【作者】 李红阳；

【导师】 刘长安；

【作者基本信息】 重庆医科大学 ， 外科学， 2013， 博士

【摘要】 肝癌是一个全球性健康问题,全世界每年因肝癌造成66万2千人死亡，而我国占一半。肝切除和肝移植被认为是治疗肝癌最佳方案,然而由于肝癌的早期症状和体征不明显,早期诊断困难，多数患者临床就诊时属于中晚期，已经远处转移，大部分不能手术切除。高强度聚焦超声(HIFU)是无创性治疗肿瘤的新方法，其机制主要是应用超声波可聚焦和穿透性，短时间内使靶区组织温度达到65～100℃，使靶组织造成凝固性坏死，HIFU是治疗中晚期肝癌安全、有效的方法。但HIFU治疗肝癌也存在一些缺陷：a.肝脏肿瘤由于肋骨遮挡，导致治疗效率的降低；b中晚期肝癌，血供丰富，热沉积效率低，聚焦点能量低，疗效差；c.由于肝脏随呼吸运动，HIFU定位不准确，可能出现治疗盲区，造成残癌的存在。由于以上原因，导致HIFU治疗肝癌后易复发可能，为了解决以上问题，可联合基因治疗，力求彻底杀灭残余肝癌细胞，弥补HIFU治疗的不足，提高HIFU治疗中晚期肝癌的疗效。单纯疱疹病毒Ⅰ型胸苷激酶/更昔洛韦(HSV1-TK/GCV)自杀基因系统，是众多肿瘤基因治疗效果明显、应用最广泛、最成熟、最为有效的基因治疗方法之一。自杀基因系统的原理：通过胸甘激酶的表达，磷酸化前药更昔洛韦，阻止肝癌细胞的DNA合成，从而起杀灭肝癌细胞的作用。自杀基因系统直接杀伤作用及旁观者效应作用显著。最近出现的一种超声波靶向爆破携基因的微泡技术(UTMD)，即当携基因的微泡造影剂经静脉注射进入肿瘤组织时，经超声声像图确定其到达靶组织后，通过低频超声波击碎携基因的微泡造影剂，产生的空化效应及机械效应，使外源性基因进入肿瘤组织，在低频的超声辐照条件下，超声既可提高靶基因的转染率，又不破坏转染基因，实现基因治疗的靶向性和准确性。前期试验在国家自然科学基金的资助下（高强度聚焦超声联合超声微泡包裹HSV-TK自杀基因靶向治疗肝癌的实验研究NO.30872977），我们证明了在小鼠移植瘤模型中，通过超声波靶向爆破携自杀基因的微泡释放，明显增加自杀基因转染率及靶向性。但我们的课题仍有些不足之处：a.由于脂质微泡不能通过血管内皮间隙，因而脂质微泡并未真正意义上进入肿瘤细胞，仅在肿瘤血管周围聚集，降低了治疗效果；b.携基因的微泡在基因治疗中无主动靶向性，仅有相对靶向性，其原理为：脂质微泡通过肿瘤区动脉压与周围动脉压压力差，随血液聚集到肿瘤区域，因此仅具有相对靶向性,不能主动识别肿瘤组织。以上缺点导致基因治疗效果不佳，如何提高基因治疗的主动靶向性，是基因治疗的热点问题。超声分子探针即靶向超声微泡(球)造影剂，是将能与靶组织上相应的受体结合的特异性配体耦联到微泡表面，使其能主动识别靶组织，达到分子水平的特异性增强显影。随着分子生物学与纳米技术发展，纳米级造影剂以其穿透力强、分子小的突出特性,有望弥补脂质微泡不能通过血管内皮间隙的不足，成为基因的新型载体。磷脂酰肌醇蛋白聚糖3(glypican-3，GPC3)，是存在于肝细胞性肝癌细胞膜表面的硫酸乙酰肝素多糖蛋白(HSPC)，参与肿瘤侵袭与转移。AFP定位于肝细胞胞浆中，在循环中可被抗体中和，不适合为抗体识别位点，GPC3是存在于细胞膜表面,可成为抗体识别部位。因此，我们选用GPC3单抗与纳米级超声造影剂结合，构建具有主动靶向性的纳米级超声分子探针，有望解决基因治疗时低转染率、靶向性差等诸多问题。本项目旨在主动投递靶向造影剂即超声分子探针于残癌组织内并在残留肝癌组织中特异性表达，利用超声分子主动靶向识别残癌组织，进而使自杀基因利用直接杀伤和旁杀作用杀灭残癌组织，力图在HIFU和联合基因靶向治疗肝癌方面取得新的进展。本课题研究内容主要包括以下三个部分：第一部分载HSV-TK自杀基因的肝癌特异性纳米级超声分子探针合成目的制备载HSV1-TK自杀基因的肝癌特异性纳米级超声分子探针.方法生物素-亲和素构建GPC3单抗与纳米级超声造影剂结合，应用超声图像定量分析仪（DFY软件分析系统）分析超声图像的视频强度改变，并描绘时间一峰值曲线，Zeta电位仪测试分析出载HSV1-TK自杀基因的肝癌特异性纳米级超声分子探针平均粒径、电位、浓度。结果超声声像图可见，注入超声分子探针（主动靶向造影剂）造影剂呈向心性强化逐渐聚集到肿瘤中心，直至肿瘤全部强化,与普通造影剂比较，超声分子探针造影峰值（VI）更高，声强更强，达峰时间缩短，持续时间更长。Zeta电位仪测试分析出载HSV-TK自杀基因的肝癌特异性纳米级超声分子探针，其平均粒径、电位、浓度符合纳米级超声分子探针要求。结论成功制备载HSV1-TK自杀基因的肝癌特异性纳米级超声分子探针,能够在裸鼠残癌模型中增强显像，是一种理想的超声分子探针。第二部分低频超声辐照载HSV1-TK基因的纳米超声分子探针靶向治疗HIFU消融后裸鼠残留肝癌实验研究目的观察低频超声辐照载HSV1-TK基因纳米超声分子探针在HIFU消融后裸鼠残留肝癌组织中的表达情况及结合前药更昔洛韦(GCV)对HIFU消融后裸鼠残留肝癌的杀伤效果。方法取荷瘤裸鼠肝癌60只，HIFU治疗仪(重庆海扶技术有限公司研制)对裸鼠肝癌消融80%后，成功建立裸鼠残留肝癌模型。随即分成六组，每组10只，A组：HIFU联合超声辐照载基因纳米级超声分子探针组（主动靶向组）（HIFU+US+HSV1–T K+NB+G PC3）；B组： HIFU联合超声辐照基因纳泡组（被动靶向组）（HIFU+US+HSV1-TK+NB）；C组：HIFU联合载基因超声分子探针组（HIFU+HSV1-TK+NB+GPC3）；D组：HIFU联合超声辐照基因组（HIFU+US+HSV1-TK）；E组：HIFU联合超声辐照超声分子探针组（HIFU+US+NB+GPC3）；F组：HIFU治疗组（空白对照组）。分别经残癌裸鼠尾静脉注入相同浓度的： HSV1-TK+NB+GPC3，HSV1-TK+NB，HSV1-TK+NB+GPC3，HSV1-TK，GPC3，PBS，A、B、D、E组分别给予超声辐照，Real-time PCR检测TK mRNA表达情况，Western-blot及免疫组化检测TK蛋白的表达情况，用TUNEL法检测肿瘤细胞凋亡情况，绘制抑瘤曲线。结果免疫组化及Real-time PCR及Western-blot及调亡指数及抑瘤率均显示A组（主动靶向组）TK的表达量明显高于被动靶向组和其他各组(P<0.05)，且能明显改善残癌裸鼠的生存时间。结论低频超声辐照载HSV1-TK基因的纳米超声分子探针，主动靶向投递HSV1-TK自杀基因于残癌组织内并在残留肝癌组织中特异性表达，提高HSV1-TK自杀基因的转染效率，增强了自杀基因对裸鼠残癌的杀伤效果。第三部分：不同浓度载HSV1-TK基因的纳米超声分子探针靶向造影剂对HIFU治疗后裸鼠残留肝癌的影响目的研究高强度聚焦超声（HIFU）治疗裸鼠残留肝癌后，观察不同浓度载HSV1-TK基因纳米超声分子探针靶向造影剂对裸鼠残留肝癌组织中TK基因表达的影响。方法建立裸鼠肝癌模型50只，随即分成五组，每组10只，使用HIFU治疗仪对裸鼠消融80%后，经裸鼠尾静脉注入不同浓度载纳米超声分子探针靶向造影剂,然后用超声辐照。免疫组化及Western-blot检测TK蛋白的表达情况，Real-time PCR检测TK mRNA表达情况，用TUNEL法检测肿瘤细胞凋亡情况，绘制抑瘤曲线。结果免疫组化及Real-time PCR及Western-blot及抑瘤率及调亡指数均显示与A组相比，B、C、D、E组有统计学意义(P<0.05)，而C、D、E组比较无统计学意义(P>0.05)。结论当携基因纳米超声分子探针靶向造影剂浓度≤0.6μg/μL时，TK mRNA、蛋白的表达及抑瘤率与纳米超声分子探针靶向造影剂呈浓度依赖，而当携基因纳米超声分子探针靶向造影剂浓度>0.6μg/μL时，基因的表达及抑瘤率与纳米超声分子探针靶向造影剂浓度无相关性，故携基因的纳米超声分子探针靶向造影剂微泡浓度为0.6μg/μL时TK基因表达最强。更多还原

