生物周期节律紊乱大鼠乳腺组织的蛋白质组学研究

Study on proteomics of mammary tissue of rats with circadian rhythm disorder

目的研究光照条件改变所致的生物周期节律紊乱大鼠乳腺组织蛋白组学,重点分析乳腺癌等疾病发生、发展相关的信号通路蛋白表达水平差异,探讨生物周期节律紊乱对乳腺癌等疾病发生、发展的影响。方法 (1)制备外界光照环境改变所致的生物周期节律紊乱大鼠动物模型,采用荧光定量PCR检测实验大鼠下丘脑组织Clock、Bmal1、Per1、Per2、Cry1、Cry2核心生物钟基因表达水平的变化,并观察大鼠的日常行为及检测体内雌二醇(E2)水平,来验证生物周期节律紊乱大鼠动物模型造模是否成功。(2)应用iTRAQ蛋白标记技术、HPLC分离以及液相色谱-质谱联用方法,检测生物周期节律紊乱实验组和对照组乳腺组织的蛋白质表达水平,并应用GO及Pathway等注释方法对实验数据进行分析。结果 (1)经荧光定量PCR方法检测,生物周期紊乱可导致主要的时钟分子表达水平异常,其中Bmal1基因的表达水平下调,其他生物钟因子的表达呈不规律性变化。实验组大鼠的行为异常,其自身昼伏夜出的节律性行为逐渐消失。(2)生物周期节律紊乱实验组大鼠血清中E2的含量为(132.8±8.7)pg/mL,对照组为(125.95±0.9)pg/mL。与对照组相比较,实验组大鼠血清中E2水平有显著升高(P<0.05)。(3)对两组大鼠的乳腺组织进行蛋白质组学研究。实验检测到1 716种蛋白,并对其中1 651种蛋白进行定量分析,结果发现有169种蛋白在两组间存在显著差异,其中60种蛋白在实验组中表达上调,有109种蛋白表达下调。经GO、Pathway等注释分析发现:Tensin1、Glutathione S transferase pi、Glutathione S-transferase theta 2、Calmodulin-like 5、Calmodulin-3等与肿瘤、内分泌等相关蛋白质表达异常。结论 (1)实验室模拟人类由于外界光照环境改变导致的生物周期节律紊乱能够改变大鼠乳腺组织中蛋白质表达模式。(2)Tensin1、Glutathione S transferase pi、Glutathione S transferase theta 2、Calmodulin-like 5、Calmodulin-3等多种与肿瘤、内分泌相关的重要生物功能分子表达水平发生改变,提示生物周期节律紊乱可能通过影响肿瘤及内分泌等信号通路参与乳腺癌等乳腺疾病发生、发展进程,增加女性患乳腺癌等疾病的风险。

Objective To study the proteomics of mammary gland tissue in rats with circadian rhythm disorder caused by changes in light conditions,thus to investigate the impact of circadian rhythm disorder on the occurrence and development of breast cancer and other diseases.Methods Animal models of circadian rhythm disorder induced by the changes of external light environment were prepared.Fluorescence quantitative PCR was used to detect the changes in Clock,Bmal1,Per1,Per2,Cry1 and Cry2 core biological Clock gene expression in the hypothalamus.The behavior of the rats and the estrogen content was determined.Using iTRAQ protein label,HPLC separation and liquid chromatography-mass spectrometry were used to assess the expression of protein in the biological cycle disorder model group and the control group,and the experimental data were analyzed.Results According to fluorescence quantitative PCR,the circadian rhythm disorder lead to abnormal expression of the main clock molecules,with down-regulation of Bmal1 and irregular expression of other biological clock.The abnormal behavior of rats in the model group gradually disappeared.The content of estradiol(E2)in the serum was 132.8±8.7 pg/mL in the biologic rhythm disorder group,and was 125.95±0.9 pg/mL in the control group.The content of E2 in the serum of the biologic rhythm disorder group was significantly increased compare to the control group.A total of 1716 proteins were detected in the proteomics of the mammary tissues of the two groups.The quantitative analysis of 1651 proteins found that there were significant differences in 169 proteins between the two groups,among which 60 proteins were up-regulated in the model group,and 109 were down-regulated.Tensin1,glutathione S transferase Pi,glutathione S-transferase theta 2,calmodulin-like 5,calmodulin-3 and other related factors were abnormal.Conclusion The biological cycle disorder caused by ambient light environment changes can change the protein expression level in the mammary gland tissue of the rats.Biologic rhythmic disorders may be involved in the development of breast cancer and other breast disease.