Translation State Array Analysis of LPS Stimulated Human Endothelial Cells. Homo sapiens

Confluent human umbilical vein endothelial cells (HUVECs) were exposed to LPS (1 micg/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, LPS monosome and LPS polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (LPS polysome/LPS monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. Keywords: Translation State Array Analysis Overall design: HUVECs were stimulated with LPS (1 micg/mL) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 uL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 uL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4°C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 uL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 u­L 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.

汇合态人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）经1 μg/mL脂多糖（lipopolysaccharide，LPS）处理2小时。通过梯度离心与分级分离技术开展核糖体谱分析，分离单体核糖体（单核糖体，即翻译不足状态）与多聚核糖体（多核糖体，即活跃翻译状态）结合的mRNA，以此作为探针检测互补DNA（complementary DNA，cDNA）芯片，该流程被称为翻译状态芯片分析（Translation State Array Analysis，TSAA）。本实验共获取4组样本：对照单体核糖体组、对照多聚核糖体组、LPS处理单体核糖体组以及LPS处理多聚核糖体组。基于GeneFilters芯片的归一化信号强度，我们计算得到翻译指数，即反映刺激条件下mRNA分子从单体核糖体组分向多聚核糖体组分转移程度的指标，计算公式如下：(LPS处理多聚核糖体信号/LPS处理单体核糖体信号)/(对照多聚核糖体信号/对照单体核糖体信号)。我们选取翻译指数大于2.5（翻译上调）或小于0.4（翻译下调）的基因开展后续研究。
关键词：翻译状态芯片分析（Translation State Array Analysis，TSAA）
整体实验设计：以1 μg/mL脂多糖处理人脐静脉内皮细胞2小时。向培养基中加入终浓度为100 ng/mL的环己酰亚胺（cycloheximide，CHX）处理5分钟，随后用含100 ng/mL CHX的预冷汉克斯平衡盐溶液（Hank's Balanced Salt Solution，HBSS）冲洗细胞，刮取细胞后以2000×g离心5分钟收集细胞沉淀。弃去上清液，将细胞重悬于375 μL低盐缓冲液（Low Salt Buffer，LSB：20 mM Tris、10 mM NaCl、3 mM MgCl₂，pH 7.4）中，加入RNasin（40 U/mL）与二硫苏糖醇（dithiothreitol，DTT，10 nM），冰上静置3分钟。随后加入125 μL LSB裂解缓冲液（LSB中添加200 mM蔗糖与1.2% Triton-X100），通过吹打使细胞裂解。将混合液转移至1.7 mL微量离心管中，以20000×g、4℃条件下离心1分钟，去除细胞核与细胞碎片。将上清液转移至新的离心管中，管内预先加入50 μL LSB、RNasin（40 U/mL）与DTT（10 nM），以及15 μL 5 M NaCl。将该混合液加至预先配制的15%-50%蔗糖LSB梯度缓冲液上层。将梯度体系以43700 rpm、4℃条件下离心90分钟，随后通过密度梯度流式细胞分级仪（Isco）进行分级分离，获取核糖体谱。收集对应单体核糖体（M）与多聚核糖体（P）的核糖体组分（见图1），将其加入Trizol LS试剂（Invitrogen）中，按照试剂盒说明书提取总RNA。以寡聚dT为引物，在33P标记的dCTP存在下对总RNA进行逆转录，分别从对照与LPS处理细胞的单体核糖体、多聚核糖体结合mRNA组分中制备带有放射性标记的cDNA探针；使用上述探针检测4张相同的Research Genetics（ResGen）人基因cDNA芯片（GF 211，Invitrogen）上的特定芯片靶点。芯片经高严谨条件洗涤后，置于磷屏成像系统中曝光，使用ResGen芯片配套的专用软件Pathways 3进行数据分析。将每张芯片的杂交信号归一化至该芯片所有杂交信号的平均强度，以校正不同芯片间全局杂交效率的差异。为判断特定杂交信号是否显著高于背景水平，首先通过将单个cDNA斑点的杂交信号除以对应芯片的背景信号，完成首轮芯片靶点筛选。背景信号水平通过Pathways 3分析软件测定：软件会测量cDNA斑点间区域的信号强度，并将该信号在全芯片范围内取平均。当杂交信号与背景信号的比值大于1.1时，判定该芯片靶点可与背景区分。每张芯片的信号强度均通过将单个cDNA斑点的原始信号强度除以该芯片的平均信号强度完成归一化。对每一个符合筛选标准的芯片靶点，计算其翻译状态（translation state，TS）：将该靶点在多聚核糖体组分中的归一化信号强度除以其在单体核糖体组分中的归一化信号强度，即TS=P/M。翻译指数（Translation Index，TLI）用于反映单个基因的翻译状态变化，其计算方式为处理组细胞的TS与对照组细胞的TS的比值：TLI = TSe/TSc，其中e代表实验/处理组，c代表对照组。在此计算体系中，翻译状态未受调控的基因其TLI值为1；翻译活跃的基因TLI值大于1，而翻译效率低下或翻译不足的基因TLI值小于1。我们选取TLI阈值2.5与0.4，将单个芯片靶点划分为翻译调控阳性基因。