显（P＜0.005）,较大癌巢比例下降为11.2%。环磷酰胺注射组肺部形成的黑色素瘤转移灶为130±36,其抑制肿瘤生长的效果更加明显,较大瘤灶的比率降低为6%。使用IONP-PLL/pNM23-GFP和环磷酰胺的联合治疗组转移灶的形成率进一步下降,仅为19±7,偶见较大瘤灶,抑制肿瘤转移和生长的效果都非常明显,取得了良好的治疗效果。生存曲线分析显示在注射生理盐水的空白对照组,小鼠的生存时间均短于28天,平均大概为15天。而在注射裸DNA组和空IONP-PLL组,生存时间与空白组相比无明显的区别。注射IONP-PLL/pNM23-GFP的治疗组和环磷酰胺治疗组均明显延长了小鼠的生存时间,分别平均达到了38天和34天,与空白对照组存在显著的区别,P＜0.05。值得注意的是,联合使用的IONP-PLL/pNM23-GFP和环磷酰胺的治疗组更加显著的延长了小鼠的生存时间,与单独应用组相比,其P值均小于0.05。而且在第100天结束观察时,联合治疗组仍有两只小鼠生存,解剖肺组织发现,其中一只小鼠基本未见黑色素瘤转移灶,另外一只小鼠仅见极少量黑色素瘤转移灶。以上结果充分说明IONP-PLL能携带pNM23-GFP质粒进入肿瘤细胞,并且NM23-H1-GFP融合蛋白在肿瘤细胞中获得表达产生了基因治疗的效果。联合使用化疗药物一环磷酰胺和IONP-PLL/pNM23-GFP基因治疗较单独使用化疗或基因治疗能更加明显的抑制肿瘤的转移和生长,延长小鼠的寿命。综上所述,IONP-PLL以类似于铁转运的方式进入体内、外细胞,能携带外源基因进入体内各器官、淋巴结组织和肿瘤组织,并使外源基因获得表达。IONP-PLL携带抑制肿瘤转移的基因进入小鼠体内后,能明显的抑制肿瘤的转移和生长,显著延长小鼠的寿命达到基因治疗的效果。而且当与常规化疗药物联合应用进行肿瘤转移的治疗能达到更好的效果。更多还原

【Abstract】 【Background of IONP-PLL】Iron oxide nanoparticles are one of the most widely studied of nanomaterials in biological research.Dr.Juan-juan Xiang developed a self-assembled non-viral gene carrier;Poly-L-Lysine modified Iron Oxide Nanoparticles（IONP-PLL）.IONP was prepared by alkaline precipitation of divalent and trivalent iron chloride in the presence of high concentration of dextran, then poly-L-lysine grafted on the surface of IONP and developed IONP-PLL.Previous study has shown that IONP-PLL could bind DNA and protect it against enzymatic degaradation as well as efficiently transfer plasmid DNA and antisense oligonucleotides into cells and have no obvious cytotoxicity in broad range of concentration.In vivo, IONP-PLL was demonstrated to deliver exogenous genes to organs such as lung,spleen,brain and kidney,the expression of exogenous genes in lung reached highest 34.9%around these organs. Taken together,IONP-PLL has ability to transfer exogenous gene into cells and get high expression in vitro and in vivo.However,what are the mechanisms about this ability of IONP-PLL? And should we use this ability in gene therapy? In this study,we will focus on the research of these two questions.【The research of mechanisms of IONP-PLL into cells】IONP-PLL was shown to internalize and accumulate at the cytoplasm and nucleus of the treated cells by transmission electron microscopy and iron stain test.This process seems like how iron gets into cells.Considering that the main elements of IONP-PLL are different valence stage iron ion complexes,are there same mechanisms between IONP-PLL and iron uptake by cells?First transferring-bound IONP-PLL accumulation in the cultured cells was investigated.The results shown that Tf-IONP-PLL uptake kept increasing in a linear manner during the whole period of incubation.By adding blockers,such as NH₄Cl,CH₃NH₂ or Trypsin,the cellular Tf-IONP-PLL uptake was decreased but it couldn’t completely suppress IONP-PLL into cells.The cells had the capacity to acquire transferring-free IONP-PLL at pH 6.5 and the uptake was time and temperature dependent.In conclusion,the mechanisms of how IONP-PLL moves into cells,much like iron,were mediated by Tf-TfR endocytosis and transferring-free uptake. 【IONP-PLL/DNA complexes distribution in organs and tumor tissue】To define again the profile of cell uptake and the tissue distribution of IONP-PLL/DNA in vivo,fluorescent protein in vivo was analyzed by fluorescent microscopy.After intravenous injection of IONP-PLL/ EGFP-C2 complexes to C57/BL6 mice,blood film was observed in different time and Fluorescence was analyzed in many organs after 48h. From blood film,IONP-PLL/DNA complexes dispersed in blood and adhered in the surface of blood cells.Fluorescent micrographs showed fluorescent signal in cells of lung,spleen and kidney.Liver,heart and stomach showed very weak GFP expression.This result was same with previous studies.Additionally,high GFP expression was observed in lymph node.B16F10 melanoma cells were injected into mice via the tail lateral vein,resulting in tiny but visible nodules on the lung surface 3 days after implantation,and 15 days after injection,the nodules were large and displayed both hemorrhage and necrosis.To define the transfection efficiency and distribution of DNA in lung tumor metastasis nodules, sections were immunohistochemically stained with an anti-GFP and anti-NM23-H1 antibody after intravenous injection of IONP-PLL/ pNM23-GFP complexes to modeling mice.High positive expression of the gene was observed in the tumor cells of metastatic nodules.The study showed that IONP-PLL may be used as a gene vector for systemic tumor gene therapy.