高强度聚焦超声联合载HSV1-TK自杀基因超声微泡造影剂治疗肝癌的实验研究
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摘要
肝细胞癌(Hepatocellular Carcinoma,HCC)在我国发病率高，危害性大，目前仍以手术为主的综合治疗，虽然外科手术如肝叶切除或肝脏移植，能提供一个较好的远期预后，但诸多因素使得适合和接受手术者仅为20%左右。近十年来，HCC局部治疗手段，特别是高强度聚焦超声(High intensity focused ultrasound, HIFU)的应用愈发广泛，因其适应证宽，对患者全身情况要求不高，对巨块型，多结节型肝癌均可治疗并能取得了较好的疗效，目前已成为最有前途的肝癌辅助治疗方法之一。但因诸多因素如肋骨遮挡、呼吸运动、肿瘤丰富的血供等影响，使得HIFU在治疗肝癌时存在肿瘤组织残存的缺陷。为了解决这一难题，目前使用的方法即联合其他的治疗手段如联合化疗、基因治疗、介入等，以弥补该方法的缺陷，使HIFU治疗肝癌更加的彻底。
目前肝癌的基因治疗根据肝癌的发病机制分为免疫基因治疗、抑癌基因治疗、反义基因治疗、自杀基因治疗、抗肿瘤血管生成基因治疗、多药耐药基因治疗等，而自杀基因疗法尤其是其中单纯疱疹病毒Ⅰ型胸苷激酶/更昔洛韦(Herpes simplexvirus type1thymidine kinase/Gancyclovir, HSV1-TK/GCV)自杀基因治疗系统成为了目前肝癌基因治疗中最有前途的治疗方法之一。
HSV1-TK/GCV自杀基因系统作为肝癌基因治疗的手段之一，其主要是通过TK蛋白将无毒的前药GCV经过多次磷酸化转变成细胞毒性的三磷酸化合物(Ganciclovir triphosphate, GCV-TP)， GCV-TP能抑制DNA聚合酶活性，阻止DNA自我复制，从而杀灭肿癌细胞；同时该系统还能将毒性产物通过细胞间隙连接或直接分泌到细胞外，通过诱导癌细胞凋亡或免疫介导等途径杀死周边未被转染的瘤细胞而发挥“旁观者效应”，而与其他治疗基因一样，外源基因载体系统的选择成为基因治疗亟待解决的问题之一。
目前基因载体系统包括两大类，即病毒载体和非病毒载体。病毒载体包括腺病毒、慢病毒、逆转录病毒等，其主要优点为转染效率高，但是其安全性有待进一步提高；非病毒载体包括脂质体、质粒DNA等，其优点是安全性较高、可反复利用，但是其转染效率较低、缺乏主动靶向性，因而限制其临床应用。
超声靶向微泡破坏(Utrasound-targeted microbubble destruction,UTMD)技术是新近出现的一种基因或药物靶向递送系统。该技术通过体外超声靶向破坏经血液循环到靶组织携带有药物或外源基因的超声微泡造影剂使药物或外源基因靶向释放，实现药物或治疗基因的靶向递送。当超声波击碎超声微泡时产生的空化效应能使肿瘤毛细血管通透性增高，增加血管内皮间隙使外源基因更容易进入肿瘤组织内，提高了外源基因的转染率，同时该技术使用的低频超声不但不会对外源基因产生任何破坏，还能增加外源基因的转染率，从而提高基因治疗肿瘤的效果。超声微泡合成材料为脂质材料，脂溶性好，对生物体的安全性高且低免疫原性，可重复使用。而超声微泡造影剂作为基因载体的同时本身具备超声造影的功能，能提高肝癌的检出率，提高治疗基因的靶向性和精准性。
基于上述超声靶向微泡破坏技术的各种优势，本课题通过制备载HSV1-TK自杀基因超声造影剂，将其应用于H22小鼠肝癌皮下移植瘤模型中观察其靶向性及治疗效果，同时为了进一步观察其联合HIFU治疗效果，将HIFU联合载HSV1-TK基因超声微泡治疗VX2肝癌，观察联合治疗效果及HSV1-TK/GCV自杀基因系统的作用机理，为HIFU和基因联合治疗肝癌方面提供可靠的实验依据。
本课题研究内容主要包括以下三个部分：
第一部分载HSV1-TK基因脂质超声微泡造影剂的制备
目的：构建携HSV1-TK自杀基因质粒并实现超声微泡造影剂与HSV1-TK基因的连接，评价其物理性质、携带基因的能力及体内显影效果。方法：通过选用真核表达载体pIRES2-EGFP和TK通过酶切、PCR扩增、重组、鉴定、测序合成pIRES2-EGFP-TK；采用静电吸附原理将TK基因吸附于自制的超声微泡造影剂上，检测微泡的大小、分布、电荷、形态、携基因量及体内显影效果。结果：重组质粒经酶切及PCR反应后电泳结果显示约1024bp附近有一特异条带，与目的基因的长度相近，测序鉴定结果与Genbank中序列相比对完全一致；载基因微泡粒径大小、分布情况、电荷情况均满足超声造影剂的要求；内体造影效果显示，载基因微泡能明显增强小鼠H22肝癌的显影，且持续时间较长，约25min。结论：成功构建pEGFP-TK质粒并实现了自制超声微泡造影剂与目的基因之间的连接，同时还实现了体内的造影需求，为后续的实验奠定了较好的基础。
第二部分超声辐照微泡介导HSV1-TK自杀基因治疗小鼠肝癌的实验研究
目的：观察超声辐照介导的单纯疱疹病毒Ⅰ型胸苷激酶基因(HSV1-TK)在小鼠肝癌细胞H22移植瘤组织中的表达情况及结合前药更昔洛韦(GCV)后对肿瘤的杀伤效果。方法：建立小鼠肝癌(H22)皮下移植瘤模型40只，随机分成四组，即A.对照组(PBS)；B.单纯质粒组(HSV1-TK)；C.质粒+超声辐照组(HSV1-TK+US)④质粒+超声辐照+微泡组(HSV1-TK+US+MB)。分别经鼠尾静脉注入PBS、HSV1-TK、HSV1-TK、HSV1-TK+微泡，每次注射200μl，每隔3d注射一次，共注射3次，C、D组分别给予1MHz，2W/cm~2，5min超声辐照，48h后各治疗组分别经腹腔注射GCV0.2ml(100mg·kg~(-1)·d~(-1))连续注射14天。48h后活杀一批荷瘤鼠采用Western-blot检测肿瘤组织中TK蛋白的表达情况，监测肿瘤大小及生存时间，分别绘制肿瘤生长曲线及生存曲线图，检测肿瘤细胞凋亡情况及生物学形态。结果：治疗组中均有TK蛋白的表达，其中D组表达量明显高于其他各组(P<0.05)；各组荷瘤鼠经治疗后A组、B组、C组、D组抑瘤率分别是0%，3.90%±1.80%，22.70%±2.86%，41.25%±3.20%。四组肿瘤体积比较，D组与其他各治疗组比较抑瘤效应更加明显，且D组与C组LSD-t=161.29，P<0.05；D组与B组LSD-t=323.71，P<0.01；C组与B组LSD-t=162.43，P<0.05；A组与B组LSD-t=28.96，P>0.05；从生存时间曲线图可以看出微泡载基因组能明显改善荷廇鼠的生存时间；TUNEL染色D组肿瘤细胞凋亡指数明显比其他各组高(P<0.05)；HE染色D组较A组坏死病变明显。结论：超声破坏包裹HSV1-TK基因的微泡定位释放，即增加了肝癌基因治疗的靶向性，又提高了外源基因的转染率，从而增强了自杀基因对小鼠肝癌的杀伤效果，这为肝癌的基因治疗提供了一个新的策略。
第三部分高强度聚焦超声联合载HSV1-TK自杀基因超声微泡治疗VX2兔肝癌的实验研究
目的：探讨联合载HSV1-TK自杀基因超声微泡能否增强高强度聚焦超声(HIFU)治疗肝癌的杀伤效果。方法：建立75只VX2兔肝癌模型，随机分成①HIFU治疗组；②HIFU+HSV1-TK组；③HIFU+HSV-TK+US组；④HIFU+HSV-TK+US+MB组；⑤HSV-TK+US+MB组。前四组均行HIFU治疗，治疗参数：频率1.0MHz，焦距156mm，辐照声功率150W。HIFU治疗结束后，分别经新西兰大白兔耳缘静脉注射生理盐水、HSV1-TK、HSV1-TK+MB，注射完后使用超声基因转染仪进行辐照，辐照参数为300KHz，2W/cm~2，辐照方式为脉冲式，即辐照10s，间歇10s，持续20分钟。辐照结束24h后开始经荷瘤兔后腿肌注前药GCV，剂量为100mg·kg~(-1)·d~(-1)，持续注射10d。转染HSV1-TK48h后活杀一批荷瘤兔检测其TK mRNA及蛋白表达情况，监测荷瘤兔肿瘤大小及荷瘤兔生存时间，治疗结束后使用TUNEL法检测肿瘤细胞凋亡情况，HE染色检测肿瘤生物学形态。结果：HIFU+HSV-TK+US+MB组与HSV-TK+US+MB组中TK mRNA表达及GFP蛋白的表达无统计学意义，而与其他各组比较均高于其他各组，且有统计学意义(P<0.05)。HIFU+HSV-TK+US+MB组抑瘤效应明显高于其他各组(P<0.05)，且能明显改善荷廇兔的生存时间；TUNEL染色HIFU+HSV-TK+US+MB组肿瘤细胞凋亡指数明显比其他各组高(P<0.05)；结论：HIFU联合载HSV1-TK自杀基因超声微泡治疗VX2兔肝癌能明显抑制其肿瘤细胞的生长，明显改善荷瘤兔的生存时间，为HIFU联合基因治疗肝癌提供了较为可靠的实验依据。
The prognosis for hepatocellular carcinoma (HCC) is poor and hashigh morbidity in our country. Surgical resection or liver transplantationcould provide better long-term survival outcome. However, less than20%of patients are currently undergoing surgery because of late clinicalpresentation and, subsequently, diagnosis, etc. Over the past ten years, localtreatment for HCC is becoming more wildly, especially in High intensityfocused ultrasound (HIFU), which as a potential noninvasive treatment, hasbeen widely used in clinical therapy of liver tumor. Because of HIFUsuitable for the majority of liver cancer patients, so it is becoming one ofmost promising approach in the treatment of liver cancer. Nevertheless,HIFU also has some unavoidable factors on clinical application of livertumor. Such as respiratory movement, abdomen gas, overlying ribs, bloodflow within tumors, and the limitation of imaging guidance in some cases,which might increase the energy depositing in overlying tissues and furthercause adverse effects, i.e., skin burns and nerve injuries or unexpected residual viable tissue in focused areas. How to reduce adverse effects andimprove effect of HIFU treatment has become a vital topic in the field ofHIFU. In order to improve the therapeutic efficacy, combining with othertreatment approach has becoming a hopeful means, such as chemotherapy,gene therapy, interventional therapy, and so on.
