Xiang Xi, Tang Yuanjiao, Leng Qianying, Zhang Lingyan, Qiu Li
Department of Ultrasound, West China Hospital of Sichuan University, Chengdu, Sichuan, China.
Department of Ultrasound, West China Hospital of Sichuan University, Chengdu, Sichuan, China.
Ultrasonics. 2016 Feb;65:304-14. doi: 10.1016/j.ultras.2015.09.011. Epub 2015 Sep 26.
The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD technique can significantly enhance the in vivo gene transfection efficiency without significant tissue damage in the synovial pannus of an AIA model. Thus, this could become a safe and effective non-viral gene transfection procedure for arthritis therapy.
本研究的目的是优化超声靶向微泡破坏（UTMD）技术，以提高抗原诱导性关节炎兔模型滑膜血管翳中增强型绿色荧光蛋白（EGFP）编码基因的体内转染效率。将微泡和质粒的混合物局部注射到抗原诱导性关节炎（AIA）兔的膝关节中。实验中改变了质粒浓度和超声条件。我们还测试了局部关节内注射和静脉注射。将兔子分为五组：（1）超声+微泡+质粒；（2）超声+质粒；（3）微泡+质粒；（4）仅质粒；（5）未处理的对照组。通过荧光显微镜和免疫组织化学染色观察每组滑膜血管翳中的EGFP表达。根据EGFP表达结果以及光学显微镜下组织损伤的有无确定最佳质粒剂量和超声参数。进行照射程序以观察滑膜血管翳及其他组织和器官中EGFP表达的持续时间，以及对正常细胞的损伤。确定最佳条件为施加1 MHz超声脉冲5分钟，功率输出为2 W/cm²，占空比为20%，同时使用300 μg质粒。在这些条件下，滑膜血管翳显示出显著的EGFP表达，而周围正常组织未受到明显损伤。局部关节内注射诱导的EGFP表达明显高于静脉注射诱导的表达。超声+微泡+质粒组滑膜血管翳中的EGFP表达明显高于其他四组（P<0.05）。表达在第5天达到峰值，在第40天仍可检测到，在第60天消失。在其他组织和器官中未检测到EGFP表达。UTMD技术可显著提高AIA模型滑膜血管翳的体内基因转染效率，且无明显组织损伤。因此，这可能成为一种用于关节炎治疗的安全有效的非病毒基因转染程序。
