超声靶向破坏微泡不同途径转染生长因子pDNA及siRNA促损伤肌腱再生及抑制粘连的实验研究

Scar healing and adhesion of wounded tendond are difficult clinical problems. So, promoting endogenous healing and suppressing exogenous healing are the key points of treating.It is a effective way to promote endogenous healing by transfecting growth factor genes which can promote regeneration into the dendons. At the same time, it maybe can suppress exogenous healing by using gene silence technology（siRNA）to suppress the expressing of growth factor causing adhesion in the granulation tissues. In prophase, we applied a new gene transfection way called ultrasound-targeted microbubble destruction(UTMD) to mediate tendon genes' tansfection at our laboratory. It proved that UTMD can successfully transfect gene into the wounded tendons and granulation tissues. UTMD has the characteristic of safety, high transfection efficiency,long lasting time and it can transfect multiple genes simultaneously.And we had an important finding that there would be different gene expression intensity in the tendons and granulation tissues as a result of different injection ways. We are trying to find out the optimized condition of ultrasound transfection for tendons and the granulation tissues,and to implement targeted transfection of IGF-1 pDNA and TGF-β siRNA into tendons and the granulation tissues, respectively, with different approaches, and to regulate tendons' healing way. In addtion, the physical treating effect of ultrasound is combined to promote tendons regeneration and suppress scars and adhesion.Our aim is to provide theoretical and experimental evidence for a new treating way to promote tendons healing and function recovery.

肌腱损伤后瘢痕愈合及粘连是临床工作难题，促进内源性愈合，减少外源性愈合是治疗关键。通过肌腱内转染促进其再生的生长因子基因成为促进内源性愈合的有效方法，同时应用基因沉默技术（siRNA）抑制导致粘连的生长因子在肉芽组织中的表达可能会抑制外源性愈合。我室前期应用超声靶向破坏微泡(UTMD)这一新的基因转染方式的实验证实：UTMD可成功体内转染基因到损伤处肌腱及肉芽组织，且安全，转染率高，持续时间较长，可同时转染多种基因，重要的是不同注射方式可导致在腱内及肉芽组织内不同基因表达强度，这为调节内源性与外源性愈合方式提供了可能。我们拟寻找最适合的肌腱及肉芽组织超声转染条件，通过不同途径分别靶向转染IGF-1 pDNA 及TGF-β siRNA到腱内及肉芽组织内，调节肌腱愈合方式，并联合超声物理治疗效应，促进肌腱再生，抑制瘢痕形成和粘连，旨在为肌腱寻找最佳愈合和功能恢复的新治疗方法提供理论和实验依据。

肌腱损伤后瘢痕愈合及粘连是临床工作难题，促进内源性愈合，减少外源性愈合是治疗的关键。通过肌腱内转染促进其再生的生长因子基因成为促进内源性愈合的有效方法，同时应用基因沉默技术（shRNA）抑制导致粘连的生长因子在肉芽组织中的表达可能会抑制外源性愈合。我室前期应用超声靶向破坏微泡(UTMD)这一新的基因转染方式的实验证实：UTMD可在体内成功转染基因到损伤处肌腱及肉芽组织，安全度高，转染率高，持续时间较长，可同时转染多种基因，这为调节内源性与外源性愈合方式提供了可能。本课题拟通过体内及体外实验寻找最适合的肌腱及肉芽组织超声转染条件，靶向转染IGF-1 pDNA 及TGF-β shRNA到肌腱内及肉芽组织内，调节肌腱愈合方式，并联合超声物理治疗效应，促进肌腱再生，抑制瘢痕形成和粘连，旨在为肌腱寻找最佳愈合和功能恢复的新治疗方法提供理论和实验依据。通过不同的条件设置对比研究表明，体内转染基因到肌腱处的最佳转染条件为输出功率2W/cm2，占空比20%，辐照时间10min。通过术后粘连指数评分、HE染色、免疫组化染色、天狼猩红染色、荧光染色、PCR以及生物力学测量对治疗后跟腱组织进行评估，研究结果显示UTMD 能够转染IGF-1 pDNA到跟腱组织并促进肌腱的内源性再生，UTMD能够转染TGF-β shRNA到跟腱组织并抑制跟腱的外源性再生，联合转染IGF-1 pDNA及TGF-β shRNA能够同时促进内源性再生及抑制外源性再生，治疗效果在肌腱损伤治疗后各阶段均最佳。本项目实验结果证明UTMD联合转染生长因子IGF-1 pDNA及TGF-βshRNA进入跟腱细胞具有一定的协同作用，能够共同促进损伤肌腱内源性再生，抑制其外源性再生导致的瘢痕及粘连形成，联合转染能提升肌腱的物理特性，以重建期明显，这为肌腱损伤的基因治疗新方法奠定了实践基础。