超声辐照联合造影剂微泡介导EGFP转染类风湿关节滑膜的实验研究

EGFP gene transfection into the joint synovial tissues of rheumatoid arthritis rats by ultrasound-mediated microbubble destruction

目的 探讨超声辐照联合造影剂微泡介导EGFP转染类风湿性关节炎（RA）大鼠关节滑膜组织的可能性.方法 28只正常清洁级Wister大鼠,建立RA大鼠动物模型后,其中4只为空白对照组,其余24只再分为4个实验组,每组6只（12个膝关节）.A组：单纯质粒组；B组：超声＋质粒组；C组：微泡＋质粒组；D组：超声＋微泡＋质粒组.各个实验组在不同的处理因素作用后3d处死大鼠,取膝关节滑膜组织,立即在荧光显微镜下观察EGFP的表达情况,同时取部分组织对EGFP进行RT-PCR定量检测分析.结果 与空白对照组比较,实验各组RA大鼠关节滑膜组织中均有EGFP表达,但各组EGFP的表达存在差异.单纯质粒组、超声＋质粒组、微泡＋质粒组3组之间两两比较EGFP表达水平差异无统计学意义（P＞0.05）；单纯质粒组、超声＋质粒组、微泡＋质粒组3组与超声＋微泡＋质粒组之间EGFP表达水平比较差异有统计学意义（P＜0.01）.结论 超声击破微泡可以提高EGFP质粒对RA大鼠关节滑膜组织的转染效率.

Objective To explore the feasibility of enhanced green fluorescent protein （EGFP） transfection into the joint synovial tissues of rheumatoid arthritis （RA） rats by ultrasound-mediated microbubble destruction.Methods Twenty-eight normal rats were established the RA rat model,four rats were control group,twenty-four rats were categorized into four experimental groups：EGFP,ultrasound ＋EGFP,microbubbles ＋ EGFP,and ultrasound ＋ microbubbles ＋ EGFP.The last group was irradiated with ultrasound for 10 min after the mixture consisting of 300 μl Sono Vue and 10 μg EGFP was injected into the joint cavity.The rats were sacrificed after 3 days and the joint synovial tissues were collected for EGFP observation under fluorescence microscopy and quantitative analysis by real-time polymerase chain reaction （RT-PCR）.Results Comparing with control group,EGFP expression was observed in the rat joint synovial tissues from all groups.However,a strong EGFP expression was observed in the ultrasound ＋ microbubbles ＋EGFP group.EGFP expression had no statistically significant difference （the P values were 0.89,0.93,and 0.82,respectively,P ＞ 0.05） in the EGFP,ultrasound ＋ EGFP and microbubbles ＋ EGFP groups.However,EGFP expression in the EGFP,ultrasound ＋ EGFP,microbubbles ＋ EGFP groups significantly differed （all P values were ＜0.01） from that in the ultrasound ＋ microbubbles ＋ EGFP group.Conclusions Ultrasound-mediated microbubble destruction can improve EGFP transfection efficiency into the joint synovial tissues of RA rats.