目的探讨单层培养细胞总RNA提取过程中先行细胞消化或离心富集对所提RNA质量是否产生影响。方法培养NGF诱导的PC12细胞,采用Trizol试剂提取RNA:离心组(A)先将贴壁生长细胞经胰酶消化、离心获得细胞沉淀,加入1 ml Trizol进行RNA提取;直接组(B)将培养液弃尽后直接加入Trizol试剂(1 ml/10 cm2瓶壁)进行RNA提取;均采用琼脂糖凝胶电泳分析RNA完整性,紫外分光光度仪测定RNA浓度和OD260/OD280比值。结果凝胶电泳结果显示,直接组出现3条清晰的条带(28S、18S和5S条带),离心组在电泳末端5S区出现粗大的条带。两组OD260/OD28比值相似,约为1.6。直接组组间样品间RNA浓度相差较大。结论采用离心法和直接法提取贴壁细胞总RNA,先行细胞离心富集增加了RNA降解机会,直接法提取的RNA质量较高,考虑对后续实验的影响,直接法更优于离心法。
Objective The aim of this study is to investigate whether microwave exposure would affect the N-methyl-D-aspartate receptor(NMDAR) signaling pathway to establish whether this plays a role in synaptic plasticity impairment. Methods 48 male Wistar rats were exposed to 30 m W/cm2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H&E staining and transmission electron microscope. PC12 cells were exposed to 30 m W/cm2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated. Results Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits(NR1, NR2 A, and NR2B) and related signaling molecules(Ca2+/calmodulin-dependent kinase II gamma and phosphorylated c AMP-response element binding protein) were examined. Conclusion 30 m W/cm2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.
XIONG LuSUN Cheng FengZHANG JingGAO Ya BingWANG Li FengZUO Hong YanWANG Shui MingZHOU Hong MeiXU Xin PingDONG JiYAO Bin WeiZHAO LiPENG Rui Yun