According to the pathogenesis of HCC, the gene therapy of HCC isdivided into immune gene therapy, tumor suppressor gene therapy,antisense gene therapy, suicide gene therapy, antiangiogenesis gene therapy,multidrug resistance gene therapy, etc. However, suicide gene therapy,especially, the herpes simplex virus type1thymidine kinase/Gancyclovir(HSV1-TK/GCV) is becoming one of most promising approach in thetreatment of liver cancer.
As one of the treatment approach of liver cancer, HSV-TK suicidetherapy system is expressing and producing thymus kinase in the tumorcells, catalyze nucleoside analogue to form monophosphate products, andfurther form a triphosphoric acid product under the effect of phosphokinasein the cell. The system can also be toxic products through the intercellularjunctions or directly secreted into the cell, by inducing apoptosis of cancercells or immune mediated peripheral pathways, which can kill tumor. It wascalled "bystander effect". However, the main problem of gene therapy islack of a safe and effective motheds for gene delivery to pecific tissue ororgan.
Current gene carrier system includes two categories, viral vectors andnon-viral vectors. Viral vectors, including adenovirus, retrovirus, lentivirus,etc. its main advantages are high transfection efficiency, but its safety needsto be further improved. Non-viral vectors include liposomes, plasmid DNA,has the advantages of high safety, repeated use, but its transfectionefficiency is low, the lack of active targeted, so it was limited the clinicalapplication for liver cancer.
Ultrasound targeted microbubble destruction (UTMD) technology is anewly gene or drug targeted delivery system. Microbubbles currently usedas ultrasound contrast agents by ultasound exposure can immediately crush,then accroding to produce cavitation effect and mechanical effect whichtransiently perforates the cell membrane or disrupts the capillary wall toallow delivery of agents into cells or interstitial space. It will be improvedthe exogenous gene transfection efficiency. Because of using the lowfrequency ultrasound, it does not have any damage to the exogenous gene,and can increase the transfection efficiency of exogenous gene. Thesynthetic material of ultrasound microbubble contrast agent is nontoxiclipid materials, so can be repeated using without immunogenicity. However,Ultrasound microbubble contrast agent was used as a gene carrier, at thesame time, was also a kind of ultrasound contrast agent. It can not onlyimprove the liver cancer detection rate, but also can improve thetherapeutic gene transfection efficiency.
All in all, based on the advantages of UTMD, our research would beby preparation gene-loaded ultrasound microbubble, which was applied inH22mice hepatoma subcutaneous transplantation tumor model to observethe therapeutic effect. At the same time, in order to further observe thetherapeutic effect of combined HIFU, HIFU combined with carryingHSV1-TK gene ultrasound-targeted microbubbles for liver tumor. At last, itwould provide reliable experimental evidences for HIFU combined withgene treatment for hepatocellular carcinoma. This research mainly includedthe following three parts:
PART ONE: EXPERIMENTAL STUDY ON PREPARATION OFHSV1-TK GENE-LOADED LIPID ULTRASOUNDMICROBUBBLES
Aim To construct EGFP and HSV1-TK co-expression vector, realizeultrasound microbubble and HSV1-TK gene connection, and to evaluatethe physical character of ultrasound microbubble and its enhancementeffects in contrast imaging on liver cancer model. Methods Through theselection of the eukaryotic expression vector pIRES2-EGFP and TK byenzyme digestion, PCR amplification, pIRES2-EGFP-TK synthesis,sequencing, identification of recombinant; Applying electrostatic adsorp-tion principle, TK gene was adsorbed on the self-made ultrasoundmicrobubble contrast agent, detection of microbubble size, distribution, morphology, charge, carrying gene and contrast imaging effect in vivo.Results After electrophoresis, recombinant plasmids by restriction andPCR responses, there was a specific band about1024bp. The DNAsequencing results was identical to HSV1-TK that reported on Gene bank.Self-made microbubble can meet the ability of ultrasound contrast. In vivoultrasound contrast imaging shown that gene-loaded microbubbles couldenhance the hepatoma mice developing, and last developing about25min.Conclusions The plasmid pEGFP-TK was constructed successfully and toachieve a self-made gene-loaded ultrasound microbubble contrast agent,while meeting the imaging needs in vivo, and laid a good foundation forthe subsequent experiments.
