Author + information
to prepare a cationic lipid microbubble and investigate its characterization and ultrasonic imaging in vivo; to explore the silencing effect of shPHD2-EGFP after its transfection into MSCs mediated by UTMD; to study if the MSCs transfected with shPHD2-EGFP can be easier to transplant into myocardial tissue by UTMD and enhances their cardioprotective effects after transplantation into infarcted hearts.
Cationic lipid microbubble was prepared by filming-rehydration and machine vibration. Its concentration, average diameter and zeta potential were measured. The binding capacity with plasmid and contrast-enhanced ultrasound effect was also assessed. The plasmid that silence PHD2 gene and carry GFP gene was constructed. Firstly, the cultured MSCs were divided into three groups, including Plasmid, Plasmid + Ultrasound and Plasmid + UTMD to prove that UTMD enhances gene transfection and find optimal transfection parameters by UTMD. Then, to demonstrate that shPHD2-EGFP silence PHD2 gene and induce HIF-1α and angiogenic factor gene, the experimental regimens included Control, EGFP and shPHD2-EGFP; to prove that PHD2 silencing enhances MSCs survival, the MSCs were divided into four groups, including Control, BMSC, BMSC-EGFP and BMSC-shPHD2-EGFP. Rat myocardial infarction models were conducted. Firstly, 30 rats were divided into three groups, including MI, MI-MSCEGFP and MI-MSCshPHD2-EGFP. The MSCs transplantation effect was tested. Then, other 120 rats were divided into four groups, including Control, MI-PBS, MI-BMSCsEGFP and MI-BMSCsshPHD2-EGFP. The echocardiography, histological character and myocardial apoptosis were evaluated.
The concentration of CMB was (4.12±0.29) × 109/ml. its average diameter and zeta potential was (1.67±0.32) μm and (29.18±1.36) mV respectively. The saturated DNA loading capacity for 1×108CMB was (3.56±0.31) μg. And enhancement effect in rat heart was good. Compared with Plasmid and Plasmid + Ultrasound groups, the gene transfection efficiency was highest. US intensity (1.0W/cm2) and exposure time (30s) were relative optimal transfection parameters. The shPHD2-EGFP down-regulated PHD2 and up-regulated HIF-1α after it was delivered into MSCs by UTMD. Meanwhile, the shPHD2-EGFP decreased MSC apoptosis. The number of MSCs for transplantation in MI-Plasmid + UTMD group was higher than MI-Plasmid and MI-Plasmid + ultrasound groups. Four weeks after transfection, compared with the other groups, myocardial apoptosis and scar size at the infarct area reduced in the MI-BMSCsshPHD2-EGFP group (P<0.05). Capillary densities in MI-BMSCsshPHD2-EGFP group were increased and left ventricular function had improved (P<0.05) over other groups.
UTMD successfully promoted transfection efficiency of plasmid DNA in vitro and increased marrow stem cell homing in vivo, thus improving the efficacy of therapeutic angiogenesis and myocardial function.