【IONP-PLL delivery of anti-metastatic NM23-H1 gene improves chemotherapy in a mouse tumor model】B16 cells were injected into C57BL/6 mouse via tail lateral vein, and subsequently formed an experimental pulmonary metastasis.The NM23-H1 gene expressed by pNM23-GFP plasmid is characterized by its ability to suppress metastatic potential in vivo.Mice were injected with tumor cells and randomized over six different treatment groups.The different treatment groups were（ⅰ） IONP-PLL/ pNM23-GFP,（ⅱ） cyclophosphamide,（ⅲ） IONP-PLL/pNM23-GFP + cyclophosphamide, （ⅳ） empty IONP-PLL,（ⅴ） free pNM23-GFP plasmid DNA,and（ⅵ） saline.These groups were treated with intravenous injections into model mice the day before B16F10 cell injection and once a week for 3 weeks thereafter.IONP-PLL/ pNM23-GFP complexes were formed as above with 30μg of pNM23-GFP plasmid DNA and 15μg of IONP-PLL. Cyclophosphamide was dissolved in 0.9%salt solution and injected at 100 mg/kg.Group（ⅲ） was treated with a combination of both IONP-PLL/ pNM23-GFP complexes and cyclophosphamide.Empty IONP-PLL and free pNM23-GFP plasmid DNA were separately injected with 15μg of empty IONP-PLL and 30μg of free pNM23-GFP plasmid DNA.First the size and number of metastatic nodules in the different treatment groups were evaluated（n=6）.The result shown:the growth of tumor nodules at the surface of the lungs was significantly suppressed after treatment with either cyclophosphamide,IONP-PLL incorporating pNM23-GFP plasmid DNA,or with the combination of these two compounds（p＜0.05）.The number of metastasis nodes in these three test groups was 62.2%,23.9%and 8.9%,respectively,of those in the mice treated with free pNM23-GFP plasmid.Of note,combined use of cyclophosphamide and IONP-PLL incorporating pNM23-GFP allowed more efficient suppression compared to using either cyclophosphamide or IONP-PLL/pNM23-GFP separately（p＜0.05）.One of the 6 mice showed no evidence of nodules on the lung at the end of experiment（15 days）. The groups of mice treated with empty IONP-PLL,free pNM23-GFP or saline all developed metastases similarly,showing evidence of hemorrhage and necrosis.Then the Kaplan-Meier survival curves was analyzed of all six animal groups after the start of treatment（n=10）.All groups were blinded to the investigators with regard to the treatment given.Disclosure of the different groups occurred at the end of the experiment（100 days）.As expected,all the control mice in the saline group died within 28 days with a mean survival of approximately 15 days.Treatment with either empty IONP-PLL or free pNM23-GFP plasmid DNA solution did not result in prolonged sirvival.Interestingly,treatment with pNM23-GFP -loaded IONP-PLL nanovectors delayed tumor growth and prolonged survival to an average survival of approximately 38 days.This was comparable to the improved survival after treatment with cyclophosphamide,which extended the mean survival to approximately 34 days.Notably, combination treatment with IONP-PLL/ pNM23-GFP and cyclophosphamide further prolonged survival significantly compared to cyclophosphamide treatment alone（p＜0.032）.In the group receiving IONP-PLL/pNM23-GFP and cyclophosphamide,2 out of 10 mice were alive at the end of the experiment（100 days）.In one of these mice,there was no evidence of pulmonary metastasis,and,in the other mouse,only small nodules were observed in the lungs.In conclusion,IONP-PLL can transfer pNM23-GFP plasmid into tumor cells and get high expression of NM23-H1-GFP fusion protein to arrive effect of gene therapy.It is noteworthy that the combination treatment of cyclophosphamide and the NM23-H1-GFP gene loaded IONP-PLL induced a more efficient suppression of metastasis and longer survival time of mice.In conclusion,IONP-PLL uptake by cells through mechanism similar to iron into cells and transfer exogenous gene into organs,lymph node and tumor tissue in vivo at physiological conditions.In the mouse model,prolonged survival and suppression of tumor metastasis were observed due to significant improvement of the transgene expression using the IONP-PLL nanoparticles.In combination with cyclophosphamide,the anti-tumor effects were observed to be enhanced. We suggest that IONP-PLL may be developed for efficient gene delivery for use in future anti-tumor therapies in the clinic.更多还原