PART TWO: THE EXPERIMENTAL STUDY OF ULTRASOUNDMICROBUBBLE CARRING HERPES SIMPLEX VIRUSTHYMIDINE KINASE FOR HEPATOCELLULAR CARCINOMA INMICE
Aim To observe the effect of microbubbles encapsulating herpessimplex virus thymidine kinase gene transfection on cancer tissue of micewith ultrasound exposure and assess the therapy efficacy by combiningGCV. Methods Kunming mice were inoculated subcutaneously in themiddle of the right flank with H22tumor cells line.40male mice bearingsubcutaneous hepatoma were randomized into4groups as follows:groupA,PBS;group B, HSV1-TK; group C,HSV1-TK+ultrasound; groupD, HSV1-TK+ultrasound+Microbubbles.The microbubbles of encap-sulatingHSV1-TK,PBS and Plasmid HSV1-TK were injected into the tail vein ofeach mouse，every3d once time, in all3times. Group C and D wereexposed by ultrasound and without group A and B. Western-blot was usedto detect the TK protein expression after48h. Subsequently, Ganciclovi(GCV) was administered in each mouse daily by intraperitoneal injection ata dose of0.2ml (100mg kg~(-1) d~(-1))in group B，group C and group D．Thetumor size was measured every2d till16d, then, all mice were killed toevaluated the histopathological changes of tumors. Results Gene could beinjected exactly into hepatocellular carcinoma with ultrasound monitor, andexpression of TK proteins was found in all4groups. Expression in group Dwas higher than others (P<0.05). The rate of tumor growth inhibition were0in group A,3.90%±1.80%in group B，22.70%±2.86%in group C，41.25%±3.20%in group D (group B vs group C, P<0.05;group D vs groupC, P<0.05;groupD vs group B, P<0.05);There was more obvious necrosisof tumor tissue in group D than group A. Conclusions Theultrasound-induced destruction of microbubbles encapsulated HSV-TKgene was release in the targeted spot, not only do these microbubbles helpto improve targeted in gene therapy of hepatocellular carcinoma, but theycan also enhance transfection efficiency of foreign gene. At last, thetherapeutic effect of suicide gene was enhanced in mice hepatoma. It wouldprovide a new strategy for liver cancer.
PART THREE: THE EXPERIMENTAL STUDY OF HIFUCOMBINED WITH HSV1-TK GENE-LOADED ULTRASOUNDMICROBUBBLE FOR VX2RABBIT LIVER TUMOR
Aim To investigate the effect of HSV1-TK gene-loaded ultrasoundmicrobubble microbubbles combined with HIFU for VX2rabbit livertumor and assess the therapy efficacy by combining gene-loaded ultrasoundmicrobubble. Methods seventy-five rabbits bearing VX2liver tumors wererandomly divided into the following five groups:①g roup HIFU;②groupHIFU+HSV1-TK;③groupHIFU+HSV-TK+US;④groupHIFU+HSV-TK+US+MB;⑤group HSV-TK+US+MB. The first four groups were treated withHIFU, HIFU treatment parameters: the frequency of1MHz, a focal lengthof156mm, radiation sound power150W. After HIFU ablation, the normalsaline, HSV1-TK gene, and ultrasound microbubble were injected viamarginal ear veins, then accept ultrasonic irradiation20min (irradiationparameters: a frequency of300KHz and an intensity of2W/cm~2with10seconds with10s pause, lasting a total of20minutes). All rabbits werereceived an intramuscular injection of100mg/kg/d ganciclovir24h afterirradiation, which last for10days. Tumor-bearing rabbits were killed48hours after transfection, testing its TK mRNA and protein expression,monitoring of tumor-bearing rabbits tumor size and survival time oftumor-bearing rabbits, using TUNEL assay for detection of tumor cellapoptosis after the end of treatment. Results The TK mRNA expression and GFP protein expression were higher in HIFU+HSV-TK+US+MBgroup and HSV-TK+US+MB group than other groups, and there wasstatistical significance (P<0.05). HIFU+HSV-TK+US+MB group was thehighest in all other groups in antitumor effect, and there was statisticalsignificance (P<0.05). It can obviously improve tumor-bearing rabbitssurvival time in HIFU+HSV-TK+US+MBs group. The tumor cell apoptoticindex was higher than other groups in HIFU+HSV-TK+US+MB group (P<0.05). Conclusions HIFU conbinated with carrying HSV-TK ultrasound-targeted microbubbles could inhibit the growth of VX2rabbit liver tumors,and improve the survival time of tumor-bearing rabbits which couldprovide a reliable experimental evidences for liver cancer treatment.
