CAS OpenIR  > 中科院上海应用物理研究所2004-2010年
M-PEIs转导OPN shRNA抑制肝癌的生长和转移
Alternative TitleM-PEIs Mediated Transfer of shRNA Against Osteopontin Suppresses Growth and Metastasis of HCC
董黎
Subtype博士
Thesis Advisor姚思德
2008-05-30
Degree Grantor中国科学院上海应用物理研究所
Place of Conferral上海应用物理研究所
Keyword聚乙烯亚胺纳米凝胶 骨桥蛋白 基因治疗 肿瘤转移 肿瘤凋亡
Abstract由于基因转导载体系统存在毒性和转染效率较低等缺陷,制约了当今基因治疗的发展和应用。骨桥蛋白(Osteopontin, OPN)是一种磷酸化的分泌型糖蛋白,其表达与各种肿瘤的发展与转移密切相关。但是OPN在肿瘤侵袭转移过程中的具体作用机制尚不明确,有待进一步研究。本研究首先评价了M-PEIs作为一种新型载体对肿瘤细胞系统应用的特性。然后我们应用M-PEIs转导质粒shRNA抑制肝癌细胞中OPN表达,观察其对肝癌细胞体内、体外抗肿瘤的效果并进行可能的机制探讨。 M-PEIs可使转导的质粒持续释放并能保护质粒避免被血清清除。用M-PEIs介导基因转染,可使细胞转基因持续表达至14天。更为重要的是,给肝癌原位荷瘤裸鼠静脉注射M-PEIs/质粒复合物,GFP报告基因大部分在肿瘤组织表达。该结果提示M-PEIs在靶向肿瘤的系统基因传递应用方面有一定潜能。 应用M-PEIs转导质粒shRNA抑制肝癌细胞中OPN表达,研究发现OPN下调抑制了肝癌细胞的生长能力,锚定非依赖性生长能力,粘附能力和侵袭能力,体内实验发现抑制了肝癌细胞的成瘤能力以及裸鼠的肺脏转移。随后的机理探讨发现OPN的沉默抑制αv, β1和β3 integrin的表达,阻断了核转录因子NF-κB的激活,抑制了Bcl-2/Bcl-xL和XIAP的表达,增加了Bax的表达,从而诱导肝癌细胞发生线粒体途径的凋亡。给肝癌细胞补充人重组OPN蛋白并不能逆转OPN沉默导致的促细胞凋亡作用。此外,OPN的下调抑制了化疗药物诱导的NF-κB的激活,使肝癌细胞对化疗药物的敏感性增加,体内实验采用M-PEIs转导OPN shRNA联合应用化疗药物5-FU,结果显示联合应用导致肝癌荷瘤裸鼠肿瘤消退。实验结果提示,OPN促使肿瘤发生和转移是通过保护肿瘤细胞避免发生凋亡来实现的。RNA干扰介导的OPN下调,通过增加化疗药物的敏感性为肝癌的治疗提供了一种新策略。
Other AbstractSuccessfully gene therapy has been hindered by vector-related limitations, including toxicity and inefficient gene delivery to tumor cells after intravenous administration. Expression of osteopontin correlates with tumor progression and metastasis. The mechanisms by which osteopontin promotes tumor cell survival remain unclear. In this study, we first evaluated the potential of spherical polyethylenimine nanogels (M-PEIs) as a novel vector for intravenous delivery of plasmids to tumor cells. Then we applied M-PEIs to deliver a short-hairpin RNA for depletion of osteopontin in HCC cells. The anti-tumor effects by osteopontin-depletion in hepatocellular carcinoma (HCC) were investigated. M-PEIs provided a sustained release of plasmids up to 14 days and were also efficient in protecting plasmids from enzymatic degradation in serum conditioned media. M-PEIs showed no obvious cytotoxicity to mammalian cells in vitro. Importantly, following intravenous administration of M-PEIs/plasmid complexes into human hepatocellular carcinoma xenograft-bearing mice, GFP reporter gene expression was predominantly found in the tumor. These results indicate that M-PEIs may be a candidate for systemic delivery of plasmids into tumors. Down-regulation of osteopontin inhibited HCC cell growth, anchorage-independent growth, adhesion with fibronectin and invasion through ECM in vitro and suppressed tumorigenicity and lung metastasis in nude mice. Osteopontin-silencing resulted in suppression of αv, β1 and β3 integrin expressions, blockade of NF-κB activation, inhibition of Bcl-2/Bcl-xL and XIAP expressions, increase of Bax expression, and induction of a mitochondria-mediated apoptosis. The pro-apoptotic effects induced by osteopontin-silencing could not be reversed by addition of recombinant human osteopontin. Furthermore, down-regulation of osteopontin inhibited drug-induced NF-κB activation and sensitized HCC cells to chemotherapeutic agents in vitro, which led to complete regression of HCC xenografts in nude mice. Osteopontin may facilitate tumorigenesis and metastasis through prevention of tumor cells from apoptosis. RNA interference-mediated depletion of osteopontin may be a promising strategy for the treatment of HCC by sensitizing the chemotherapeutic drugs.
Pages105
Language中文
Document Type学位论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/7251
Collection中科院上海应用物理研究所2004-2010年
Recommended Citation
GB/T 7714
董黎. M-PEIs转导OPN shRNA抑制肝癌的生长和转移[D]. 上海应用物理研究所. 中国科学院上海应用物理研究所,2008.
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