三氯乙烯诱导人L-02肝细胞适应性反应的差异蛋白质组学分析

目的研究三氯乙烯(TCE)诱导L-02肝细胞的适应性反应及蛋白质表达改变。方法利用噻唑蓝(MTT)比色法,以0.5%二甲基亚砜(DMSO)作为溶剂对照组,确定对细胞增殖无明显作用的1μmo.lL-1TCE作为TCE诱导L-02肝细胞适应性反应的预刺激浓度,预刺激时间12 h,预刺激后再刺激浓度(适应组)为30μmo.lL-1TCE,刺激时间24 h。然后选取0,1,30和1+30μmo.lL-1TCE(1μmo.lL-1TCE预刺激12 h后,再次接受30μmo.lL-1TCE刺激24 h)4组平行进行双向凝胶电泳分离细胞总蛋白,银染显色,图像分析后,挑选差异蛋白进行基质辅助激光解吸电离飞行时间串联质谱鉴定。结果1μmo.lL-1TCE预刺激L-02肝细胞后,能降低30μmo.lL-1TCE攻击产生的细胞毒性。与单独用30μmo.lL-1TCE攻击相比,细胞增殖能力增加明显,细胞死亡减少。分析比较4组二维电泳图谱,发现15个蛋白质斑点表达发生改变,对其中5个差异较明显的蛋白点进行质谱分析鉴定,分别为Ku 86自体抗原相关蛋白1、透明质酸蛋白(hyaluronan)介导细胞运动受体、谷胱甘肽转移酶ω1、白细胞弹性蛋白酶抑制剂和空泡ATP合酶亚单位B。结论TCE可诱导L-02肝细胞产生适应性反应,引起L-02肝细胞蛋白表达改变,差异蛋白大多参与机体保护。     

Effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis: preliminary experimental study with rats

The aim of this paper is to study the effects of Fuzheng Huayu Decoction on the plasma proteome in cirrhotic rats. Twenty-six male Sprague-Dawley (SD) rats were randomly divided into three groups: cirrhotic model group (n = 10), treated with CCl₄ (CCl₄/olive oil: v/v = 1:1); Fuzheng Huayu Decoction intervention group (n = 10), treated with CCl₄ + Fuzheng Huayu Decoction; and normal control group (n = 6), treated with olive oil only. After 8 weeks, blood samples were collected from the inferior vena cava to undergo bi-dimensional electrophoresis (2DE) and analysis by PDQuest 7.3 software. Differential protein spots were cut, enzyme hydrolysis was conducted, and peptide fragments extracted from the mixture underwent mass spectrometry (MS) with MALDI-TOF-TOF-MS. The liver fibrogenesis was assessed using a digital image analysis instrument of Masson’s trichrome stained sections. The fibrosis area of the Fuzheng Huayu Decoction was (8.9 ± 3.7)%, significantly smaller than that of the cirrhotic model group [(12.4 ± 4.7)%, P<0.05]. Ten markedly changed protein spots were identified by MALDI-TOF-TOF-MS. Eight of the 10 proteins, including plasma glutathione peroxidase, plasma glutathione peroxidase precursor, prealbumin, haptoglobin, apolipoprotein A-IV precursor, complement C4, inter-alpha-inhibitor H4 heavy chain, and serine/threonine-protein kinase microtubule-affinity regulating kinase 1 (MARK1) were expressed very lowly in the cirrhotic model group while they were expressed highly in the Fuzheng Huayu Decoction group. The expression of liver regeneration-related protein LRRG03 and vimentin increased in the cirrhotic model group, and reduced in the Fuzheng Huayu Decoction group. Some proteins related to oxidative stress, cell proliferation and transformation have changed in the plasma of cirrhosis induced by CCl₄. Fuzheng Huayu Decoction promotes protein synthesis and plays an anti-fibrotic role by anti-oxidation and accommodation of cell proliferation and transformation.     

在固相pH梯度4~7范围内海洛因成瘾者血浆蛋白质组分析(英文)

目的比较鉴定海洛因成瘾者和正常人血浆蛋白质组差异,为研究海洛因成瘾相关血浆蛋白提供线索。方法海洛因成瘾者(n=5)和正常对照者(n=5)血浆蛋白经剔除白蛋白和免疫球蛋白IgG后,以固相pH梯度4~7胶条等电聚焦为第一向,SDS聚丙烯酰胺凝胶电泳为第二向,进行蛋白双向电泳。图像分析软件Image Master Elit 5.0分析蛋白质2维图谱。手工挖取组间相差1.5倍的差异点,串联质谱分析鉴定。结果每张图谱平均检测到350±21个蛋白(亚基)斑点,其中5个蛋白点在2组图谱中差异1.5倍以上,鉴定结果分别为γ纤维蛋白原、人α1B糖蛋白、α1-抗胰蛋白酶原、视黄醇结合蛋白载体蛋白四聚体单体和铜蓝蛋白。结论海洛因成瘾者血浆与正常人血浆对比存在蛋白质组差异。某些差异蛋白可能与海洛因成瘾造成的神经损伤相关。     

蛋白质组学技术的研究

本文讨论了蛋白组学对人体疾病研究的意义。采用双向聚丙烯酰胺凝胶电泳、激光捕获显微切割、质谱技术等研究方法。结合数据库和分析软件对实验结果的分析,探索在蛋白质水平上的作用模式、功能机理、调节、调控,以及蛋白质群组内相互作用。为各种全身疾病的早期诊断,进展监测,新靶点的确定提供了重要依据。     

金线莲水提物给药前后肿瘤细胞蛋白质组的差异比较

目的:建立利用二维液相色谱法分离肿瘤细胞全细胞裂解液的分析方法,进而研究金线莲水提物的抗肿瘤作用以及肿瘤细胞蛋白质组的差异。方法:将金线莲水提物给药后的肿瘤细胞裂解样品及对照样品用初始缓冲液置换后,进行一维色谱聚焦分离,然后先对二维色谱条件进行优化和重复性分析,再将一维收集的pH4.0～8.5之间的组分分别进行二维无孔硅胶HPLC分离,利用ProteoVue软件将UV图转换成胶图,分析差异。结果:一维色谱聚焦分离pH4.0～8.5之间组分共收集到16个,每个组分的二维UV图转换成PI/UV胶图,结果表明金线莲水提物给药前后肿瘤细胞蛋白质组差异具显著性。结论:二维液相色谱分离法是一种有效的分离肿瘤细胞裂解液的方法,给药后的肿瘤细胞蛋白质组和对照相比差异具显著性。     

The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry

Aim:

To investigate the proteome composition and function of human neonatal arterial umbilical cord.

Methods:

Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Results:

A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel biomarkers, such as alpha-fetoprotein and periostin that were specific to newborn infants.

Conclusion:

These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.     

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.