The Study Of HSV1-TK Geng-load Nansize Ultrasound Molecular Probe Kill The Residual Liver Cancer After HIFU Treatmnt

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.