UTMD靶向释放分泌型PTD4-Apoptin治疗肝癌的实验研究

Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus.The nuclear translocation of apoptin is largely controlled by its phosphorylation. Apoptin protein harbors tumor-selective cell death activity, which makes it a potential anticancer therapy candidate. However, the therapeutic gene is not easy to penetrate into the liver cells far from blood vessels because of hepatoma complicated physiological barrier. So hepatoma complicated physiological barrier is still a bottleneck restricting the efficacy and clinical application of hepatoma gene therapy. Specific protein domains known as protein transduction domains (PTDs) can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. Ultrasound targeted microbubble destruction (UTMD) has evolved as a novel system for non-invasive, organ- and tissue-specific drug and gene delivery. Some studies showed that injection of PTD4-apoptin fusion protein had significant anticancer effect in human hepatoma epidermis xenografts. Thus, we will build a secretary PTD4-GFP-Apoptin recombinant plasmid and specifically transfect it into the hepatic sinusoidal endothelial cells by UTMD with SonoVue microbubble. Based on PTD4-mediated transduction of protein, secreted PTD4-apoptin fusion protein enters adjacent hepatic cells and play a "bystander" tumor-selective killing effect. Our studies will reveal that secretary PTD4-Apoptin recombinant plasmid delivery by UTMD with SonoVue microbubble constitutes a novel target, efficient and safe anticancer gene therapy

凋亡素(Apoptin)的“肿瘤选择性”凋亡诱导效应，使其成为极具应用前景的特异性抗肿瘤生物制剂。超声靶向微泡破坏(UTMD)是新兴起的无创靶向基因传输方法。然而肝癌复杂的生理屏障仍是制约肝癌基因治疗的瓶颈。在前期研究基础上，本研究拟构建分泌型带穿膜肽PTD4的Apoptin重组质粒，经UTMD联合声诺维微泡，高效靶向转染至肝原位移植瘤模型鼠的肝血窦内皮细胞中，并表达分泌Apoptin融合蛋白，进而PTD4穿膜引导Apoptin蛋白进入邻近未转染肝细胞，发挥“旁观者”肿瘤选择性杀伤效应。本项目充分利用无创、可重复输注的UTMD靶向转基因技术、PTD4穿膜功能和Apoptin特异性肿瘤杀伤作用，可巧妙突破常规基因治疗难以突破肝癌复杂的生理屏障这一瓶颈，为实现Apoptin对原位肝癌安全、高效、靶向特异性治疗提供一种新方法。该项目的顺利进行和即将取得的成果可进一步推广到其他肿瘤的治疗中。

肝癌被称为“癌中之王”，严重危害我国人民的生命健康。肝癌复杂的生理屏障仍是制约药物和基因靶向治疗肝癌的瓶颈。本项目构想利用超声靶向微泡破坏技术（UTMD），高效靶向转染分泌型apoptin重组质粒入肝血窦内皮细胞，并表达分泌Apoptin蛋白，结合PTD4穿膜肽技术，突破肝癌复杂的生理屏障，到达邻近肿瘤细胞发挥“旁观者”杀瘤效应，从而达到治疗较大肝癌实体瘤的效果。.我们构建了分泌型带穿膜肽PTD4的Apoptin重组质粒（pSecTag2-PTD4-GFP-Apoptin），将其有效转染入HUVEC中，经荧光显微镜和WB检测结果表明：转染48h后的HUVEC可高效表达PTD4-GFP-Apoptin融合蛋白，WB可检测到培养上清中亦含有Apoptin融合蛋白。收集该培养上清作为PTD4-Apoptin条件培养液，分别孵育人肝癌细胞HepG2及人正常肝细胞L02，荧光显微镜观察到HepG2细胞浆及胞核内有聚集性绿色荧光。MTT法及流式细胞术检测结果显示：Apoptin融合蛋白对HepG2具有凋亡诱导杀伤效应，而对正常肝细胞L02无显著影响。随后，我们确定了 SonoVue微泡包载该重组质粒的最佳比率及UTMD靶向转染至鼠肝的最佳参数，结果显示：以20ul SonoVue包裹1ug质粒的比例实验，UTMD靶向转染的最佳参数可确定为超声强度1.5 W/cm2，辐照时间180秒。于辐照后不同时间点，取鼠肝组织、各主要脏器组织，经荧光显微镜及HE染色检测，结果显示UTMD转染具有一定靶向性、安全性。最后，我们建立了不同肝癌细胞系的裸小鼠肝原位移植瘤模型，麻醉瘤径大于0.5cm的裸鼠，尾静脉缓慢注射载质粒声诺维微泡，选用最佳参数超声辐照肿瘤灶，辐照后不同时间点随机处死每组中的7只，取肝癌、癌旁及各主要脏器组织，进行疗效观察。其余裸鼠继续观察并绘制生长曲线。结果显示：基因治疗干预组，1）肿瘤体积显著减小，2）Apoptin融合蛋白在肝脏中分布，3）Caspase3、Caspase9在mRNA和蛋白水平显著增加，4）肝癌组织的凋亡、坏死情况较对照组显著增强。可初步判断UTMD促进Apoptin重组质粒转染对裸鼠原位肝癌移植瘤具有一定的靶向抑制效应，为实现Apoptin基因无创、安全、高效的肝癌靶向性治疗提供有力实验依据。