目前肝癌的基因治疗根据肝癌的发病机制分为免疫基因治疗、抑癌基因治疗、反义基因治疗、自杀基因治疗、抗肿瘤血管生成基因治疗、多药耐药基因治疗等，而自杀基因疗法尤其是其中单纯疱疹病毒Ⅰ型胸苷激酶/更昔洛韦(Herpes simplexvirus type1thymidine kinase/Gancyclovir, HSV1-TK/GCV)自杀基因治疗系统成为了目前肝癌基因治疗中最有前途的治疗方法之一。
HSV1-TK/GCV自杀基因系统作为肝癌基因治疗的手段之一，其主要是通过TK蛋白将无毒的前药GCV经过多次磷酸化转变成细胞毒性的三磷酸化合物(Ganciclovir triphosphate, GCV-TP)， GCV-TP能抑制DNA聚合酶活性，阻止DNA自我复制，从而杀灭肿癌细胞；同时该系统还能将毒性产物通过细胞间隙连接或直接分泌到细胞外，通过诱导癌细胞凋亡或免疫介导等途径杀死周边未被转染的瘤细胞而发挥“旁观者效应”，而与其他治疗基因一样，外源基因载体系统的选择成为基因治疗亟待解决的问题之一。
目前基因载体系统包括两大类，即病毒载体和非病毒载体。病毒载体包括腺病毒、慢病毒、逆转录病毒等，其主要优点为转染效率高，但是其安全性有待进一步提高；非病毒载体包括脂质体、质粒DNA等，其优点是安全性较高、可反复利用，但是其转染效率较低、缺乏主动靶向性，因而限制其临床应用。
超声靶向微泡破坏(Utrasound-targeted microbubble destruction,UTMD)技术是新近出现的一种基因或药物靶向递送系统。该技术通过体外超声靶向破坏经血液循环到靶组织携带有药物或外源基因的超声微泡造影剂使药物或外源基因靶向释放，实现药物或治疗基因的靶向递送。当超声波击碎超声微泡时产生的空化效应能使肿瘤毛细血管通透性增高，增加血管内皮间隙使外源基因更容易进入肿瘤组织内，提高了外源基因的转染率，同时该技术使用的低频超声不但不会对外源基因产生任何破坏，还能增加外源基因的转染率，从而提高基因治疗肿瘤的效果。超声微泡合成材料为脂质材料，脂溶性好，对生物体的安全性高且低免疫原性，可重复使用。而超声微泡造影剂作为基因载体的同时本身具备超声造影的功能，能提高肝癌的检出率，提高治疗基因的靶向性和精准性。
基于上述超声靶向微泡破坏技术的各种优势，本课题通过制备载HSV1-TK自杀基因超声造影剂，将其应用于H22小鼠肝癌皮下移植瘤模型中观察其靶向性及治疗效果，同时为了进一步观察其联合HIFU治疗效果，将HIFU联合载HSV1-TK基因超声微泡治疗VX2肝癌，观察联合治疗效果及HSV1-TK/GCV自杀基因系统的作用机理，为HIFU和基因联合治疗肝癌方面提供可靠的实验依据。
本课题研究内容主要包括以下三个部分：
第一部分载HSV1-TK基因脂质超声微泡造影剂的制备
目的：构建携HSV1-TK自杀基因质粒并实现超声微泡造影剂与HSV1-TK基因的连接，评价其物理性质、携带基因的能力及体内显影效果。方法：通过选用真核表达载体pIRES2-EGFP和TK通过酶切、PCR扩增、重组、鉴定、测序合成pIRES2-EGFP-TK；采用静电吸附原理将TK基因吸附于自制的超声微泡造影剂上，检测微泡的大小、分布、电荷、形态、携基因量及体内显影效果。结果：重组质粒经酶切及PCR反应后电泳结果显示约1024bp附近有一特异条带，与目的基因的长度相近，测序鉴定结果与Genbank中序列相比对完全一致；载基因微泡粒径大小、分布情况、电荷情况均满足超声造影剂的要求；内体造影效果显示，载基因微泡能明显增强小鼠H22肝癌的显影，且持续时间较长，约25min。结论：成功构建pEGFP-TK质粒并实现了自制超声微泡造影剂与目的基因之间的连接，同时还实现了体内的造影需求，为后续的实验奠定了较好的基础。
第二部分超声辐照微泡介导HSV1-TK自杀基因治疗小鼠肝癌的实验研究
目的：观察超声辐照介导的单纯疱疹病毒Ⅰ型胸苷激酶基因(HSV1-TK)在小鼠肝癌细胞H22移植瘤组织中的表达情况及结合前药更昔洛韦(GCV)后对肿瘤的杀伤效果。方法：建立小鼠肝癌(H22)皮下移植瘤模型40只，随机分成四组，即A.对照组(PBS)；B.单纯质粒组(HSV1-TK)；C.质粒+超声辐照组(HSV1-TK+US)④质粒+超声辐照+微泡组(HSV1-TK+US+MB)。分别经鼠尾静脉注入PBS、HSV1-TK、HSV1-TK、HSV1-TK+微泡，每次注射200μl，每隔3d注射一次，共注射3次，C、D组分别给予1MHz，2W/cm~2，5min超声辐照，48h后各治疗组分别经腹腔注射GCV0.2ml(100mg·kg~(-1)·d~(-1))连续注射14天。48h后活杀一批荷瘤鼠采用Western-blot检测肿瘤组织中TK蛋白的表达情况，监测肿瘤大小及生存时间，分别绘制肿瘤生长曲线及生存曲线图，检测肿瘤细胞凋亡情况及生物学形态。结果：治疗组中均有TK蛋白的表达，其中D组表达量明显高于其他各组(P<0.05)；各组荷瘤鼠经治疗后A组、B组、C组、D组抑瘤率分别是0%，3.90%±1.80%，22.70%±2.86%，41.25%±3.20%。四组肿瘤体积比较，D组与其他各治疗组比较抑瘤效应更加明显，且D组与C组LSD-t=161.29，P<0.05；D组与B组LSD-t=323.71，P<0.01；C组与B组LSD-t=162.43，P<0.05；A组与B组LSD-t=28.96，P>0.05；从生存时间曲线图可以看出微泡载基因组能明显改善荷廇鼠的生存时间；TUNEL染色D组肿瘤细胞凋亡指数明显比其他各组高(P<0.05)；HE染色D组较A组坏死病变明显。结论：超声破坏包裹HSV1-TK基因的微泡定位释放，即增加了肝癌基因治疗的靶向性，又提高了外源基因的转染率，从而增强了自杀基因对小鼠肝癌的杀伤效果，这为肝癌的基因治疗提供了一个新的策略。
第三部分高强度聚焦超声联合载HSV1-TK自杀基因超声微泡治疗VX2兔肝癌的实验研究
目的：探讨联合载HSV1-TK自杀基因超声微泡能否增强高强度聚焦超声(HIFU)治疗肝癌的杀伤效果。方法：建立75只VX2兔肝癌模型，随机分成①HIFU治疗组；②HIFU+HSV1-TK组；③HIFU+HSV-TK+US组；④HIFU+HSV-TK+US+MB组；⑤HSV-TK+US+MB组。前四组均行HIFU治疗，治疗参数：频率1.0MHz，焦距156mm，辐照声功率150W。HIFU治疗结束后，分别经新西兰大白兔耳缘静脉注射生理盐水、HSV1-TK、HSV1-TK+MB，注射完后使用超声基因转染仪进行辐照，辐照参数为300KHz，2W/cm~2，辐照方式为脉冲式，即辐照10s，间歇10s，持续20分钟。辐照结束24h后开始经荷瘤兔后腿肌注前药GCV，剂量为100mg·kg~(-1)·d~(-1)，持续注射10d。转染HSV1-TK48h后活杀一批荷瘤兔检测其TK mRNA及蛋白表达情况，监测荷瘤兔肿瘤大小及荷瘤兔生存时间，治疗结束后使用TUNEL法检测肿瘤细胞凋亡情况，HE染色检测肿瘤生物学形态。结果：HIFU+HSV-TK+US+MB组与HSV-TK+US+MB组中TK mRNA表达及GFP蛋白的表达无统计学意义，而与其他各组比较均高于其他各组，且有统计学意义(P<0.05)。HIFU+HSV-TK+US+MB组抑瘤效应明显高于其他各组(P<0.05)，且能明显改善荷廇兔的生存时间；TUNEL染色HIFU+HSV-TK+US+MB组肿瘤细胞凋亡指数明显比其他各组高(P<0.05)；结论：HIFU联合载HSV1-TK自杀基因超声微泡治疗VX2兔肝癌能明显抑制其肿瘤细胞的生长，明显改善荷瘤兔的生存时间，为HIFU联合基因治疗肝癌提供了较为可靠的实验依据。
The prognosis for hepatocellular carcinoma (HCC) is poor and hashigh morbidity in our country. Surgical resection or liver transplantationcould provide better long-term survival outcome. However, less than20%of patients are currently undergoing surgery because of late clinicalpresentation and, subsequently, diagnosis, etc. Over the past ten years, localtreatment for HCC is becoming more wildly, especially in High intensityfocused ultrasound (HIFU), which as a potential noninvasive treatment, hasbeen widely used in clinical therapy of liver tumor. Because of HIFUsuitable for the majority of liver cancer patients, so it is becoming one ofmost promising approach in the treatment of liver cancer. Nevertheless,HIFU also has some unavoidable factors on clinical application of livertumor. Such as respiratory movement, abdomen gas, overlying ribs, bloodflow within tumors, and the limitation of imaging guidance in some cases,which might increase the energy depositing in overlying tissues and furthercause adverse effects, i.e., skin burns and nerve injuries or unexpected residual viable tissue in focused areas. How to reduce adverse effects andimprove effect of HIFU treatment has become a vital topic in the field ofHIFU. In order to improve the therapeutic efficacy, combining with othertreatment approach has becoming a hopeful means, such as chemotherapy,gene therapy, interventional therapy, and so on.
