【作者】 苏燕胜；

【导师】 王禾； 秦卫军；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 前列腺癌是男性发病率第二位的恶性肿瘤，我国前列腺癌发病率也逐年升高，其发病增长率已居泌尿生殖系肿瘤之首。前列腺癌具有潜伏性长、发病率高的特点。在我国70岁以上男性人群中，高达25%的人患有前列腺癌。但临床上前列腺癌早期诊断率低，因而多数患者失去了早期手术根治的机会。通过siRNA下调Notch1基因可以有效抑制人类前列腺癌细胞的生长。然而如何靶向输送siRNA进入特定细胞是临床应用此技术的主要障碍。最近有研究表明，单链抗体介导的靶向输送系统是高效下调相应基因的有效方法。为了解决RNA干扰技术在体内应用缺乏靶向性的难题，我们拟通过抗前列腺癌单链抗体的融合基因，并予以优化，进行表达、纯化后获得融合蛋白scFv-Fdt-HA2-tp(sFH-tP)，构建成siRNA靶向输送系统。利用FITC-siRNA在体内外验证这一系统的有效性；随后验证靶向输送的针对Notch1基因抑制效果最佳的siRNA在体内外对前列腺癌细胞生长的抑制作用。利用这一靶向输送系统不但能克服siRNA在体内输送效率低下，还避免siRNA大量应用时所产生的“脱靶”效应、非特异性免疫反应等问题，实现对肿瘤生长的高效抑制，为RNA干扰技术在前列腺癌的临床治疗中的应用提供理论、实验依据。目前前列腺特异性膜抗原（PSMA）被认为是一个较明确的前列腺抗原。我们的前期研究工作表明Notch信号通路中的Notch1在前列腺癌的发生、发展中发挥重要作用，应用siRNA抑制前列腺癌细胞内Notch1基因的表达，引起细胞周期阻滞、细胞凋亡，表明肿瘤细胞的生长受到有效的抑制，因此通过siRNA抑制Notch1基因有望成为一种有效的针对前列腺癌的基因治疗方法。但由于缺乏有效的靶向给药输送系统，常常出现药物的“脱靶”现象，限制了此技术的进一步应用。本课题是在获得高亲和力的特异性抗前列腺癌单链抗体(scFv)的基础上，拟将其与不同的基因片段融合并予以优化，构建成融合蛋白。使单链抗体介导的靶向输送系统与特异性的siRNA结合后，在抗体的介导下到达靶细胞，随抗体内化入细胞到达靶点，从而高效抑制Notch1基因的表达。利用抗体介导siRNA可以特异性的到达肿瘤部位，从而实现特异、高效的抗肿瘤作用，同时避免siRNA大量应用时所产生的“脱靶”效应，此策略将为RNAi技术在肿瘤研究、治疗中的应用开辟新途径。本研究的目的是评估单链抗体及两种融合蛋白是否可以有效的将特异性siRNA输送到PSMA阳性的前列腺癌细胞内。融合蛋白由抗PSMA单链抗体(scFv)、鱼精蛋白截短体(tp)构成。将弗林蛋白酶(Furin)、流感病毒的融合肽(HA2)片段序列插入到单链抗体及鱼精蛋白截短体之间构成另一融合蛋白。我们的结果表明，siRNA能够被融合蛋白特异性的输送到PSMA阳性的前列腺癌细胞内，sFH-tP融合蛋白效果更明显。应用前列腺癌细胞系及前列腺癌荷瘤裸鼠模型，验证靶向输送系统引导下siRNA是否能够在体内实现对Notch1的高效抑制，实现抗肿瘤效果。进一步的研究证实了siNotch1融合蛋白可以在体内及体外有效地抑制前列腺癌细胞的增殖。本研究成果将开创一种高效、特异、安全可行的治疗前列腺癌新疗法，具有重大的临床意义与广阔的应用前景。更多还原

【Abstract】 Prostate cancer is the second leading cause of death in America and isbecoming morecommon in China. Prostate cancer is characterized with the latency and high incidence.InChina,25percent of men over70suffer from Prostate cancer,but only a few can becomethe patients with clinical symptoms.The down-regulation of Notch1by small interfering RNA (siRNA) can significantlyinhibit human prostate cancer cell growth. The delivery of siRNA into specific cells is akey requirement for its clinical application. Recent reports have indicated thatantibody-mediated siRNA delivery is an effective approach for targeted knockdown ofspecific genes in appropriate cells. Prostate-specific membrane antigen (PSMA) isregarded as an ideal target for the delivery of therapeutic agents to prostate cancer cells.Our previous work showed that Notch1take a great role in prostate cancer developmentand siRNA targeting Notch1inhibition result in cell cycle arrest. So it is an effectivemethod of prostate cancer gene therapy by siRNA of Notch1if we can target delivery ofsiRNA effectively. We plan to fuse the human truncated protamine (tp) frigment sequenceto the3’terminus of the specific high DNA binding affinity scFv, which we have synthesized before. After optimization, the specific Notch1targeting siRNA can deliver toprostate cancer cell and internalization. So under the leading of fusion protein, siRNA caninhibitthe specific tumor cell with high performance and without "target off" effect. Thismethod will open new avenues for future targeted therapies of this tumor.The purpose of the present study was to evaluate whether siRNA can be efficientlydelivered into PSMA-positive prostate cancer cells using two fusion proteins, s-tP andsFH-tP. These fusion proteins are composed of an anti-PSMA single chain antibody (scFv,abbreviated as an “s”) and a truncated protamine (tp); and in sFH-tP a furincleavage site and an HA2fragment sequence (FH) were inserted between the scFv and tPdomains. Our results showed that siRNA can be specifically delivered into PSMA-positiveLNCaP cells by these two fusion proteins, with the sFH-tP fusion protein being moreeffective. Efficient knockdown of Notch1by siNotch1delivered by either fusion proteinwas observed in PSMA-positive LNCaP cells and in LNCaP xenografted nude mice.Further experiments confirmed that the fusion protein-delivered siNotch1could efficientlyinhibit PSMA-positive LNCaP cell proliferation and promote apoptosis both in vitro andin vivo. Our data describe a promising strategy for the targeted delivery of siRNA toPSMA-positive prostatecancer cells using anti-PSMA scFv fusion proteins.更多还原