| Summary: | 碩士 === 國立臺北科技大學 === 機電整合研究所 === 101 === Ultrasonic microbubble-mediated gene delivery has shown to be a feasible method in gene delivery. Endothelial progenitor cells (EPCs) have emerged as potentially useful substrates for neovascularization, tissue repair, and bioengineering. The responsiveness of different physical parameters of ultrasonic microbubble transfection (UMT) on EPCs remained unclear.
EPCs originated from porcine peripheral blood were perfused with solution containing luciferase reporter gene and suspended microbubbles (1.5 ~ 2.5 μm in diameter) followed by exposure to therapeutic ultrasound (US) with different transfection parameters. The physical parameters included US exposure timing (24, 48 and 72 hours after cell seeding), DNA concentration (5, 15 and 30 μg/ml), microubble concentration (3 % and 10 % (v/v)), transfection temperature (23, 30, 37 and 45℃), and ultrasonic parameters (intensity, duty cycle and US exposure time). Luciferase gene expression was evaluated 48 hours post transfection. In parallel, a series of ultrasonic parameters were conducted to investigate the delivery of pDNA into EPCs.
In the presence of microbubbles, the relatively low intensity of ultrasonic parameters of 1 W/cm2, duty cycle 5 % and exposure time 30 seconds, which yielded a relatively high luciferase expression up to 200 LU/cell (n = 4 ~ 7, p < 0.001 vs control), were selected for the gene delivery. The use of physical transfection factors of ultrasound exposure timing (72 hours after cell seeding), DNA concentration (15 μg/ml), microubble concentration (3 % (v/v)), and temperature (37℃) can significantly improve gene expression (all p < 0.05). Cell viability post transfection is 60 % ~ 70 % in average (all p > 0.05 vs individual control).
UMT successfully delivers reporter gene into porcine EPCs by using different sets of US parameters. Among physical factors, ultrasound exposure timing (72 hours after cell seeding, P < 0.001), DNA concentration (15 μg/ml), microubble concentration (3 % (v/v)), temperature (37℃), and ultrasonic parameters (intensity 1 W/cm2, DC 10 %, and exposure time 30 sec) are optimal for ultrasonic microbubble-mediated gene delivery into EPCs. By using the optimized US parameters, the luciferase protein expression can be enhanced up to 3.5 fold (P < 0.001), compared to the initial parameters. Our findings provide a practical basis to achieve relatively high delivery efficiency together with a cell viability more than 60 % ~ 70 %.
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