Department of Medical Ultrasound, The Third Affiliated Hospital of Guangzhou Medical University, PR China.
J Exp Clin Cancer Res. 2010 Nov 23;29(1):152. doi: 10.1186/1756-9966-29-152.
Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection in vivo by the combination of ultrasound-targeted microbubble destruction (UTMD) with polyethylenimine (PEI) in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA) interference therapy targeting human survivin by this novel technique.
Two different expression vectors (pCMV-LUC and pSIREN) were incubated with PEI to prepare cationic complexes (PEI/DNA) and confirmed by the gel retardation assay. Human cervical carcinoma (Hela) tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected.
Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression.
This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration effectively without causing any apparently adverse effect, and might be a promising candidate for gene therapy. Silencing of survivin gene expression with shRNA could be facilitated by this non-viral technique, and lead to significant cell apoptosis.
非侵入性和组织特异性的基因转染技术在临床基因治疗中具有重要价值。本研究旨在探讨超声靶向微泡破坏（UTMD）联合聚乙烯亚胺（PEI）能否增强肿瘤异种移植体内的基因转染，并通过靶向人存活素的短发夹 RNA（shRNA）干扰治疗来阐明该新技术对基因沉默和细胞凋亡诱导的影响。
将两种不同的表达载体（pCMV-LUC 和 pSIREN）与 PEI 孵育，制备阳离子复合物（PEI/DNA），并通过凝胶阻滞实验进行确认。将人宫颈癌（Hela）肿瘤种植于裸鼠双侧胁部皮下。肿瘤荷瘤小鼠经尾静脉给予 PBS、质粒、质粒和 SonoVue 微泡、PEI/DNA 和 SonoVue 微泡。一个肿瘤接受超声辐射，另一个作为对照。研究了 UTMD 和 PEI 促进的靶向递药和组织特异性的可行性。此外，还检测了基因沉默和细胞凋亡诱导的免疫组织化学分析。
电泳实验表明，PEI 可以有效地浓缩 DNA。UTMD 的应用显著增加了组织转染。两种表达载体均显示，接受超声照射的肿瘤所有切片均有基因表达，而对照肿瘤则无。更重要的是，转染基因的表达增加与超声联合 PEI 有关。沉默存活素基因可以通过下调存活素和 bcl-2 的表达有效诱导细胞凋亡，同时导致 bax 和 caspase-3 的表达上调。
本研究采用的非侵入性、新型的 UTMD 联合 PEI 方法可有效增强静脉注射时肿瘤异种移植的靶向基因传递和基因表达，且无明显不良反应，可能成为基因治疗的一种有前途的候选方法。该非病毒技术可促进 shRNA 沉默存活素基因表达，并导致显著的细胞凋亡。
