Sun Xiu-Ning, Liu Zhi-Jun, Guan Zhi-Yu, Liang Rui-Wen, Zhang Hao-Yun, Wu Xiao-Yan, Yu Li, Guan Ying-Jun
Department of Parasitology, Weifang Medical College, Weifang 261053, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Aug 30;30(4):253-7.
To investigate the effect of Toxoplasma gondii infection on the proliferation, differentiation and migration of the embryonic neural stem cells (NSCs) in early pregnancy of rat.
Twelve pregnant Sprague-Dawley rats were randomly divided into control and infection groups. Rats in the infection group were each inoculated intraperitoneally with 1 x 10(5) T. gondii RH strain tachyzoites at day 1 (E1 day). Same amount of physiological saline was intraperitoneally injected for rats in control group. At E5 day, blood samples were taken from caudal vein and Giemsa staining of blood cells was performed to find T. gondii. At E9, E10 and E11 day, two rats in each group per time point were sacrificed and reverse transcription PCR (RT-PCR) was performed to detect B1 gene expression of T. gondii in amniotic fluid to confirm T. gondii infection. NSCs were cultured in vitro. The proliferation level was detected by methyl thiazolyl tetrazolium (MTT) assay. After differentiation culture of NSCs, the immunofluorescence assay was conducted to detect the expression of nestin, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) to calculate the ratio of NSCs which differentiated to neurons and astrocytes. The embryonic nerve tissues at E9, E10 and E11 day in each group were taken to make frozen sections. The immunofluorescence assay was carried out to detect the expression of neuronal cell adhesion molecule (NCAM) in the nerve tissues at different developmental stages.
Both the results of blood smears and RT-PCR confirmed that the pregnant rats and embryos were all infected by T. gondii in infection group. The morphology of the cultured NSCs under microscope was consistent with the characteristics of the normal NSCs. In addition, the NSC biomarker nestin protein was stained positive. The MTT assay showed that the proliferation level was lower in infection group than that of the control, and statistical differences were found between the two groups at day 3 and 4 after passages (P < 0.05). The immunofluorescence staining of MAP2 and GFAP showed that the percentage of neuron differentiation was 15.15% (55/363) in control group and 8.73% (31/355) in infection group, respectively, with a statistical difference (P < 0.05), and the percentage of astrocyte differentiation was 53.35% (199/374) and 67.48% 249/369), respectively (P > 0.05). In both groups, NCAM protein was found expressed at E9, E10 and E11 day in embryo nerve tissues. The fluorescence became stronger with time. The expression level in control group was significantly higher than that in infection group (P < 0.01).
T. gondii infection at early gestation may inhibit the proliferation, differentiation and migration of neural stem cells in rats.
探讨弓形虫感染对大鼠妊娠早期胚胎神经干细胞（NSCs）增殖、分化及迁移的影响。
将12只妊娠的Sprague-Dawley大鼠随机分为对照组和感染组。感染组大鼠于妊娠第1天（E1天）腹腔注射1×10⁵个弓形虫RH株速殖子。对照组大鼠腹腔注射等量生理盐水。于E5天，从尾静脉取血，进行血细胞吉姆萨染色以查找弓形虫。于E9、E10和E11天，每个时间点每组处死2只大鼠，进行逆转录聚合酶链反应（RT-PCR）检测羊水中弓形虫B1基因表达以确认弓形虫感染。体外培养NSCs。采用甲基噻唑基四氮唑（MTT）法检测增殖水平。NSCs分化培养后，进行免疫荧光检测巢蛋白、微管相关蛋白2（MAP2）和胶质纤维酸性蛋白（GFAP）的表达，以计算分化为神经元和星形胶质细胞的NSCs比例。取每组E9、E10和E11天的胚胎神经组织制作冰冻切片。进行免疫荧光检测不同发育阶段神经组织中神经细胞黏附分子（NCAM）的表达。
血涂片和RT-PCR结果均证实感染组妊娠大鼠及胚胎均被弓形虫感染。显微镜下培养的NSCs形态与正常NSCs特征一致。此外，NSC生物标志物巢蛋白染色呈阳性。MTT法显示感染组增殖水平低于对照组，传代后第3天和第4天两组间差异有统计学意义（P<0.05）。MAP2和GFAP免疫荧光染色显示，对照组神经元分化百分比为15.15%（55/363），感染组为8.73%（31/355），差异有统计学意义（P<0.05），星形胶质细胞分化百分比分别为53.35%（199/374）和67.48%（249/369）（P>0.05）。两组胚胎神经组织在E9、E10和E11天均检测到NCAM蛋白表达。荧光随时间增强。对照组表达水平显著高于感染组（P<0.01）。
妊娠早期弓形虫感染可能抑制大鼠神经干细胞的增殖、分化及迁移。
