Study On Effects Of Ultrasound-Mediated Microbubbles Contrast Agent On Cells, Capillaries And Transfection Efficiency Of Gene In HUVEC

IntroductionWith the development in biotechnology and gene technology, gene therapy has been advancing gradually. The application of ultrasound mi-crobubbles contrast agent (hereinafter USMBCA for short. ) has brought innovations in diagnosis and its potential value in therapy has drawn great attention, which shows a broader application field. In gene therapy, the genetic materials reach the target cell, enter cell nucleus, transform into protein and take effects. Transfer gene methods cover direct injection and vein injection. The genetic materials, after being injected in vein, are u-sually decomposed by the enzymes in serum unless their stability is ensured through certain methods. It is difficult that the genetic materials pass through endothelial cells. So it is necessary to avoid decomposition of genetic materials in blood. Various materials as gene vectors in vein injection have been tried to improve genetic materials transfer. Recent studies show that USMBCA could be a safe and effective vector of gene therapy. Gene therapy of ultrasound — mediated microbubbles contrast agent (hereinafter MBCA. for short) destruction may be influenced by many factors, such as transfected tissues, types of genes, the energy and time of ultrasound irradiation, the ratio of gene and MBCA. Gene therapy of ultrasound— mediated microbubbles destruction can also generate some potentially hazardous influence on body.The aim of this study is to evaluate preliminarily the effects of ultra-sound—mediated microbubbles upon cultured HUVECs in vitro, the effects of ultrasound — mediated microbubbles injected into rat vein upon its heart capillary vascular and cardiac muscle fiber and the effect of ultrasound— mediated microbubbles upon enhancing transfection rates of gene — EPGFP after being lipofectamine—mediated to HUVECs in vitro.Materials and Methods1. HUVECs were cultured and MBCA—SonoVue was confected.2. Effects of ultrasound —mediated microbubbles contrast agents of different concentrations on cultured viable HUVEC quantity through MTT experiment104 cells of HUVECs were dropped into the well of 150//1 culture medium of 96 wells plate, 50/^1 MBCAs of different concentrations were dropped into each well. Cultured HUVECs were divided into 6 groups upon being dropped MBCA s — SonoVue of different concentrations. Group A: SonoVue.concentration, 50mg/ml;Group B: SonoVue concentration, 25mg/ml;Group C: SonoVue concentration, 17mg/ml;Group D: SonoVue concentration, 12. 5mg/ml;Group E: control group,0. 9% Nacl 50ptl per well and Group F: Neither HUVECs nor MBCA dropped, but 200/il culture medium per well. Each group occupied 8 wells of 96 — well plate. After ultrasound irradiation, HUVECs were cultured for 24 hours. After 10/nl MTT dropped in per well, HUVECs were cultured for another 4 hours. HUVEC s were quaked for 10 minutes after 150/Jtl DMSO being dropped in per well. The measurements of the ray absorbed were recorded. MTT experiment was repeated three times. Cell viability calculation via MTT: assuming cell livability with group of 0. 9% Nacl was 100%, the livability of every well cells in different concentration SonoVue was calculated: ( cell absorbency of every well cells in different concentration SonoVue/cell absorbency every well cells in 0. 9% Nacl group ) X 100%.3. Ultrasound — mediated microbubble contrast agents of differentconcentrations on cultured HUVEC apoptosis and HUVEC death quantitieslX106/ml HUVECs of lml were dropped into a single—well culture plate of diameter 35mm. Cultured HUVECs were divided into 5 groups upon being dropped MBCA s of different concentrations. Group A: SonoVue concentration, 50mg/ml, 300fil/per well;Group B: 25mg/ml, 300fil/per well;Group C:17mg/ml, 300/Ltl/per well;Group D:12. 5mg/ ml, 300^1/per well;Group E: control group,300/xl 0. 9% Nacl dropped into per plate. 6 single—well culture plates of diameter 35mm per group. After ultrasound irradiation, HUVECs were cultured for 24 hours. The experiment was conducted under the manufacturer's recommendations (Annexin—v — FITC and PI). Then, under fluorescence microscope (hereinafter FMS for short) , the situation of apoptosis and dead cells of HUVEC were observed after different concentration MBCAs being added. The cells in green were early apoptosis ones and the ones in red were the cells of late apoptosis and death. The cell apoptosis and viability were evaluated via flow cytometry (hereinafter FCMfor short).4. Effects of ultrasound —mediated microbubbles contrast agent on cultured HUVEC epicyteCultured HUVECs were divided into 4 groups: Group A: control group, 1 plate;Group B: ultrasound irradiation group, 1 plate;Group C: adding microbubble contrast agent SonoVue, 1 plate;Group D: both ultrasound irradiation and microbubble contrast agent SonoVue dropped, 2 plates, lml of lxlO6/ml HUVECs were dropped into single —well culture plate of diameter 35mm. 300/ul MBCA SonoVue of 17mg/ml was dropped into Group C and D. After ultrasound irradiation, HUVECs were scanned via an electron microscope. After ultrasound irradiation, HUVECs in another plate in Group D were cultured for 24 hours and scanned via an electron microscope.5. Animal experiment16 male Wistar rats were divided randomly into 4 groups, 4 rats per group: control group (Group A), ultrasound irradiation group (GroupB), MBCA group (Group C), both ultrasound irradiation and MBCA dropped group (Group D). 