【Abstract】 Hepatocellular carcinomais a global health problem. There is a highincidence of Hepatocellular carcinoma (HCC) in China, Each yearworldwide liver cancer caused the deaths of660,002people, half of them inChina. HCC is a heterogeneous malignancy with multiple etiologies, highincidence, and high mortality. The standard surgical management forpatients with HCC consists of locoregional ablation, surgical resection, orliver transplantation, depending on the background state of the liver. Most ofpatients initially presenting with HCC are unresectable, because of distantmetastasis.HIFU is a new and noninvasive treatment for HCC. The mechanism ofapplication of HIFU is ultrasonic focus and penetrating may cause thetemperature of the target tissue reach of65~100℃, which lead tocoagulation necrosis about the target tissue. Treatment of unsatisfactory inHIFU, ribs obscured; advanced hepatocellular carcinoma has a rich bloodsupply; liver with breathing exercises, which resulting in reduced treatmentefficiency and and further cause the presence of residual tumor. HSV1-TK/GCV is used with mature program. Principle of dutch actgene system: by thymidine kinase expression, phosphorylation of theprodrug ganciclovir, prevent hepatocellular carcinoma cells DNAsynthesis, and kill the carcinoma cells.It is significant to the role of Suicidegene system that directly kill function and bystander effect.Ultrasonic damage with gene microbubble technology （UTMD) is anewly gene targeted delivery system.The basic principle is using ultrasonicbreak microbubble target within the organization in particular space and time.In the low-frequency ultrasonic crushing carrying gene microbubble, whichcan cause cavitation effect and mechanical effect, the exogenous gene intothe tumor. Ultrasound can improve the rate of transfection of targetgene, and does not destroy the gene transfection, to achieve argeting andaccuracy in gene therapy.With the help of the natural science fund support NO.30872977,Weconfirmed Ultrasonic damage with gene microbubble technology (UTMD)can not only improve suicide gene microbubble targeting release.But the subject is still some deficiencies: a. due to lipid microbubblecan’t through the endothelial gap, and lipid microbubble do not really senseinto tumor cells, only in the tumor blood essels gathered round to reduceclinical application effect; b gene micro bubble in gene therapy withoutactive targeting property, only relative targeting property, its principle isdifferential from pressure in the tumor area arterial pressure and peripheral artery pressure, lipid microbubble along with blood gathered to tumor area,therefore it’s only relative targeting