Gene Therapy Of Endostatin On Liver Cancer Mediated By Ultrasound Microbubble Destruction

PART Ⅰ: Preparation of microbubbles loaded with endostatingeneSECTION ⅠConstruction of human endostatin gene ofrecombinant plasmid vectorObjective To construct the expression vector of endostatin gene andprepare efficient ultrasound microbubbles for gene delivery. MethodsHuman endostatin gene was acquired by polymerase chain reaction (PCR)after the primers were designed. The DNA segment and pEZ-M46vectorwere cut and connected by enzymes, so the recombinant plasmid ofpEZ-M46-ES was constructed. The recombinant was then transformed intoJM109, and then the PCR of colony, restriction analysis, and DNAsequencing were performed for the sequence identification. Results Thesequence of the cloned DNA segment was basically identical to humanendostatin gene reported on gene bank. The sequence of amino acids codedby the protein was identical to endostatin. Conclusions The recombinant plasmid of human endostatin gene was successfully constructed.SECTION Ⅱ Experimental study on preparation and characteristicsof lipid microbubbles carrying geneObjective To develop stable and efficient ultrasound microbubblescarrying endostatin gene. Methods Lipid microbubbles and nanoliposomeswere prepared firstly by hydrated film formation, film extrusion andmechanic oscillation, and then were connected by biotin-actin method toconstruct an efficient gene vector. DC-cholesterol was added to the recipefor film formation to create positive charge and cationic microbubbles werethen developed by mechanic oscillation. The microbubbles were observedin the microscope and their basic characteristics (concentration, size,surface charge), ability of enhancing ultrasound imaging and carrying genewere investigated. Results In the microscope, nanoliposomes weresuccessfully coupled to the surface of microbubbles. However, the finalconcentration of nanoliposomes-microbubble was particular low due to theinefficient reaction. The cationic lipid microbubbles were also successfullydeveloped and appeared as regular and round shape, uniform size and gooddispersion. The mean diameter, surface charge and concentration were1.8±0.29μm,21.07±5.30mV,3.13±0.26109/ml, respectively. In thecontrast mode, the cationic microbubbles showed durative and long timeenhancement to ultrasound imaging. These microbubbles also exhibited ahigh gene loading efficiency owing to the large amount of positive surface charge as compared to the common microbubbles. The binding rates togene of cationic and common microbubbles were31.45±5.16%and4.33±1.47%, respectively. Conclusions Nanoliposomes-mciroubbbledidn't meet the demand of ultrasound contrast agent and couldn't carry theendostatin gene for target therapy. The cationic lipid microbubblespossessed the basic characteristics of ultrasound contrast agent and couldbecome an efficient gene carrier.PART Ⅱ: Gene transfection of endostatin mediated by UTMDin vitroSECTION ⅠThe effect of endostatin gene transfection on thegrowth and angiogenesis of HUVEC cell mediated by UTMDObjective To explore the optimal acoustic condition for UTMDmediated gene therapy and study the inhibition effect of endostatin genetransfection on the growth and angiogenesis of HUVEC cell mediated byUTMD. Methods The recombinant plasmid pEZ-M46-ES was transformedinto JM109and extracted, amplified, purified and adjusted to a finalconcentration of0.1g/l for transfection. HUVEC cells in logarithmicphase were collected and treated with ultrasound exposure at differentintensities (0.1,0.2,0.3,0.5W/cm2) and microbubble concentration (1,3,5,7108/ml). Twenty four hours later, the cell survival rate was thendetermined. The cells were divided into the groups for transfection asfollows:①blank control(C),②plasmid+lipofectamine (P+L),③ plasmid+ultrasound exposure(P+US),④plasmid+cationic microbubble(P+US),⑤plasmid+cationic microbubble+ultrasound exposure(P+MB++US). The first group was used as a negative control with emptyplasmid. The second group was a positive control with the application oflipofectamine2000. The effect of ultrasound exposure or cationicmicrobubble alone on transfection was investigated in the third and thefourth group. In the fifth group, the effect of the combination of cationicmicrobubble carrying endostatin gene with ultrasound exposure on genetherapy was studied.(1)The cells were observed in the convertedfluorescence microscope and collected to determine transfection rate byflow cytometry at24h after treatment. MTT method was used to assay thecell inhibition rate.(2)The expression of endostatin protein in cells wasdetected by Western blot.