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人巨细胞病毒体外感染SD乳鼠海马神经元细胞模型的建立及钙代谢的初步研究 被引量：4

Establishment of SD neonatal rat hippocampal neuron models with human cytomegalovirus infection in vitro and preliminary study on calcium metabolism
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摘要 目的探讨人巨细胞病毒（HCMV）感染对原代培养sD乳鼠海马神经元细胞内钙离子的影响及其机制。方法取24h内出生的SPF级SD乳鼠雌雄各10只，建立原代乳鼠海马神经元单层细胞；在体外培养的第8天将生长良好的单层神经元细胞随机分为HCMV感染组、HCMV＋MK-801组、MK-801组和对照组，每组设10个复孔；在处理后24h用Fluo-3AM钙离子探针进行荧光染色，在激光共聚焦显微镜下检测各组神经元游离钙离子的荧光强度值。结果接种HCMV24h后，神经元逐渐变圆、肿胀，4d后大部分细胞裂解消失；通过免疫组织化学在海马神经细胞的培养物中可见HCMV产生早期蛋白，呈棕黄色颗粒，苏木精复染后呈棕褐色。HCMV组神经元细胞内游离钙离子荧光强度值为215．5±14．9，高于对照组（116．4±5．9），差异有统计学意义（t=15．2，P〈0．01）；HCMV＋MK-801组神经元的荧光强度值（135．5±8．6）较HCMV组（215．5±14．9）明显下降，下降率为（37．0±3．4）％，差异有统计学意义（t=11．3，P〈0．01）；MK-801组的荧光强度值为88．1±4．5；较对照组（116．4±5．9）明显下降，下降率为（24．0±6．7）％，差异有统计学意义（t=-9．3，P〈0．01）。结论HCMVADl6株感染体外培养大鼠海马神经元可致细胞内的钙超载，N-甲基-D-天冬氨酸受体通道介导的钙离子内流可能是HCMV感染后神经元内钙超载的主要机制之一。 Objective To investigate the influence of primary cultured neonatal rat hippocampal neurons caused by human cytomegalovirus （ HCMV AD169 ） infection on intracellular calcium and its mechanism. Methods Twenty SPF SD rats born within 24 hours（ 10 cases of male and 10 cases of female） were assigned to establish the pri- mary rat hippocampal neuronal monolayer cells; After cultured 8 days in vitro, the eligible cells were randomly divided into HCMV infection group, HCMV ＋ MK-801 group, MK-801 group and control group,with 10 wells in each group. The fluorescence intensity values of the intracellular free calcium were detected after 24 hours of treatment with Fluo-3AM fluorescence staining. Results Inoculation of HCMV neurons after 24 h turned to round and swollen gradually, and 4 days later, most of the cells disappeared ; by immunohistochemistry in cultures of hippocampal neurons in HCMV, visi- ble early proteins, brownish yellow granules, hematoxylin were found after being stained with brown pigment. The fluo- rescence intensity values of neuronal intracellular calcium （215.5 ± 14.9 ） in HCMV group was higher than that of con- trol group （ 116.4 ± 5.9 ） （ t = 15.2, P 〈 0.01 ） , whilerise, that in M K-801 group （ 88.1± 4.5 ） was significantly lower than that of control group,with decreased rate of （24.0 ± 6.7 ） % （ t = - 9.3, P 〈 0.01 ）. The fluorescence intensity values of neuronal intracellular calcium in HCMV ＋ MK-801 group （ 135.5 ＋ 8.6） was significantly decreased com- pared with that of HCMV group （ 215.5 ± 14.9 ）, with decreased rate of （ 37.0 ± 3.4 ） % （ t = 11.3, P 〈 0.01 ）. Conclu- sions Intracellular calcium overload of cultured rat hippocampal neurons in vitro with HCMV AD16 strains infection can be detected. One of its main mechanisms is the N-methyl-D-aspartic acid receptor channel-mediated calcium influx.
作者 田克印 吴德 杨李 陈玉萍 唐久来
机构地区 安徽医科大学第一附属医院小儿神经康复中心
出处 《中华实用儿科临床杂志》 CAS CSCD 北大核心 2014年第6期455-458,共4页 Chinese Journal of Applied Clinical Pediatrics
基金 国家自然科学基金（81270665）
关键词 激光共聚焦显微镜 人巨细胞病毒 大鼠神经元 细胞模型 钙超载 Laser scanning confocal microscope Human cytomegalovirus Rat neuron Cell model Calcium o- verload
分类号 R373 [医药卫生—病原生物学]
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参考文献14

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共引文献2

同被引文献51

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中华实用儿科临床杂志
2014年 第6期

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