羊膜腔注射Ad-eGFP对显性脊柱裂胎鼠脊髓缺损区域转染效果的研究  

Transfection efficacy of intra-amniotic injection of Ad-eGFP in fetal rat with spina bifida aperta

作　　者：张超楠 马巍 曹嵩颖 魏晓伟 顾卉 罗文婷 薛佳 赵连帅 黄天楚 袁正伟[1,2] Zhang Chaonan;Ma Wei;Cao Songying;Wei Xiaowei;Gu Hui;Luo Wenting;Xue Jia;Zhao Lianshuai;Huang Tianchu;Yuan Zhengwei(Key Laboratory of Health Ministry for Congenital Malformation, Shenyang 117004, China;Department of Pediatric Surgery, Affiliated Shengjing Hospital, China Medical University, Shenyang 110004, China)

机构地区：[1]卫生部小儿先天畸形重点实验室,沈阳117004 [2]中国医科大学附属盛京医院小儿外科,沈阳110004

出　　处：《中华小儿外科杂志》2019年第2期172-176,共5页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(81671469、81370717).

摘　　要：目的对胚胎进行羊膜腔注射腺病毒空白载体(Ad-eGFP),观察腺病毒作为基因治疗的载体在显性脊柱裂胎鼠组织的荧光分布位置以及对显性脊柱裂胚胎神经元相关标记物的影响,筛选显性脊柱裂进行基因治疗的载体。方法运用全反式维甲酸诱导显性脊柱裂胎鼠模型,于胚胎发育第15天(E15)向羊膜腔内注射适量Ad-eGFP,在胚胎发育第20天(E20)剖腹取出胎鼠,体视荧光显微镜下观察其体表GFP荧光表达情况。取新鲜脊髓组织,提取RNA,用Real-timePCR法检测脊髓组织神经元标记物相关基因mRNA表达变化。结果羊膜腔内注射Ad-eGFP的胎鼠,E20体视荧光显微镜下可见GFP荧光集中表达在胎鼠脊柱裂皮肤缺损区域以及脊柱中线处;同时与对照组相比,Ad-eGFP注射组脊髓组织中神经元标记物synapsin1、tau、tubulin的表达量虽然有所下调,但差异并没有统计学意义。结论运用羊膜腔注射的方法进行基因治疗,腺病毒载体主要表达在脊髓缺损区域,可以提高基因治疗的导入效率和靶向性。同时在本实验中并未发现腺病毒作为载体对神经元有显著影响。Objective To observe the distribution of adenovirus and the expressions of neuron-related markers in fetal rat with spina bifida aperta after injecting adenovirus vector (Ad-eGFP) into intra-amniotic cavity. MethodsThe spina bifida aperta rat fetus model was induced by all trans retinoic acid.Injecting Ad-eGFP into intra-amniotic cavity was performed at E15 and the distribution of green fluorescent protein (GFP) examined by fluorescent microscope at E20.RNA was extracted from fresh spinal cord tissue.And mRNA expressions of neuronal-related marker genes in spinal cord tissue were examined by real-time polymerase chain reaction (PCR). ResultsAfter an injection of Ad-eGFP into amniotic cavity, the expression of GFP was observed in defective region and central spine of rat fetuses with spina bifida aperta under fluorescent microscope.As compared with control group, the expressions of neuron-related markers synapsin1, tau and tubulin were lower in Ad-eGFP injection group.However, there were no statistical significances. ConclusionsFor fetuses with spina bifida aperta, an intra-amniotic injection of Ad-eGFP is feasible for gene therapy without obvious harm to neural development.

关 键 词：基因疗法 显性脊柱裂 羊膜腔穿刺术 

分 类 号：R450[医药卫生—治疗学] R726.8[医药卫生—临床医学]