property; can’t initiative to identifycancer. The above disadvantages lead to gene therapy effect is not good, it isthe hot issue how to improve the gene therapy of active targeting property.Ultrasonic molecular probe is specific ligand coupling ultrasonicmicro bubble surface, specific ligand initiative identify target organizationalcorresponding receptors, make targeted contrast agent to stay in theorganization, to the molecular level of specificity enhance development.nanoscale contrast medium with its strong penetrating power andoutstanding small molecular characteristics, it is expected to make up for theinsufficient that lipid microbubble can’t through the endothelial gap andbecome a new gene delivery means.Phosphatidylinositol proteoglycan3(glypicana3,GPC3), phosph-atidylinositol is the one anchor of the surface of cell membrane proteoglycanand participate in tumor cell differentiation, proliferation, migration andadhesion, etc. AFP located in liver cell plasma, it can be antibodyneutralization in the cycle, which is not fit for antibody recognition site,GPC3can be antibody recognition site is due to sulfuric acid acetyl liverrope glycoprotein (HSPC)is in cell membrane surface. Therefore, we chooseGPC3Monoclonal Antibody (McAb) and nanoscale ultrasound contrastagent combination, build an active targeting nanoscale ultrasound molecularprobes, gene therapy is expected to solve low transfection rate and poor target of many problems.This project aims to deliver active ultrasonic molecular probe is inresidual carcinoma tissues and specificity expression in the residual livertissue, the use of ultrasonic molecular active target recognition residualcarcinoma tissue, and suicide gene using direct damage and side effect to killresidual carcinoma tissue. PART ONE: PREPARATION OF NANSIZE ULTRASOUNDMOLECULAR PROBE ENCAPSULATING HSV-TK GENEObjective to preparation nansize ultrasound molecularprobe encapsulating HSV-TK gene.Methods Nansize ultrasound molecular probe to GPC3monoclonalantibody were prepared by the biotin-avider interaction. Application ofultrasonic image quantitative analysis (DFY software) analysis ofultrasonic image video intensity change, drawing time-VI curves, theaverage particle size and Potential and concentration were measured andanalyzed by Zeta Potential instrument test in nansize ultrasound.Results From Ultrasonographic，it can be seen that, the ultrasoundmolecular probe (active targeting contrast agent) is centripetal enhancementgradually gathered into the center of tumor. Compared with the general agent,ultrasound molecular probe contrast peak (VI) and sound intensity (SI) ishigher the peak time is shortened, the longer duration. The average particlesize were measured and analyzed by Zeta Potential instrument test in nansizeultrasound, the result shows that the molecular probe to reach nanometerlevel standards.Conclusions The nansize ultrasound molecular probe encapsulatingHSV-TK gene were successfully prepared, which enhanced the contrastagent enhancement in nude mice