Establishment of SD neonatal rat hippocampal neuron models with human cytomegalovirus infection in vitro and preliminary study on calcium metabolism / 中华实用儿科临床杂志

ABSTRACT

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 primary 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 4days later,most of the cells disappeared; by immunohistochemistry in cultures of hippocampal neurons in HCMV,visible early proteins,brownish yellow granules,hematoxylin were found after being stained with brown pigment.The fluorescence intensity values of neuronal intracellular calcium (215.5 ± 14.9) in HCMV group was higher than that of control group (116.4 ± 5.9) (t =15.2,P ＜ 0.01),whilerise,that in MK-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 compared with that of HCMV group (215.5 ± 14.9),with decreased rate of (37.0 ± 3.4) ％ (t =11.3,P ＜ 0.01).Conclusions 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.