According to the pathogenesis of HCC, the gene therapy of HCC isdivided into immune gene therapy, tumor suppressor gene therapy,antisense gene therapy, suicide gene therapy, antiangiogenesis gene therapy,multidrug resistance gene therapy, etc. However, suicide gene therapy,especially, the herpes simplex virus type1thymidine kinase/Gancyclovir(HSV1-TK/GCV) is becoming one of most promising approach in thetreatment of liver cancer.
As one of the treatment approach of liver cancer, HSV-TK suicidetherapy system is expressing and producing thymus kinase in the tumorcells, catalyze nucleoside analogue to form monophosphate products, andfurther form a triphosphoric acid product under the effect of phosphokinasein the cell. The system can also be toxic products through the intercellularjunctions or directly secreted into the cell, by inducing apoptosis of cancercells or immune mediated peripheral pathways, which can kill tumor. It wascalled "bystander effect". However, the main problem of gene therapy islack of a safe and effective motheds for gene delivery to pecific tissue ororgan.
Current gene carrier system includes two categories, viral vectors andnon-viral vectors. Viral vectors, including adenovirus, retrovirus, lentivirus,etc. its main advantages are high transfection efficiency, but its safety needsto be further improved. Non-viral vectors include liposomes, plasmid DNA,has the advantages of high safety, repeated use, but its transfectionefficiency is low, the lack of active targeted, so it was limited the clinicalapplication for liver cancer.
Ultrasound targeted microbubble destruction (UTMD) technology is anewly gene or drug targeted delivery system. Microbubbles currently usedas ultrasound contrast agents by ultasound exposure can immediately crush,then accroding to produce cavitation effect and mechanical effect whichtransiently perforates the cell membrane or disrupts the capillary wall toallow delivery of agents into cells or interstitial space. It will be improvedthe exogenous gene transfection efficiency. Because of using the lowfrequency ultrasound, it does not have any damage to the exogenous gene,and can increase the transfection efficiency of exogenous gene. Thesynthetic material of ultrasound microbubble contrast agent is nontoxiclipid materials, so can be repeated using without immunogenicity. However,Ultrasound microbubble contrast agent was used as a gene carrier, at thesame time, was also a kind of ultrasound contrast agent. It can not onlyimprove the liver cancer detection rate, but also can improve thetherapeutic gene transfection efficiency.
All in all, based on the advantages of UTMD, our research would beby preparation gene-loaded ultrasound microbubble, which was applied inH22mice hepatoma subcutaneous transplantation tumor model to observethe therapeutic effect. At the same time, in order to further observe thetherapeutic effect of combined HIFU, HIFU combined with carryingHSV1-TK gene ultrasound-targeted microbubbles for liver tumor. At last, itwould provide reliable experimental evidences for HIFU combined withgene treatment for hepatocellular carcinoma. This research mainly includedthe following three parts:
PART ONE: EXPERIMENTAL STUDY ON PREPARATION OFHSV1-TK GENE-LOADED LIPID ULTRASOUNDMICROBUBBLES
Aim To construct EGFP and HSV1-TK co-expression vector, realizeultrasound microbubble and HSV1-TK gene connection, and to evaluatethe physical character of ultrasound microbubble and its enhancementeffects in contrast imaging on liver cancer model. Methods Through theselection of the eukaryotic expression vector pIRES2-EGFP and TK byenzyme digestion, PCR amplification, pIRES2-EGFP-TK synthesis,sequencing, identification of recombinant; Applying electrostatic adsorp-tion principle, TK gene was adsorbed on the self-made ultrasoundmicrobubble contrast agent, detection of microbubble size, distribution, morphology, charge, carrying gene and contrast imaging effect in vivo.Results After electrophoresis, recombinant plasmids by restriction andPCR responses, there was a specific band about1024bp. The DNAsequencing results was identical to HSV1-TK that reported on Gene bank.Self-made microbubble can meet the ability of ultrasound contrast. In vivoultrasound contrast imaging shown that gene-loaded microbubbles couldenhance the hepatoma mice developing, and last developing about25min.Conclusions The plasmid pEGFP-TK was constructed successfully and toachieve a self-made gene-loaded ultrasound microbubble contrast agent,while meeting the imaging needs in vivo, and laid a good foundation forthe subsequent experiments.
PART TWO: THE EXPERIMENTAL STUDY OF ULTRASOUNDMICROBUBBLE CARRING HERPES SIMPLEX VIRUSTHYMIDINE KINASE FOR HEPATOCELLULAR CARCINOMA INMICE
Aim To observe the effect of microbubbles encapsulating herpessimplex virus thymidine kinase gene transfection on cancer tissue of micewith ultrasound exposure and assess the therapy efficacy by combiningGCV. Methods Kunming mice were inoculated subcutaneously in themiddle of the right flank with H22tumor cells line.40male mice bearingsubcutaneous hepatoma were randomized into4groups as follows:groupA,PBS;group B, HSV1-TK; group C,HSV1-TK+ultrasound; groupD, HSV1-TK+ultrasound+Microbubbles.The microbubbles of encap-sulatingHSV1-TK,PBS and Plasmid HSV1-TK were injected into the tail vein ofeach mouse，every3d once time, in all3times. Group C and D wereexposed by ultrasound and without group A and B. Western-blot was usedto detect the TK protein expression after48h. Subsequently, Ganciclovi(GCV) was administered in each mouse daily by intraperitoneal injection ata dose of0.2ml (100mg kg~(-1) d~(-1))in group B，group C and group D．Thetumor size was measured every2d till16d, then, all mice were killed toevaluated the histopathological changes of tumors. Results Gene could beinjected exactly into hepatocellular carcinoma with ultrasound monitor, andexpression of TK proteins was found in all4groups. Expression in group Dwas higher than others (P<0.05). The rate of tumor growth inhibition were0in group A,3.90%±1.80%in group B，22.70%±2.86%in group C，41.25%±3.20%in group D (group B vs group C, P<0.05;group D vs groupC, P<0.05;groupD vs group B, P<0.05);There was more obvious necrosisof tumor tissue in group D than group A. Conclusions Theultrasound-induced destruction of microbubbles encapsulated HSV-TKgene was release in the targeted spot, not only do these microbubbles helpto improve targeted in gene therapy of hepatocellular carcinoma, but theycan also enhance transfection efficiency of foreign gene. At last, thetherapeutic effect of suicide gene was enhanced in mice hepatoma. It wouldprovide a new strategy for liver cancer.
