Comparative Time-Dependent Analysis of Potential Inflammation Biomarkers in Lymphoma-Bearing SJL Mice

SJL mice colonized with RcsX lymphoma cells undergo a rapid inflammatory response associated with biological and physiological effects including increased nitric oxide production and mutations in spleen DNA. By 2 weeks postcolonization, these changes were accompanied by both up- and down-regulation of a number of plasma proteins. In the experiments reported here, plasma from individual SJL mice was analyzed at several time-points over the 2-week period to determine if there were sets of proteins whose expression varied in concert and thus might serve as early biomarkers for inflammation-related disorders. Samples were collected just prior to injection of the RcsX cells and then after 4, 8, and 12 days. Albumin and immunoglobulins were depleted, and the samples were resolved by 1D gel electrophoresis. The gels were cut into 20 slices, and the proteins were digested in-gel with trypsin. The digests were treated with iTRAQ reagents and then analyzed using LC/MS/MS. The resulting data were processed with two software packages, that is, ProQuant and Spectrum Mill, and then subjected to K-means cluster analysis (K = 4). The four clusters revealed a set of highly up-regulated proteins, a set of progressively up-regulated proteins, a set with no major changes, and a set that declined. The first cluster included haptoglobin and serum amyloid A; the second included groups with several functions including protease inhibition, cell motility, and transport. The iTRAQ results for a selection of the up-regulated proteins, including haptoglobin, hemopexin, serum amyloid P component, and ceruloplasmin, were confirmed with Western blots. Prominent down-regulated proteins included esterase-1, paraoxonase, and α-2-macroglobulin. Approximately 50% of the up-regulated proteins are canonical acute phase proteins, while the remainder are regulated by the Nrf2 transcription factor. Keywords: inflammation • SJL mouse • lymphoma • iTRAQ • tumor progression • biomarker • cluster analysis • acute phase proteins定植有RcsX淋巴瘤细胞的SJL小鼠会出现快速炎症反应，伴随一氧化氮生成增加、脾脏DNA突变等一系列生物学与生理学效应。定植后2周时，这类变化会伴随多种血浆蛋白的上调与下调表达。在本研究报道的实验中，我们在2周周期内的多个时间点对单只SJL小鼠的血浆进行分析，以探究是否存在表达协同变化的蛋白组，进而有望作为炎症相关疾病的早期生物标志物。样本采集时机包括RcsX细胞注射前，以及注射后第4、8、12天。首先去除样本中的白蛋白与免疫球蛋白，随后通过一维凝胶电泳对样本进行分离。将凝胶切割为20个条带，对凝胶内的蛋白进行胰蛋白酶消化。消化产物经同位素标记相对和绝对定量（iTRAQ）试剂标记后，采用液相色谱-串联质谱（LC/MS/MS）进行分析。所得数据通过ProQuant与Spectrum Mill两款软件进行处理，随后进行K-means聚类分析（聚类数K=4）。四个聚类分别为：一组显著上调的蛋白、一组渐进式上调的蛋白、一组无显著变化的蛋白，以及一组表达下调的蛋白。第一聚类包含触珠蛋白与血清淀粉样蛋白A；第二聚类涵盖功能多样的蛋白组，涉及蛋白酶抑制、细胞运动与物质转运。针对部分上调蛋白（包括触珠蛋白、血液结合素、血清淀粉样P成分以及铜蓝蛋白）的iTRAQ标记结果，经蛋白质免疫印迹（Western blots）实验得到验证。显著下调的蛋白包括酯酶-1、对氧磷酶与α-2-巨球蛋白。约50%的上调蛋白为经典急性期反应蛋白（acute phase proteins），其余蛋白则受Nrf2转录因子（Nrf2 transcription factor）调控。关键词：炎症 • SJL小鼠 • 淋巴瘤 • iTRAQ • 肿瘤进展 • 生物标志物 • 聚类分析 • 急性期反应蛋白