(3) HUVEC cells were seeded into the transwellsto establish the angiogenesis model and then treated as above groups. Thenumber of formed vessels in each group was calculated at24h aftertreatment. Results The cell survival rate was not affected when theultrasound intensity was below. It declined at the intensity of0.3W/cm2.More than half of the cells died when the ultrasound intensity went up to0.5W/cm2. At the intensity of0.1W/cm2, the cell survivate rate was notaffected when the microbubble concentration was not more than5108/ml.No fluorescence cells were found in the negative control group and onlyfew were found in P+L and P+US group. Large quantities of fluorescence cells were shown in P+L and P+MB++US group but no significantdifference was found between them. The transfection efficiencies inexperimental groups were31.24±8.3%,12.57±0.67%,3.14±0.72%,30.64±8.5%as determined by flow cytometry assay. The results of MTTshowed that high inhibition rates were found in both P+L and P+MB++USgroup but difference between them was no significant. Expression ofendostatin protein was detected in cells transfected with pEZ-M46-ESplasmid by western blot. The numbers of vessels in various groups were28.47±3.52,9.17±1.62,22.19±4.33,25.26±5.31,4.42±1.52, respectively.Conclusions The best acoustic condition for gene transfection was0.1W/cm2with5108/ml. Compared to the group of plasmid combiningwith ultrasound or microbubbles alone, UTMD application can enhance thetransfection efficiency of endostatin gene, but no significant difference wasfound as compared to that by Lipofectamine2000. After transfected withendostatin gene, the growth and angiogenesis of HUVEC cells weremarkedly inhibited.SECTION Ⅱ The effect of endostatin gene transfection on themigration and invasion of HepG2cell mediated by UTMDObjective To study the migration, invasion, cloning formation andinducing angiogenesis in HepG2cells when transfected with endostatingene mediated by UTMD. Methods HepG2cells were routinely culturedand transfected with pEZ-M46-ES plasmid as follows:①blank control(C),②plasmid+ultrasound(P+US),③plasmid+MB(P+MB)④plasmid+MB+ultrasound (P+MB++US). After treatment, wound healingassay Matrigel and Transwell were employed to test the cell migration andinvasion ability. Soft agar was used to investigate the cell clone formationin each group. After transfected with endostatin gene, HepG2and HUVECcell were co-cultured in Transwell, the angiogenesis of HVEC cell wasobserved in the microscope. In addition, the VEGF expression in culturedsupernatant of HepG2cell was assayed by ELISA. Results Aftertransfected with pEZ-M46-ES plasmid, the transferred cells in migrationand invasion experiments in P+MB+US group were much less than thosein any other group. The number of cell clones in this group was also thesmallest. When co-cultured with HUVEC cell, the formed the vesselsinduced by HepG2cell in this group were less than those in any othergroup. The number of the migrated cells, cell clones and induced vesselswas reduced in the P+US group but not in the P+MB group as compared tothe control group. Conclusions Gene transfection with endostatin mediatedby UTMD can remarkably inhibit the migration, invasion, clone formationof HepG2cells and angiogenesis induced by their cultured supernatant.PART III The effect of endostatin gene transfection mediated byUTMD on the growth of implanted H22tumor in miceObjective To investigate the inhibitory effect of endostatin genetransfection mediated by UTMD on the implanted H22tumor in mice and explore the relevant mechanisms. Methods (1) The cationic microbubbleswere loaded with endostatin gene by incubation with pEZ-M46-ES plasmid.H22cells were cultured in suspension and collected in logarithmic phase.The cells suspension in PBS were adjusted to5.3×107/ml and inoculatedsubcutaneously into the dorsal flank area of the mice. The injection volumewas0.2ml.(2)0.2ml cationic microbubbles loaded with endostatin genewere injected through the tail veil of mice and the enhanced ultrasoundimaging in tumor sites was observed.(3)Twenty tumor bearing mice weredivided into five groups and treated with①saline control,②P+US,③unloaded MB+US,④P+MB,⑤MB loaded with plasmid+ultrasound(P+MB+US), respectively. Ultrasound exposure was intermittentlyperformed at an intensity of2W/cm2with10seconds on followed by5seconds off, lasting for totally5minutes post-injection. The mice weretreated two times a week for consecutive three weeks. The tumor size wasmeasured by vernier calipers before the first treatment and two times aweek after that. The tumor volume was calculated and the growth curvewas depicted. At the end of the treatment the mice were sacrificed, thetumors were excised and weighed. Tumor inhibition rate was thencalculated. Tumor masses were fixed in4%paraformaldehyde in PBS aftermeasurement, paraffin embedded and sliced in serial sections. Themicrovessel density (MVD) was evaluated by immunohistochemistrystaining. Results The ultrasound imaging of tumor was greatly enhanced5 secnods after the injection of the cationic microbubbles loaded withendostatin gene. The enhanced imaging lasted more than10minutes. Fromthe tumor growth curve, the group of MB loaded with endostatin gene plusultrasound exhibited a notably stronger antitumor effect than any othergroup. The tumor in this group grew much slower compared to othergroups (P<0.01) and the tumor inhibition rate was the highest (70.1%). Theresults of immunohistochemistry showed that the MVD expression was thelowest in the group of MB loaded with endostatin plus ultrasound. It wasalso reduced in the group of P+US and P+MB. No antitumor effect wasfound in the group and unloaded MB+US. Conclusions The cationicmicrobubbles loaded with endostatin gene can enhance the ultrasoundimaging of H22tumor in mice which provides evidence for the furthertargeted tumor therapy. The H22tumor in mice was markedly inhibited byendostatin gene therapy mediated by UTMD and the angiogenesis wassuppressed. It may become a novel means for the gene therapy of livercancer based on anti-angiogenesis in the future.