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High Intensity Focused Ultrasound Combined With Carrying Herpes Simplex Virus Thymus Kinase Gene Ultrasound-targeted Microbubbles For Liver Tumor

Posted on:2013-02-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:S J ZhouFull Text:PDF
GTID:1114330374978429Subject:Surgery
Abstract/Summary:PDF Full Text Request
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 ULTRASOUNDMICROBUBBLESAim 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 INMICEAim 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 TUMORAim 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/cm2with10seconds 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.
Keywords/Search Tags:Hepatocellular Carcinoma, HSV1-TK, Ultrasoudmicrobubble contract agents, gene therapy, HIFU
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