with residual cancer imaging. It is a goodmolecular probe. PART TWO: THE STUDY OF HSV1-TK GENG-LOAD NANSIZEULTRASOUND MOLECULAR PROBE KILL THE RESIDUALLIVER CANCER AFTER HIFU TRRATMNTObjective To study of HSV1-TK load ultrasoundmolecular probe kill the residual liver cancer after HIFU treatmentMethods HIFU treatment60hepatocellular carcinoma of nudemice were randomized into six groups as follows: Group A：active targeting（HIFU+US+HSV1-TK+NB+GPC3; Group B： Passive targeting（HIFU+US+HSV1-TK+NB）; Group C：HIFU+HSV1-TK+NB+GPC3GroupD:HIFU+US+HSV1–TK; GroupE： HIFU+US+NB+GPC3; GroupF：HIFU. The nansize ultrasound molecular probe of encap-sulatingHSV1-TK+NB+GPC3, HSV1-TK+NB, HSV1-TK+NB+GPC3，HSV1–TK，GPC3，PBS，were injected into the tail vein of Nude mice hepatocellularcarcinoma. The nansize ultrasound molecular probe of encap-sulating,HSV1-TK+NB+GPC3, HSV1-TK+NB, HSV1–TK+N B+GPC3，HSV1-TK，GPC3，PBS，were injected into the tail vein of Nude micehepatocellular carcinoma, Group A and B and D and E were exposed byDZC ultrasound irradiation1MHz，2W/cm2，irradiation5min．Ganciclovi(GCV) was injectioned in each Nude mice hepatocellular carcinoma whichlast for14days. TK gene expression was detected by RT-PCR, IHC,WB anddetected Nude mice hepatocellular carcinoma survival time and size. Results Expression of TK proteins in group A（active targeting）（HIFU+US+HSV1-TK+NB+GPC3） was higher than others (P<0.05).The tumor cell RT-PCR, IHC, WB, and apoptotic index was higher thanother groups in group A（active targeting）(P <0.05). The rate of tumorgrowth inhibition group A were higher than others (P<0.05). It can obviouslyimprove Nude mice hepatocellular carcinoma survival time in group A（active targeting）（HIFU+US+HSV1-TK+NB+GPC3）Conclusions HSV1-TK geng-load nansize ultrasound molecular probekill the residual liver cancer after HIFU treatment, Which active targeteddelivery HSV-TK suicide gene in the residual tumor tissue and specificexpression. To further improve the suicide gene targeting and transfectionrate, With more fully after the treatment of HIFU kill the residual cancercells, Improve HIFU curative effect, In HIFU and the gene combination totargeted therapy liver cancer has the new breakthrough. PART THREE: Effect of ultrasound nansize ultrasound molecularprobe targeted contrast agent encapsulating HSV-TK gene in Nudemice hepatocellular carcinoma tissue after treatment of HIFUObjective To observe the effect of differentconcentrations of nansize ultrasound molecular probe targeted contrast agentloaded HSV1-TK gene expression in Nude mice hepatocellular carcinomatissue after treatment of HIFU.Methods50Nude mice hepatocellular carcinoma after HIFU wererandomized into5groups, ultrasound was used at1MHz，2W/cm2，irradiation5min. RT-PCR, IHC detected TK gene expression.Results Western-blot and PCR and immunohistochemical detectionshowed the TK gene expression of group A than group B、C、D、E (P<0.05),Conclusions the best concentration of nansize ultrasound molecular probetargeted contrast agent loaded gene is0.6μg/μL.更多还原