Saito Masazumi, Mazda Osam, Takahashi Kenji A, Arai Yuji, Kishida Tsunao, Shin-Ya Masaharu, Inoue Atsuo, Tonomura Hitoshi, Sakao Kei, Morihara Toru, Imanishi Jiro, Kawata Mitsuhiro, Kubo Toshikazu
Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
J Orthop Res. 2007 Oct;25(10):1308-16. doi: 10.1002/jor.20392.
The purpose of this study was to investigate the usefulness of sonoporation method on in vivo transduction of plasmid DNA (pDNA) and small interfering RNA (siRNA) into joint tissue. pGEG.GL3 plasmid was mixed with microbubble and injected into knee joints of rats. Ultrasound sonication was performed percutaneously. Three days after injection, GL3 expression of synovial tissue was determined by luciferase assay and RT-PCR. siRNA specific for GL3 (siGL3) or nonspecific siRNA were mixed with pGEG.GL3 plasmid and transduced by sonoporation. siRNA specific for EGFP (siEGFP) was transduced into the knee joints of EGFP transgenic rats, and gene silencing effects for endogenous gene were examined. To determine the localization of transduced siRNA, fluorescently labeled siRNA was transduced into joints. The expression of GL3 in the synovium was significantly enhanced by sonoporation. The gene expression was only seen in the synovium of the knee joint. The expression of GL3 was remarkably suppressed by co-transduction of siGL3, but not suppressed by nonspecific siRNA. siEGFP transduced by sonoporation attenuated green fluorescence on the surface layer of synovium of EGFP transgenic rats. The fluorescently labeled siRNA was seen in the synovium around the patella, femur, and tibia. Sonoporation is examined as a recent, novel, gene transduction method, and the advantage of this technique is minimal invasiveness. In this study, we showed that pDNA/siRNA can be transduced specifically into the joint synovium using sonoporation. The present method may be useful in nucleic acid therapy for joint disorders.
本研究的目的是探讨声孔效应法在体内将质粒DNA（pDNA）和小干扰RNA（siRNA）转导至关节组织中的有效性。将pGEG.GL3质粒与微泡混合后注入大鼠膝关节。经皮进行超声处理。注射后三天，通过荧光素酶测定和逆转录聚合酶链反应（RT-PCR）测定滑膜组织的GL3表达。将针对GL3的特异性siRNA（siGL3）或非特异性siRNA与pGEG.GL3质粒混合，并通过声孔效应进行转导。将针对增强绿色荧光蛋白（EGFP）的特异性siRNA（siEGFP）转导至EGFP转基因大鼠的膝关节中，并检测对内源基因的基因沉默效果。为了确定转导的siRNA的定位，将荧光标记的siRNA转导至关节中。通过声孔效应，滑膜中GL3的表达显著增强。基因表达仅在膝关节的滑膜中可见。共转导siGL3可显著抑制GL3的表达，但非特异性siRNA则无此抑制作用。通过声孔效应转导的siEGFP减弱了EGFP转基因大鼠滑膜表层的绿色荧光。在髌骨、股骨和胫骨周围的滑膜中可见荧光标记的siRNA。声孔效应作为一种最新的新型基因转导方法进行了研究，该技术的优点是微创性极小。在本研究中，我们表明使用声孔效应可将pDNA/siRNA特异性转导至关节滑膜中。本方法可能对关节疾病的核酸治疗有用。