16 male Wistar rats were anesthetized via abdomen injection of 10% Chlorali Hydras (1 —2ml/kg). Rats in Group C and Group D were injected intravenously with self—made MBCA(0. 5ml/ per rat) for 1 minute. Intermittent harmonic imaging was performed in the left chest area with the HP55OO Sonos system, S4 probe for 6 minutes and destructed MBCA in myocardium. Mechanical index was set to 1. 6, the depth and focus were adjusted to 4 cm. End — systolic images, triggered by ECG R wave were obtained at pulse intervals of 7 cycles. The adjustments remained unchanged throughout the process. Transmission electron microscope (hereinafter TEMS for short) investigation: The rats in Group B and Group D were irradiated by ultrasound and myocardium of left ventricle was removed from the rats, which were investigated via TEMS.6. Gene transfectionCultured HUVECs were divided into 5 groups: Group A: control group, HUVECs .dropped;Group B: HUVECs, EPGFP and Lipo-fectamine 2000;Group C: HUVECs, EPGFP, Lipofectamine 2000 and MBCA;Group D: HUVECs, EPGFP, Lipofectamine2000 and ultrasound irradiation;Group E: HUVECs, EPGFP, Lipofectamine 2000, MBCA and ultrasound irradiation, lml of 1 X 106/ml HUVEC s was dropped into the single — well culture plate of diameter 35mm. 300/zl MBCA SonoVue of 17mg/ml was dropped into Group C and Group E. Lipofectamine was mixed with AfMg of EPGFP and transfected to HUVECs. Each group occupied 5 plates, with or without ultrasound irradiation of different characteristics. After ultrasound irradiation, HUVECs were cultured for 24 hours. Expression of the marker gene EPGFP was observed by FMS and quantified by FACS analysis.Results1. MTT experiment Ultrasound — mediated microbubble contrast a-gents of different concentrations exerted different effects on cultured HUVECs. The cells in ultrasound —mediated Group C produced significantly more viable cells than those of Group A or Group B. Compared with Group C, the cells in ultrasound— mediated Group D did not produce significantly more viabLe cells.2. Ultrasound — mediated microbubble contrast agents of different concentrations exerted different effects on cultured HUVECs. When the agent was diluted 2 times, the rate of viable cells increased and the rates of late apoptosis and dead cells decreased, obviously different from the previous one respectively (P0. 05). When the agent diluted 3 times, the rate of early apoptosis cells decreased obviously, quite different from the previous ones respectively in primary micro-bubble contrast agent and the agent diluted 2 times (P0. 05).3. In ultrasound — mediated microbubbles contrast agent group, nearly 23% of the cells exhibited pores of diameter about 2/^m in the cell surface. The cells returned to normal appearance right after being cultured for 24 hours.4. With TEMS investigation, in ultrasound — mediated microbubble contrast agent group, endothelial cell epicyte of rat's myocardia vessels was broken and endothelial cell mitochodria of ones was vacuolar degene-nation, heterochromatin in nucleolus was condensation;rat's myocardial capillary vessels burst, red blood cells oozed;myocardial fiber broke and was dissolved.5. After Ultrasound irradiation and being transfected by EPGFP for 24 hours, under FMS, particular EPGFP expression was observed inplates of experimental groups, the expression amount of EPGFP in ultrasound and microbubbles group significantly increased. The analysis results via FCM indicated that ultrasound — mediated MBCA destruction produced a more significantly increased transfection rate of lipofectamine -mediated EPGFP to HUVECs.Conclusions1. Ultrasound — mediated microbubbles contract agent destruction enhances significantly the transfection rate of EPGEP after being lipofectamine—mediated to HUVECs. Ultrasound microbubbles contract a-gent can be used as vector in gene therapy.2. SonoVue enhances ultrasound cavitation. USMBCA produces small transiet pores in the surface of HUVEC, which increases permeability of the cell epicyte. Cavitation effect is one of the mechanisms for ultrasound—mediated MBCA to enhance gene transfection rate.3. The technology of targeted transfer via ultrasound — mediated MBCA makes endothelial cell epicyte of rat's myocardia vessels broke and endothelial cell mitochodria of ones be vacuolar degenenation, and makes heterochromatin in nucleolus be condensation;rat's myocardial capillary vessels burst, red blood cells ooze into between myocardial fibers;myocardial fiber break and dissolved.4. Ultrasound — mediated microbubble contrast agents of different concentrations produce different effects on HUVEC. Ultrasound—mediated microbubble contrast agent of higher concentration decreases the quantity of viable cells and increases the quantities of cell apoptosis and death.