干燥综合征患者细胞因子途径、Jak-Stat信号途径以及神经激活受体配体途径相关基因表达的初步分析

目的分析干燥综合征患者的细胞因子途径、Jak-Stat信号途径及神经激活受体配体途径相关基因的表达情况并分析意义。方法取3例干燥综合征患者及3例健康对照者的外周血单个核细胞,提取总RNA反转录至cDNA后,分别用Cy5及Cy3荧光标记干燥综合征组及对照组的cDNA样品,与寡核苷酸基因芯片杂交。采用图像分析软件提取芯片图像数据,以Cy5/Cy3的比值(SAM软件)寻找差异表达基因,并进一步用分子功能注释软件(MAS系统)进行处理。结果干燥综合征患者细胞因子途径、Jak-Stat信号途径及神经激活受体配体作用途径相关基因的差异表达有统计学意义(P<0.05)。以上途径中基因PF4、GZMA表达下调,基因IL-2RA、IL-10表达上调。结论干燥综合征的发病可能是多基因调控异常所致,细胞因子途径、Jak-Stat信号途径及神经激活受体配体途径的差异表达基因可能为研究该病的发病机制及新的治疗靶向提供了重要依据。

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus （WSSV） in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based mieroarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the mieroarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62μg/mL, with a woven long shelf life for 6 months at 4℃ or 8 months at -20℃. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.

基于GEO芯片数据挖掘和网络药理学探讨芪归抵挡汤调控糖尿病肾病“代谢记忆”的作用机制

目的:基于基因芯片数据挖掘和网络药理学研究芪归抵挡汤治疗糖尿病肾病(DKD)分子机制。方法:利用相关数据平台,挖掘芪归抵挡汤治疗DKD的活性成分和作用靶点,并进行GO功能和KEGG通路富集分析。最终,通过体内实验验证芪归抵挡汤调控糖尿病肾病的作用机制。结果:芪归抵挡汤共获得349个潜在靶点,GEO数据库分析获得具有显著性差异基因有841个,将二者取交集共获得45个交集靶点,经复合网络筛选出槲皮素、山柰酚、芦荟大黄素、常春藤皂苷元和β-谷甾醇等重要成分;CASP3、EGF、CXCL8、CCL2及ICAM1等治疗DKD的重要核心靶点。GO功能富集显示,药物反应、炎症反应和对缺氧的反应等在生物过程中富集度较高,质膜、细胞溶质和细胞外空间等在细胞组分中富集度高,蛋白质结合、受体结合和整合素结合等在分子功能中富集度较高;KEGG分析表明该方药效基础主要与AGE-RAGE、甲型流感、卡波西肉瘤相关疱疹病毒感染、EB病毒感染和NF-κB等信号通路有关。体内研究发现,与正常对照组比较,模型对照组小鼠FBG含量明显升高,血清Scr、BUN含量明显增加,肾组织中AGE、RAGE、Caspase-3蛋白表达上调(P<0.01);与模型对照组比较,芪归抵挡汤3.34 g/kg组可有效改善db/db小鼠血糖和肾功能(P<0.01),显著减轻肾脏病理损伤,下调AGEs-RAGE信号通路相关的AGE、RAGE、Caspase-3蛋白的表达(P<0.01);与白藜芦醇组比较,芪归抵挡汤3.34 g/kg组小鼠治疗6 w、12 w FBG含量,Scr含量明显降低,肾组织AGE、RAGE、Caspase-3蛋白表达明显下调(P<0.05或P<0.01)。结论:芪归抵挡汤可有效降低糖尿病肾病小鼠的血糖水平、改善肾脏功能及病理损伤,并可调控AGEs-RAGE信号通路,具有清除糖尿病肾病“代谢记忆”潜能。