载BDNF基因阳离子超声微泡联合超声靶向微泡破裂技术治疗大鼠急性不完全脊髓损伤的实验研究  

BDNF-loaded cationic nanobubbles for targeted therapy of acute spinal cord injury in rats

作　　者：宋昭君 胡侦明[2] 叶永杰[1] 郑佳状[1] 汪凡栋[1] 陈宇[1] 刘元彬[1] 张智[1] Song Zhaojun;Hu Zhenming;Ye Yongjie;Zheng Jiazhuang;Wang Fandong;Chen Yu;Liu Yuanbin;Zhang Zhi(Department of Trauma Surgery,Suining Central Hospital;Department of Orthopaedics,The First Affiliated Hospital of Chongqing Medical University)

机构地区：[1]四川省遂宁市中心医院骨科中心,遂宁629000 [2]重庆医科大学第一附属医院骨科,重庆400016

出　　处：《重庆医科大学学报》2018年第10期1310-1317,共8页Journal of Chongqing Medical University

基　　金：四川省卫生和计划生育委员会科研课题资助项目(编号:16PJ523)

摘　　要：目的:探讨超声靶向破裂技术介导载脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因阳离子微泡(brain-derived neurotrophic factor-loaded cationic nanobubbles,BDNF/CNBs)治疗大鼠急性不完全脊髓损伤的可行性及效果。方法:96只成年雄性SD大鼠构建急性不完全脊髓损伤模型(Allen法)后随机分为4组:生理盐水(normal saline,NS)组;载BDNF基因阳离子微泡(BDNF/CNBs)组;BDNF基因+超声(brain-derived neurotrophic factor+ultrasound,BDNF+US)组;载BDNF基因阳离子微泡+超声(BDNF/CNBs+US)组,每组24只。经大鼠尾静脉注射药物后再按上述分组进行处理,在不同时间点采用HE染色观察脊髓损伤后的病理变化;Nissl染色观察神经元存活及再生情况;TUNEL染色法检测神经元凋亡情况;采用RTPCR和Western blot检测BDNF基因和蛋白的表达情况;通过倒置荧光显微镜观察偶联绿色荧光蛋白的表达情况;最终采用BBB法(Basso,Beattie,and Bresnahan test,BBB)评估大鼠神经功能恢复情况。结果:本实验所制备的BDNF/CNBs阳离子超声微泡的平均粒径为(339.8±210.3)nm,Zeta电位为(24.30±6.24)mV。BDNF/CNBs+US治疗组能有效减轻脊髓组织损伤,明显增加BDNF基因及BDNF蛋白的表达(0.61±0.10 vs. 0.70±0.13 vs. 0.83±0.15 vs. 1.55±0.19,P=0.000;31.65±1.30 vs. 45.62±1.50 vs. 49.55±1.20 vs. 75.83±2.10,P=0.000);与对照组相比,BDNF/CNBs+US治疗组尼氏小体数量明显增多(51.00±4.95vs. 90.80±6.87 vs. 99.60±7.50 vs. 159.40±8.56,P=0.000),神经元凋亡数明显减少(60.19±1.84 vs. 54.97±2.40 vs. 36.70±2.23 vs. 17.08±1.42,P=0.000);且最终促进脊髓损伤后神经功能的恢复,即表现为明显增高的BBB评分(10.10±0.33 vs. 10.60±0.43 vs. 11.70±0.36 vs. 17.20±0.45,P=0.000)。结论:载BDNF阳离子超声微泡联合超声靶向微泡破裂治疗能有效地将BDNF基因转染入损伤脊髓组织,并能促进脊髓损伤的功能恢复。以BDNF/CNBs为基础的超声辐照联合基因治疗在治疗脊髓损伤�Objective：To explore the effect of ultrasound（US）-mediated destruction of cationic nanobubbles（CNBs） expressing brain-derived neurotrophic factor（BDNF/CNBs） on nerve regeneration in rats following spinal cord injury（SCI）. Methods：Ninety-six adult male sprague-dawley rats were randomly divided into four treatment groups after Allen hit models of SCI being established. The groups were：normal saline（NS） group,BDNF/CNBs group,BDNF and US group,BDNF/CNBs and US group. Histological changes after SCI were observed by hematoxylin and eosin staining. Neuron viabi-lity was determined by Nissl staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling（TUNEL） staining was used to examine cell apoptosis. BDNF gene and protein expression were detected by quantitative reverse-trans-cription PCR and Western blot. Green fluorescent protein（GFP） expression in the spinal cord was examined using an inverted fluorescence microscope. The recovery of neural function was determined using the Basso, Beattie, and Bresnahan（BBB） test. Results：The mean diameter of the BDNF/CNBs was （339.8±210.3） nm and the zeta potential was （24.30±6.24） mV. BDNF therapy using US-mediated BDNF/CNBs destruction significantly increased BDNF expression（0.61±0.10 vs. 0.70±0.13 vs. 0.83±0.15 vs. 1.55±0.19,P=0.000;31.65±1.30 vs. 45.62±1.50 vs. 49.55±1.20 vs. 75.83±2.10,P=0.000）, attenuated histological injury, improved the expression of Nissl body and decreased neuron loss（51.00±4.95 vs. 90.80±6.87 vs. 99.60±7.50 vs. 159.40±8.56,P=0.000）,inhibited neuronal apoptosis in injured spinal cords（60.19±1.84 vs. 54.97±2.40 vs. 36.70±2.23 vs. 17.08±1.42,P=0.000）,and increased BBB scores in SCI rats（10.10±0.33 vs. 10.60±0.43 vs. 11.70±0.36 vs. 17.20±0.45,P=0.000）. Conclusions：UTMD-mediated BDNF/CNBs destruction effectively transfects the BDNF gene into target tissues and has a significant effect on the injured spinal cord. The combination of US irradiation an

关 键 词：阳离子微泡 脑源性神经营养因子 脊髓损伤 细胞凋亡 基因治疗 

分 类 号：R651.2[医药卫生—外科学]