PART THREE: THE EXPERIMENTAL STUDY OF HIFUCOMBINED WITH HSV1-TK GENE-LOADED ULTRASOUNDMICROBUBBLE FOR VX2RABBIT LIVER TUMOR
Aim To investigate the effect of HSV1-TK gene-loaded ultrasoundmicrobubble microbubbles combined with HIFU for VX2rabbit livertumor and assess the therapy efficacy by combining gene-loaded ultrasoundmicrobubble. Methods seventy-five rabbits bearing VX2liver tumors wererandomly divided into the following five groups:①g roup HIFU;②groupHIFU+HSV1-TK;③groupHIFU+HSV-TK+US;④groupHIFU+HSV-TK+US+MB;⑤group HSV-TK+US+MB. The first four groups were treated withHIFU, HIFU treatment parameters: the frequency of1MHz, a focal lengthof156mm, radiation sound power150W. After HIFU ablation, the normalsaline, HSV1-TK gene, and ultrasound microbubble were injected viamarginal ear veins, then accept ultrasonic irradiation20min (irradiationparameters: a frequency of300KHz and an intensity of2W/cm~2with10seconds with10s pause, lasting a total of20minutes). All rabbits werereceived an intramuscular injection of100mg/kg/d ganciclovir24h afterirradiation, which last for10days. Tumor-bearing rabbits were killed48hours after transfection, testing its TK mRNA and protein expression,monitoring of tumor-bearing rabbits tumor size and survival time oftumor-bearing rabbits, using TUNEL assay for detection of tumor cellapoptosis after the end of treatment. Results The TK mRNA expression and GFP protein expression were higher in HIFU+HSV-TK+US+MBgroup and HSV-TK+US+MB group than other groups, and there wasstatistical significance (P<0.05). HIFU+HSV-TK+US+MB group was thehighest in all other groups in antitumor effect, and there was statisticalsignificance (P<0.05). It can obviously improve tumor-bearing rabbitssurvival time in HIFU+HSV-TK+US+MBs group. The tumor cell apoptoticindex was higher than other groups in HIFU+HSV-TK+US+MB group (P<0.05). Conclusions HIFU conbinated with carrying HSV-TK ultrasound-targeted microbubbles could inhibit the growth of VX2rabbit liver tumors,and improve the survival time of tumor-bearing rabbits which couldprovide a reliable experimental evidences for liver cancer treatment.
引文
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[10] Tang Y, Li J, Zhao S, et al. Killing effect of the herpes simplex virus thymidinekinase/ganciclovir enzyme/prodrug system on human nasopharyngeal carcinomacells [J]. J Int Med Res.2007,35(4):433~41
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[12] Beltinger C, Fulda S, Kammertoens T, et al. Mitochondrial amplification of deathsignals determines thymidine kinase/ganciclovir-triggered activation of apoptosis[J]. Cancer Res.2000,60(12):3212~7
[13]蔡宇，廖伟，周世骥，等.全反式维甲酸增强超声微泡包裹的单纯疱疹病毒胸苷激酶自杀基因治疗荷肝癌裸鼠的效果研究[J].第三军医大学报.2011,33(20):2112~2115
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[15] Omori Y, Li Q, Nishikawa Y,et al. Pathological significance of intracytoplasmicconnexin proteins: implication in tumor progression [J]. J Membr Biol.2007,218(1-3):73~7
[16]廖伟，李生伟，刘长安，等.全反式维甲酸增强携单纯疱疹病毒胸苷激酶自杀基因的超声微泡对SMMC-7721细胞的效应[J].第三军医大学学报.2011,33(15):1582~1585
[17]Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasound-mediated micro-bubbledestruction enhances VEGF gene delivery to the infracted myocardium in rats [J].Clinical Imaging.2004,28(6):395~398
[18] Suzuki R, Takizawa T, Negishi Y. Gene delivery by combination of novelliposomal bubbles with perfluoropropane and ultrasound [J]. J Control Release.2007,117:130~136
[19] Aoi A, Watanabe Y, Mori S, et al. Herpes simplex virus thymidine kinase-mediatedsuicide gene therapy using nano/microbubbles and ultrasound [J]. Ultrasound MedBiol.2008,34:425~434.
[20] Pitt WG, Husseini GA,Staples BJ. Ultrasonic drug delivery—A general review [J].Expert Opin Drug Deliv.2004,1:37~56
[1] Lau WY, Lai EC. The current role of radiofrequency ablation in the management ofhepatocellular carcinoma: a systematic review [J]. Ann Surg.2009,249(1):20~25
[2] Zhang L, Zhu H, Jin C, et al. High-intensity focused ultrasound (HIFU): effectiveand safe therapy for hepatocellular carcinoma adjacent to major hepatic veins [J].Eur Radiol.2009,19(2):437~45
[3] Zhang L, Wang ZB. High-intensity focused ultrasound tumor ablation: review of tenyears of clinical experience [J]. Front Med China.2010,4(3):294~302
[4] Kennedy JE, ter Haar GR, Wu F, et al. Contrast-enhanced ultrasound assessment oftissue response to high-intensity focused ultrasound [J]. Ultrasound Med Biol.2004,30:851~854
[5] Daniel Portsmouth, Juraj Hlavaty, Matthias Renner. Suicide genes for cancertherapy [J]. Mol Aspects Med.2007,28(1):4~41
[6] Haberkorn U, Khazaie K, Morr I, et a1. Ganciclovir uptake in human mammarycarcinoma cells expressing herpes simplex virus thymidine kinase [J]．Nucl MedBiol.1998,25(4):367~373
[7] Zhou S, Li S, Liu Z, et al. Ultrasound-targeted microbubble destruction mediatedherpes simplex virus-thymidine kinase gene treats hepatoma in mice [J]. J Exp ClinCancer Res.2010,29(1):170
[8] Pitt WG, Husseini GA, Roeder BL, et al. Preliminary results of combining lowfrequency low intensity ultrasound and liposomal drug delivery to treat tumors inrats [J]. J Nanosci Nanotechnol.2011,11(3):1866-70
[9] Milowska K. Ultrasound--mechanisms of action and application in sonodynamictherapy [J]. Postepy Hig Med Dosw.2007,61:338~49
[10] Yu T, Xu C. Hyperecho as the indicator of tissue necrosis during microbubble-assisted high intensity focused ultrasound: sensitivity, specificity and predictivevalue [J]. Ultrasound Med Biol.2008,34(8):1343~7
[11] Li JJ, Xu GL, Gu MF, et al. Complications of high intensity focused ultrasound inpatients with recurrent and metastatic abdominal tumors [J]. World J Gastroenterol.2007,21:2747~51
[12] Li YY, Sha WH, Zhou YJ, et al. Short and long term efficacy of high intensityfocused ultrasound therapy for advanced hepatocellular carcinoma [J]. JGastroenterol Hepatol.2007,22:2148~54
[13] Jolesz FA, McDannold N. Current status and future potential of MRI-guidedfocused ultrasound surgery [J]. J Magn Reson Imaging.2008,27:391~9
[14] Fillat C, Carrio M, Cascante A, et al. Suicide gene therapy mediated by the HerpesSimplex virus thymidine kinase gene/Ganciclovir system: Fifteen years ofapplication [J]. Curr Gene Ther.2003,3:13~26
[15] Freeman SM, Abboud CN, Whartenby KA, et al. The “bystander effect”: Tumorregression when a fraction of the tumor mass is genetically modified [J]. CancerRes.1993,53:5274~5283
[16] Beltinger C, Fulda S, Kammertoens T, Uckert W, Debatin KM. Mitochondrialamplification of death signals determines thymidine kinase/ganciclovir-triggeredactivation of apoptosis [J]. Cancer Res.2000,60(12):3212~7
[17] Freeman SM, Ramesh R, Marrogi AJ. Immune system in suicide-gene therapy [J].Lancet.1997,349(9044):2~3
[18] Bekeredjian R, Kroll RD, Fein E, et al. Ultrasound targeted microbubbledestruction increases capillary permeability in hepatomas [J]. Ultrasound Med Biol.2007,33:1592~8
[19] Zhigang W, Zhiyu L, Haitao R, et al. Ultrasound-mediated microbubble destructionenhances VEGF gene delivery to the infracted myocardium in rats [J]. Clin Imaging.2004,28:39~8
[20] Zhang KJ, Qian J, Wang SB, et al. Targeting Gene-Viro-Therapy with AFP drivingApoptin gene shows potent antitumor effect in hepatocarcinoma [J]. J Biomed Sci.2012,19:20
[21] Dai M, Liu J, Chen DE, et al. Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice [J].
