Real-time quantitative PCR analysis of human astrocytes exposed to pressure-driven flow and/or Direct Current Stimulation in vitro. Real-time quantitative PCR analysis of human astrocytes exposed to pressure-driven flow and/or Direct Current Stimulation in vitro

The objective of this study was to investigate the effect of Direct Current Stimulation (DCS) on the gene expression of human astrocytes (HA). DCS at 0.1 or 1mA was applied to monolayers of HA for 10 minutes. Expression of a set of neuroactive genes was assessed immediately of 1 hour after DCS using RT-qPCR. Because DCS can produce electroosmotic flow and fluid shear stress known to influence cell function, we compared three interventions: pressure-driven flow across the monolayer alone (conP), pressure-driven flow plus DCS (DCS P), and DCS alone with flow blocked (DCS S). Overall design: HA were obtained from Cell Applications. DCS was applied with (DCS P) or without (DCS S) a pressure differential that induced fluid flow across the cell monolayer. Monolayers exposed only to convective flow (conP) were also examined. A static control (conS) kept in an incubator for the duration of the experiment was used as the calibrator sample for gene expression analysis. Total RNA was collected either immediately after DCS, or 1 hour after DCS using the RNeasy Mini kit from Qiagen, and reversed transcribed using the High-capacity cDNA Reverse Transcription kit from Thermo Scientific. Real time RT-qPCR was performed on the 7300 Real Time PCR System from Applied Biosystems. Samples were run in duplicates.

本研究旨在探究直流电刺激（Direct Current Stimulation, DCS）对人星形胶质细胞（human astrocytes, HA）基因表达的影响。将0.1 mA或1 mA的直流电刺激施加于HA单层培养物，刺激时长为10分钟。于直流电刺激结束即刻及1小时后，采用逆转录实时定量聚合酶链式反应（RT-qPCR）检测一组神经活性基因的表达水平。鉴于直流电刺激可产生已知可影响细胞功能的电渗流与流体切应力，本研究设置了三类干预方式进行对比：仅对单层培养物施加压力驱动流的对照组（conP）、联合压力驱动流与直流电刺激的实验组（DCS P），以及阻断流体流动仅施加直流电刺激的实验组（DCS S）。

实验整体设计如下：人星形胶质细胞购自Cell Applications公司。直流电刺激分别在存在诱导单层细胞流体流动的压力差（DCS P组）与不存在该压力差（DCS S组）的条件下施加。同时设置仅接受对流流的单层培养物（conP组）作为对照。另设置静态对照组（conS），全程置于培养箱中，作为基因表达分析的校准样本。

总RNA采集分别于直流电刺激结束即刻或刺激后1小时进行，使用Qiagen的RNeasy Mini试剂盒完成提取，随后采用Thermo Scientific的高容量cDNA反转录试剂盒进行反转录。实时定量RT-qPCR实验在Applied Biosystems的7300实时荧光定量PCR系统上开展，所有样本均设置复孔。