Echocardiography Experimental Study Of Porcine Chronic Ischemic Myocardium Before And After Angiogenin-Transfected Bone Marrow Stromal Cells Transplantation

Objective:Ischemic heart disease (IHD) has become a major and common disease with the development of the society and the improvement of living standard. It's reported that every year, nearly 5,000,000 patients die of this disease in China. Therefore, how to treat IHD and how to detect the changes of IHD has been attached great importance by people. Up to now, cell transplantation and gene transfer have become the hotspots for IHD treatment.Many factors have been used for therapeutic angiogenesis, within which, Angiogenin (ANG) is a special angiogenic growth factor to improve ischemic myocardium. At the same time, several immature and stem cells are used for implantation in the inured myocardium. Studies show that Bone marrow stem cells (BMSCs) are more robust than other cells and in the native myocardial circumstance they can transdifferentiate into cardiomyogenic cells after transplantation.With the rapid development of computer and electronic technology, ultrasound new technology also improved. Integrated Backscatter (IBS) is analyzing scattering signal of myocardium tissue. Doppler tissue imaging (DTI) is analyzing frequency shift signal of myocardium tissue. Acoustic quantification (AQ) is using real time computer automatic border detection technology to track endocardium. Color kinesis (CK) provides real-time information on the timing and magnitude of endocardial displacement. Dynamic three-dimensional echocardiography (DTDE) reconstruct left ventricular stereo structure. Real-time myocardium contrast echocardiography (RMCE) using microbubbles to assess myocardial perfusion and reperfusion.In this study, we constructed recombinant adenovirus coding for ANG gene, BMSCs isolation, culture and transfer, set up procine chronic ischemic model, use ultrasound new technology to detect LV echo and function changes before and after genetherapy, with other imaging examine. Methods:1. The construction of recombinant adenovirus coding for ANG gene (Ad. ANG) and culture of BMSCs and transfer.The mRNA was isolated from embryonic brain tissue. RT-PCR was performed to synthesize and amplify the ANG DNA. Human embroynic kidney 293 cells were transfected with DNA-TPC and ANG by the calcium phosphate method. BMSCs were harvested from pig ilium and isolated by combination of gradient centrifugation of Percoll and preplating treatment, then were cultured in EMDM medium. The cells were identified by the immunohistology staining and flow cytometer of CD71, etc. BMSCs were transferred by Ad.ANG.2. chronic ischemic myocardium model was set up and cell transplantation and gene transfer therapy.An Ameroid constrictor was placed around every proximal left circumflex artery (LCX) of 25 normal pigs. Then they were divided into 4 groups, group I for transplantation of BMSCs transfected by ANG gene, group II for BMSCs, group III for ANG gene transfer and group IV for control. Four weeks after administration, all animals were killed and Histological and Pathological investigation of their hearts was performed.3. Ultrasound new technology study of porcine chronic ischemic myocardium before and after ANG-transfected BMSCs transplantation. Every examine was performed at normal state, 4 weeks after Ameriod placement and 4 weeks after administration.(1) IBS and DTI comparative study: HP sonos 5500, all of the machine settings of IBS were the same. The cyclic variation of integrated backscatter (CVIB) in endocardium and epicardium of lateral-posterior wall (LPW) of left ventricular papillary muscle level short axis view (LVPM-SAM) in every stage was calculated. Then turn the machine settings to DTI-mode, to study the spectrum in endocardium and epicardium of LPW of LVPM-SAM. The relation between CVIB-TGI and VS-TGI was studied.(2) AQ study: Turning to second harmonic imaging and AQ system, we selectstandard parasternal four chamber view (PA-4CV) and make the AQ curve accord with endocardium. Then the machine identified automatically boundary of LV and show the volume-time curve, calculate volume and function index of LV.(3) CK study: Based on AQ system, turning to CK system, systole CK images were obtained in LVPM-SAM views. CK systole index of every stages were analyzed by ICK software.(4) DTDE study: The image acquisition was commanded by HP transoesophageal omniplane probe. All the 3D datas was transferred to TomTec 4D LV-analysis, 4D Echo-view and 4D Cadio-View software. In 4D LV-analysis, three-dimensional echocardiographic (3DE) reconstruction with Automated border detection algorithms allows display the entire and local LV volume and systole function and multiple colored slices can be obtained form the base to the apex of the heart to evaluate wall motion. In 4D Echo-view, parallel or rotatory cut allowed CT like observation of LV. In 4D Cadio-View, manual tracing of endocardial border in eaxh cross-sectional image at end-diastole and end-systole allows accurate quantification of LV volume and EF.(5) RMCE study: Real-time perfusion and reperfusion images were with an Siemens Acusion Sequoia 512 and 3V2c transducer. We use SonoVue intravenous infusion. The results of contrast perfusion were analysed with CUSQ 1.4 software.4. Other related imaging technology examinationDSA, MRI and Pathology examine before and after therapeutic angiogenesis and cell transplantation of chronic ischemic myocardial. Results:1. An efficient and reliable method of constructing recombinant Ad vectors was established. Replication-deficient adenovirus vectors coding for ANG DNA were generated in high titer. Chronic ischemic myocardial big animal model was successful set up.2. Ultrasound technology(1) IBS and DTI study: CVIB-TGI and Vs-TGI are all decreased 4w after Ameroid placement. After administration, group I , II, III were all increased, among which, group I shared the best improvement. There was a linear correlation between the CVIB-TGI and Vs-TGI in each stage.(2) AQ study: Comparing LV systole function, EDV and ESV were all increased and EF, PER were all decreased. After administration, EDV and ESV were all decreased and EF, PER were all increased in group I , II, III, among which, group I shared the best improvement. Comparing LV diastole function,(3) CK study: Regional fraction area change (RFAC) were all decreased 4w after Ameroid placement, mainly in the middle and late period of the systole. After administration, group I , II, III were all increased, among which, group I shared the best improvement, mainly in the middle and late period of the systole.(4) DTDE study: Left ventrical (LV) remodeled and weakness of lateral-posterior wall (LPW) motion emerged 4w after Ameroid placement. The end-diastolic volume of left ventricle (EDV) are all increased and the ejection fraction (EF) all decreased (P<0.01). After administration, group IV remained increased volumes and decreased EF compared with that before administration (PO.05). group I -III have improvement of LV remodeling, decrease regional EDV of LPW and increase EF (PO.05~0.01). The greatest increase of EDV in base and middle PW and that of EF in base PW were found in Group I (PO.05).(5) TMCE study: The real-time MCE can show myocardial perfusion changes of every group radiantly. Quantitation analysis show that A and P are all decreased 4 weeks after Ameroid placement and increased 4 weeks after administration. The increasing of A and J3 in group I is most obvious(PO.Ol). The increasing of group II and III are also more obvious than group IV(PO.Ol).3. Other related imaging technology (MRI, DSA) and Pathology examine show improved angiogenesis and wall movement in group I -III, especially in group I,...