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2. U.koppelhus, P.E.Nielsen. Cellular delivery of peptide nucleic acid (PNA)[J],Advanced Drug Delivery Reviews.2003,55(2):267-80
3. Zhao YZ, Luo YK, Zhang Y, et al. Property and contrast-enhancement effects of lipidultrasound contrast agent: a preliminary experimental study [J]. Ultrasound MedBiol.2005,31:537-543
4. Lanza GM, Abendschein DR, Hall CS, et al. In vivo molecular imaging ofstretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles [J].Am Soc Echocardiogr.2000,13(6):608-614
5. Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasoun-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium in rats [J].Clinical Imaging.2004,28(6):395-398
6. Zhang QX, Wang ZG, Ren H, et al. Enhanced gene delivery into skeletalmuscles withultrasound and microbubble techniques [J]. Acad Radiol.2006,(3):363-367
7.张群霞，王志刚，冉海涛，等.不同超声强度及微泡对基因和组织作用的实验研究[J].中华超声影像学杂志.2005,14(4):304-306
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14. Skyba DM, Kaul S, et al. Advances in microbubble technology [J]. Coron ArteryDis.200011(3):211-9
15.程文，张青萍，贡雪灏，等.外周静脉注入超声造影剂介导p53基因治疗大鼠肝癌的实验研究[J].中国超声医学杂志.2003,19(8):561-564
16.张群霞，王志刚，冉海涛，等.超声微泡促进肿瘤细胞报告基因转染[J].临床超声医学志.2005,7(2):78-80
17.程文，张青萍，贡雪灏，等.超声引导下瘤体内注入超声造影剂和p53基因治疗大鼠肝癌的初步研究[J].中华超声影像学杂志.2004,13(7):543-54
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[1]汪朝霞，王志刚，许川山，等.一种载基因及多聚赖氨酸的脂质超声微泡造影剂制备的实验研究[J].中国超声医学杂志.2009,25(2):101~104
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[11] Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasound-mediated microbubbledestruction enhances VEGF gene delivery to the infracted myocardium in rats [J].Clinical Imaging.2004,28(6):395~39
[1] Thomas MB, Jaffe D, Choti MM, et al. Hepatocellular carcinoma: consensusrecommendations of the National Cancer Institute Clinical Trials Planning Meeting[J]. J Clin Oncol.2010,28:3994~4005
[2] Pelletier SJ, Fu S, Thyagarajan V, et al. An intention-to-treat analysis of liver trans-plantation for hepatocellular carcinoma using organ procurement transplantnetwork data [J]. Liver Transpl.2009,15:859~868
[3] Smerdou C, Menne S, Hernandez-Alcoceba R, et al. Gene therapy for HCV/HBV-induced hepatocellular carcinoma [J]. Curr Opin Investig Drugs.2010,11(12):1368~77
[4]周世骥,刘长安,龚建平,等.超声微泡包裹单纯疱疹病毒Ⅰ型胸苷激酶自杀基因靶向治疗小鼠肝癌的效果[J].中华肝脏病杂志.2010,18(4):276~279
[5] Portsmouth D, Hlavaty J, Renner M. Suicide genes for cancer therapy [J]. MolAspects Med.2007,28(1):4~41
[6] Wang ZG, Ling ZY, Ran HT, et a1. Ultrasound—mediated microbubble destructionenhances VEGF gene delivery to the infracted myocardium in rats [J]. Clin Imaging.2004,28(6:395~398
[7] Deng CX, Sieling F, Pan H, et al. Ultrasound-induced cell membrane porosity [J].Ultrasound Med Biol.2004,30(4):519~26
[8]刘晓艳，丘泰球，刘石生，等.超声对细胞膜通透性的影响及应用[J].应用声学.2002,21(2)76~79
[9] Tsuchiyama T, Kaneko S, Nakamoto Y, et al. Enhanced antitumor effects of abicistronic adenovirus vector expressing both herpes simplex virus thymidine kinaseand monocyte chemoattractant protein-1against hepatocellular carcinoma [J].Cancer Gene Ther.2003,10(4):260~9
[10] Tang Y, Li J, Zhao S, et al. Killing effect of the herpes simplex virus thymidinekinase/ganciclovir enzyme/prodrug system on human nasopharyngeal carcinomacells [J]. J Int Med Res.2007,35(4):433~41
[11] Haberkorn U, Khazaie K, Morr I, et a1. Ganciclovir uptake in human mammarycarcinoma cells expressing herpes simplex virus thymidine kinase [J]. Nucl MedBiol.1998,25(4):367~373
[12] Beltinger C, Fulda S, Kammertoens T, et al. Mitochondrial amplification of deathsignals determines thymidine kinase/ganciclovir-triggered activation of apoptosis[J]. Cancer Res.2000,60(12):3212~7
[13]蔡宇，廖伟，周世骥，等.全反式维甲酸增强超声微泡包裹的单纯疱疹病毒胸苷激酶自杀基因治疗荷肝癌裸鼠的效果研究[J].第三军医大学报.2011,33(20):2112~2115
[14] Freeman SM, Ramesh R, Marrogi AJ. Immune system in suicide-gene therapy [J].Lancet.1997,349(9044):2~3
[15] Omori Y, Li Q, Nishikawa Y,et al. Pathological significance of intracytoplasmicconnexin proteins: implication in tumor progression [J]. J Membr Biol.2007,218(1-3):73~7
[16]廖伟，李生伟，刘长安，等.全反式维甲酸增强携单纯疱疹病毒胸苷激酶自杀基因的超声微泡对SMMC-7721细胞的效应[J].第三军医大学学报.2011,33(15):1582~1585
[17]Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasound-mediated micro-bubbledestruction enhances VEGF gene delivery to the infracted myocardium in rats [J].Clinical Imaging.2004,28(6):395~398
[18] Suzuki R, Takizawa T, Negishi Y. Gene delivery by combination of novelliposomal bubbles with perfluoropropane and ultrasound [J]. J Control Release.2007,117:130~136
[19] Aoi A, Watanabe Y, Mori S, et al. Herpes simplex virus thymidine kinase-mediatedsuicide gene therapy using nano/microbubbles and ultrasound [J]. Ultrasound MedBiol.2008,34:425~434.
