超声微泡造影剂介导EGFP质粒转染大鼠骨骼肌

目的 探讨增强型绿色荧光蛋白质粒(EGFP)在大鼠骨骼肌微血管通透性增加的条件下表达的可行性.方法 20只清洁级Sprague-Dawley(SD)大鼠,随机均分为裸质粒组(P)、质粒 超声组(P U)、质粒 微泡组(P M)和质粒 超声 微泡组(P U M)共4组进行实验.选择增强绿色荧光蛋白质粒与白蛋白微泡相混合,以超声介导白蛋白微泡破裂的方法 对大鼠骨骼肌行基因转染,转染7 d后,荧光显微镜下观察质粒在大鼠脊斜肌组织中的表达情况.结果 P、P M组大鼠脊斜肌组织中均未见增强型绿色荧光蛋白表达;P U组可见少量微弱绿色荧光,荧光强度较P和P M组明显增强(P<0.05),P U M组可见明显特异性增强型绿色荧光蛋白表达,荧光强度约为P、P M组的10倍,P U组的3倍(P<0.05);P U M组转染率为(42.72±10.07)%较之P U组(13.62±6.17)%明显增高(P<0.05).结论 超声介导微泡造影剂破裂造成的脊斜肌组织毛细血管通透性的增加,是血管内基因成功跨越内膜屏障的主要途径之一.

Trasferring EGFP gene into skeletal muscle of rats mediated by microbubbles

Objective To explore the feasibility of enhanced green fluorescent protein (EGFP) transfected into skeletal muscle of rats under the condition of the increasing in vascular permeability. Methods Twenty Sprague-Dawley (SD) rats were randomly divided into four groups, i.e.plasmid group (P), plasmid+ultrasound group (P+U), plasmid+microbubble (P+M) and plasmid+ultrasound+microbubble group (P+U+M). Microbubbles were attached with the naked plasmid DNA of EGFP and followed the way of ultrasound-mediated microbubbles destruction to trasfect gene into skeletal muscle of rats. Seven days after gene transfer, the EGFP expression in the muscle was observed under the fluorescence microscope. Results EGFP expression was not observed in the rats of the P groups nor P+M groups, though a small amount of faint green fluorescence was seen in P+U group. Visibility of apparent specificity EGFP expression was observed in P+U+M group, and the fluorescence intensity of P+U+M group was 10 times higer than that of P and P +M groups, but 3 times than that of P+U group (P<0.05). The transfection rate of P+U+M group was (42.72±10.07)%, signifiantly higher than that of P+U group (13.62±6.17)%, (P<0.05). Conclusion The increasing of capillary permeability caused by destruction of ultrasound-mediated microbubble contrast agent is one of the major ways for intravascular gene penetrating endometrial barriers successfully in spinotrapezius muscle of rats.