ROCK2-shRNA转染对VaD大鼠海马神经元的保护作用  

The protective effect of ROCK2-shRNA transfection on the hippocampal neurons of VaD rats

作　　者：向军军[1] 秦红玲[1] 卢健锋 杨璧璘 邓秋媚 陈炜[1] 胡跃强[1] XIANG Junjun;QIN Hongling;LU Jianfeng;YANG Bilin;DENG Qiumei;CHEN Wei;HU Yueqiang(The First Affiliated Hospital of Guangxi Medical University,Nanning 530023,China;Nanning First People's Hospital,Nanning 530023,China;Guangxi Hezhou Hospital of Traditional Chinese Medicine,Hezhou 542899,Guangxi,China;Guangxi University of Chinese Medicine,Nanning 530023,China)

机构地区：[1]广西中医药大学第一附属医院,广西南宁530023 [2]南宁市第一人民医院,广西南宁530023 [3]广西贺州市中医医院,广西贺州530023 [4]广西中医药大学,广西南宁530023

出　　处：《西部医学》2023年第6期812-816,共5页Medical Journal of West China

基　　金：国家自然科学基金项目(81860842);广西高等学校高水平创新团队及卓越学者计划(桂教人才202006);广西中医脑病临床研究中心项目(桂科AD20238028)。

摘　　要：目的探讨不同滴度ROCK2-shRNA腺相关病毒(AAV9-ROCK2-shRNA)转染对血管性痴呆(VaD)大鼠海马神经元的保护作用。方法选取成年SPF级健康雄性SD大鼠40只,随机分为正常组、PBS对照组、低滴度组、中滴度组、高滴度组(n=8)。采用双侧颈总动脉永久性结扎法制备VaD模型,将AAV9-ROCK2-shRNA按原液(高滴度)、2倍(中滴度)、10倍(低滴度)稀释后,采用脑立体定位术注射至大鼠海马组织周围。于1周及4周后,进行Morris水迷宫测试大鼠学习和记忆能力变化,并于第4周行为学观察后取材,采用HE与尼氏染色观察神经元形态、分布及数量变化,免疫组化检测IL-1β阳性细胞数,荧光显微镜检测绿色荧光蛋白(GFP)的转染情况。结果与正常组比较,PBS对照组术后1周与4周逃避潜伏期延长、跨越平台次数显著减少,神经元损伤明显,尼氏小体显著减少,差异有统计学意义(均P<0.05);与PBS对照组比较,术后1周,低、中、高滴度组大鼠逃避潜伏期、跨越平台次数差异无统计学意义(均P>0.05);术后4周,低、中、高滴度组较PBS对照组逃避潜伏期时间缩短、跨越平台次数增加,神经元排列整齐、形态更规则,尼氏小体增多,IL-1β阳性细胞数明显减少(均P<0.05);与中滴度组比较,术后1周,低、高滴度组的潜伏期、跨越平台次数差异无统计学意义(均P>0.05);术后4周,低、高滴度组大鼠逃避潜伏期延长、跨越平台次数减少,海马神经元受损更严重,尼氏小体数减少,IL-1β阳性细胞数增多,GFP转染率显著降低(均P<0.05)。结论中滴度AAV9-ROCK2-shRNA能高效、稳定、低毒地转染大鼠海马组织,对VaD大鼠海马神经元保护作用最优。Objective To explore the protective effect of different titers of ROCK2-shRNA adeno-associated virus(AAV9-ROCK2-shRNA)transfection on hippocampal neurons in vascular dementia(VaD)rats.Methods 40 SD rats were randomly divided into Normal group,PBS control group,low titer adeno-associated virus,medium and higher titer groups(n=8).The vascular dementia model was prepared by permanent ligation of bilateral common carotid arteries.The AAV9-ROCK2-shRNA diluted in the original solution(higher titer),2 times(medium titer),and 10 times(low titer)was injected into the hippocampus tissue of the rats by stereotactic brain technique.After 1 and 4 weeks,using Morris water maze to test the learning and memory abilities of rats.After 4 week,materials were taken after behavioral observation.HE staining and Nissl staining was used to observe changes in neuron morphology,distribution and number.The immunohistochemistry was used to detect the number of IL-1βpositive cells.The fluorescence microscope was used to detect the transfection of green fluorescent protein(GFP).Results Compared with normal group,PBS control group in postoperative 1 week and 4 weeks to escape the incubation period extended,cross platform times was significantly reduced,neurons damage wass apparent,nissl body significantly reduced,the difference was statistically significant(both P<0.05).Compared with PBS control group,postoperative 1 week,low,medium and high degree of group rat escape latent period,there was no statistically significant difference across platform number(both P>0.05).4 weeks after surgery,low,medium and high drop degree group escape latency time shorten the PBS control group,cross platform number increase,neat rows of neurons,shape rules,nissl body increase,IL-1 beta positive cells was significantly reduced(P<0.05).Compared with drops of group,postoperative 1 week and incubation period of low and high drop degree group,there was no statistically significant difference across platform number(both P>0.05).4 weeks postoperatively,low,high degree

关 键 词：血管性痴呆 海马神经元 Rho关联卷曲螺旋蛋白激酶2 腺相关病毒 

分 类 号：R749.1[医药卫生—神经病学与精神病学]