[20] Pitt WG, Husseini GA,Staples BJ. Ultrasonic drug delivery—A general review [J].Expert Opin Drug Deliv.2004,1:37~56
[1] Lau WY, Lai EC. The current role of radiofrequency ablation in the management ofhepatocellular carcinoma: a systematic review [J]. Ann Surg.2009,249(1):20~25
[2] Zhang L, Zhu H, Jin C, et al. High-intensity focused ultrasound (HIFU): effectiveand safe therapy for hepatocellular carcinoma adjacent to major hepatic veins [J].Eur Radiol.2009,19(2):437~45
[3] Zhang L, Wang ZB. High-intensity focused ultrasound tumor ablation: review of tenyears of clinical experience [J]. Front Med China.2010,4(3):294~302
[4] Kennedy JE, ter Haar GR, Wu F, et al. Contrast-enhanced ultrasound assessment oftissue response to high-intensity focused ultrasound [J]. Ultrasound Med Biol.2004,30:851~854
[5] Daniel Portsmouth, Juraj Hlavaty, Matthias Renner. Suicide genes for cancertherapy [J]. Mol Aspects Med.2007,28(1):4~41
[6] Haberkorn U, Khazaie K, Morr I, et a1. Ganciclovir uptake in human mammarycarcinoma cells expressing herpes simplex virus thymidine kinase [J]．Nucl MedBiol.1998,25(4):367~373
[7] Zhou S, Li S, Liu Z, et al. Ultrasound-targeted microbubble destruction mediatedherpes simplex virus-thymidine kinase gene treats hepatoma in mice [J]. J Exp ClinCancer Res.2010,29(1):170
[8] Pitt WG, Husseini GA, Roeder BL, et al. Preliminary results of combining lowfrequency low intensity ultrasound and liposomal drug delivery to treat tumors inrats [J]. J Nanosci Nanotechnol.2011,11(3):1866-70
[9] Milowska K. Ultrasound--mechanisms of action and application in sonodynamictherapy [J]. Postepy Hig Med Dosw.2007,61:338~49
[10] Yu T, Xu C. Hyperecho as the indicator of tissue necrosis during microbubble-assisted high intensity focused ultrasound: sensitivity, specificity and predictivevalue [J]. Ultrasound Med Biol.2008,34(8):1343~7
[11] Li JJ, Xu GL, Gu MF, et al. Complications of high intensity focused ultrasound inpatients with recurrent and metastatic abdominal tumors [J]. World J Gastroenterol.2007,21:2747~51
[12] Li YY, Sha WH, Zhou YJ, et al. Short and long term efficacy of high intensityfocused ultrasound therapy for advanced hepatocellular carcinoma [J]. JGastroenterol Hepatol.2007,22:2148~54
[13] Jolesz FA, McDannold N. Current status and future potential of MRI-guidedfocused ultrasound surgery [J]. J Magn Reson Imaging.2008,27:391~9
[14] Fillat C, Carrio M, Cascante A, et al. Suicide gene therapy mediated by the HerpesSimplex virus thymidine kinase gene/Ganciclovir system: Fifteen years ofapplication [J]. Curr Gene Ther.2003,3:13~26
[15] Freeman SM, Abboud CN, Whartenby KA, et al. The “bystander effect”: Tumorregression when a fraction of the tumor mass is genetically modified [J]. CancerRes.1993,53:5274~5283
[16] Beltinger C, Fulda S, Kammertoens T, Uckert W, Debatin KM. Mitochondrialamplification of death signals determines thymidine kinase/ganciclovir-triggeredactivation of apoptosis [J]. Cancer Res.2000,60(12):3212~7
[17] Freeman SM, Ramesh R, Marrogi AJ. Immune system in suicide-gene therapy [J].Lancet.1997,349(9044):2~3
[18] Bekeredjian R, Kroll RD, Fein E, et al. Ultrasound targeted microbubbledestruction increases capillary permeability in hepatomas [J]. Ultrasound Med Biol.2007,33:1592~8
[19] Zhigang W, Zhiyu L, Haitao R, et al. Ultrasound-mediated microbubble destructionenhances VEGF gene delivery to the infracted myocardium in rats [J]. Clin Imaging.2004,28:39~8
[20] Zhang KJ, Qian J, Wang SB, et al. Targeting Gene-Viro-Therapy with AFP drivingApoptin gene shows potent antitumor effect in hepatocarcinoma [J]. J Biomed Sci.2012,19:20
[21] Dai M, Liu J, Chen DE, et al. Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice [J].
Cancer Gene Ther.2012,19(2):77~83
1. Heitmiller RF, Forastier AA, Kleinberg L, et al. Clinical Oncology [M]. Philadelphia,PA, ChurchilI Living stone.2000,1532-1533
2. U.koppelhus, P.E.Nielsen. Cellular delivery of peptide nucleic acid (PNA)[J],Advanced Drug Delivery Reviews.2003,55(2):267-80
3. Zhao YZ, Luo YK, Zhang Y, et al. Property and contrast-enhancement effects of lipidultrasound contrast agent: a preliminary experimental study [J]. Ultrasound MedBiol.2005,31:537-543
4. Lanza GM, Abendschein DR, Hall CS, et al. In vivo molecular imaging ofstretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles [J].Am Soc Echocardiogr.2000,13(6):608-614
5. Wang Zhigang, Ling Zhiyu, Ran Haitao, et al. Ultrasoun-mediated microbubbledestruction enhances VEGF gene delivery to the infarcted myocardium in rats [J].Clinical Imaging.2004,28(6):395-398
6. Zhang QX, Wang ZG, Ren H, et al. Enhanced gene delivery into skeletalmuscles withultrasound and microbubble techniques [J]. Acad Radiol.2006,(3):363-367
7.张群霞，王志刚，冉海涛，等.不同超声强度及微泡对基因和组织作用的实验研究[J].中华超声影像学杂志.2005,14(4):304-306
8. Suzuki R, Taki zawa T, Negishi Y, et al. Gene delivery by combination of novelliposomal bubbles with perfluoropropane and ultrasound [J]. J Control Release.2007,117(1):130-6
9. Christiansen JP, French BA, Klibanov AL, et a1. Targeted tissue transfection withultrasound destruction of plasmid bearing cationic microbubbles [J]．UltrasoundMed Biol.2003,29(12):1759-1767
10.Frenkel PA, Chen S, Thai T, et a1. DNA—loaded albumin microbubbles enhanceultrasound—mediated transfection in vitro [J]. Ultrasound Med Biol.2002,28(6):817-822
11.Kowabata K, Takakura Y, Hashida M. The fate of plasmid DNA after intravenousinjection in mice.involvement of scavenger receptors in its hepatic uptake [J].Pharm Res.1995,12(6):825-830
12. Ai-Na Bian,Yun-Hua Gao,Kai-Bin Tan,et al. Preparation of human hepatocellularcarcinoma-targeted liposome microbubbles and their immunological properties [J].World J Gastroenterol.2004,10(23):3424-3427
13.Schmitz G. Ultrasonic imaging of molecular targets. Basic Res Cardiol [J].2008,103(2):174-181
14. Skyba DM, Kaul S, et al. Advances in microbubble technology [J]. Coron ArteryDis.200011(3):211-9
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