Abstract： Objective To study the molecular mechanism of pathologic states related to cardiopulmonary bypass (CPB) and screen the differential proteins from the serum before and after CPB in the open heart surgery patients. Methods By the twodimensional gel electrophoresis (2DE), we took the blood samples from each of the sixteen open heart surgery patients 30 minutes before CPB, 1 hour after CPB, and 24 hours after CPB. The protein spots were analyzed by the PDQuest image analysis software and the differential protein spots were identified by matrixassisted laser desorption/ionizationtime of flightmass spectrometry (MALDITOF-MS). Then, enzymelinked immunosorbent assay (ELISA) was used to determine the expression level of serum amyloid A protein (SAA) in the serum of healthy people and the enrolled patients before and after CPB. Results Through 2DE in combination with massspectrometry, 7 proteins altered in expression were identified, including SAA, haptoglobin (HPT), leucinerich alpha2-glycoprotein (A2GL), hemoglobin subunit beta (HBB), serine/threonineprotein phosphatase 2A -regulatory subunit B″ subunit gamma (P2R3C), transthyretin (TTHY), and T-complex protein 11-like protein2 (T11L2). ELISA analysis showed that SAA levels in healthy people and the open heart surgery patients 30 minutes before CPB were not statistically different (t=-1.955, P=0.056), while the SAA level rose from 54.47±48.32 μg/ml 30 min before CPB to 1 017.78±189.92 μg/ml 24 hours after CPB in the serum of open heart surgery patients． Conclusion The results of this pilot study illustrate that SAA, HPT, A2GL, HBB, P2R3C, TTHY and T11L2 may be the molecule markers of pathologic state related to CPB. Acute phase reaction happens intensively after CPB in human body.
Objective To identify proteins that have expressed in human eyes from adults and two-month old infants by proteomics approach, so as to build a two-dimensional gel electrophoresis (two-DE) reference map for human retina. The difference of proteomics between the retinas of adults and two-month old infants are also studied. Methods Human retina tissues were collected from donor eyes (nine adults and two infants). Proteins were separated by two-DE. The gels were analyzed by image software. Protein spots were excised from the gels and detected by matrix assisted laser desorption ionization time off light mass spectrometry (MALDI-TOF-MS). Results A total of 1179 spots and 1295 spots were detected respectively on two-DE gels of Coomassie-stained adults and two-month old infants retina, of which 1039 spots were matched in the position. Five spots up-regulated were successfully identified. Human serum albumin and 4 guanylate kinase 1 (GUK1) were identified in adult retina. beta;2-tubulin, transaldolase1 and alpha A-crystallin were identified in infant retina. Conclusion The two-DE reference map for retina proteomics is successfully established. This study provides an evidence of changes in retinal protein levels between adults and infants and biochemical pathways for future studies of human retina development.
Objective To find a new specific marker that can be used to early diagnose hepatic fibrosis by detecting the change of serum protein in patients with hepatic fibrosis. Methods This research adopted 50 SD rats (25 males and 25 females), and from which 6 rats were selected randomly (3 males and 3 females) as control group, last 44 rates were divided into four groups according to four pathological stages as hepatic fibrosis model group (experimental group). Distinct proteins in serum were detected by surface-enhanced laser desorption/ionization-time of flight-mass spectra (SELDI-TOF-MS). Radioimmunoassay was used to measure four parameters of hepatic fibrosis which were hyaluronidase (HA), precollagen Ⅲ (PCⅢ), laminin (LN) and collagen Ⅳ (Ⅳ-C). Results Distinct proteins in serum were detected in 8 cases of stage Ⅰ of hapatic fibrosis, 5 cases of stage Ⅱ, 5 cases of stage Ⅲ, 6 cases of stage Ⅳ, and 5 cases of control by SELDI-TOF-MS. Three protein peaks were found (M/Z: 4 203, 4 658, and 7 400). The peaks of M/Z 4 658 and 4 700 proteins were obviously increased in the stage Ⅰ of hepatic fibrosis （Plt;0.05）, while the changes of hepatic fibrosis four parameters appeared in stage Ⅳ of hepatic fibrosis. Conclusion This method shows great potential for early diagnosing of hepatic fibrosis and finding better biomarkers to hepatic fibrosis.
Objective Toa nalyzed ifferentialp roteine xpressiono fc holangiocarcinomai np eripheralb loodb yproteomics technology, and to investigate the significance of proteomics technology in early diagnosis of bile ductmalignancy.M ethods Serum proteinf rom 58p atientsw ithc holangiocarcinomaa nd5 8c ontrols( 20p atientsw ithcholecystolithiasis and 38 healthy people) were detected by surface enhanced laser desorption/ionization-time offlight-mass spectrometry (SELDI-TOF-MS). Ciphergen protein chip software was used to identify proteinic spectra.R esults Comparedw itht hes pectrao fs erum proteini nc ontrolg roup,t herew ere1 0d ifferentiallye xpressedproteins in bile duct carcinoma group, among which three proteins with relative molecular masses of 5. 900 X 10’，9.08 0X 1 0’a nd1 1.86 3X 1 0’w ereu p-regulated( Plt;1 0-’)ands evenp roteinsw ithr elativem olecularm asseso f6.9 59X 1 0’，14.0 00X 1 0’，14.1 29X 1 0’，14.3 02X 1 0’，17.5 57X 10’，17.6 90X 1 0’a nd2 8.5 52X 1 0’w ered ownregulated(Plt; 10-’)。The average concentration of protein with the relative molecular mass of 11. 863 X 10’ incholangiocarcinoma group was eight times more than that in controls group. At the stage I of cholangiocarcinoma,thee xpressiono fp roteinp ointw itha r elativem olecularm asso f5 .90 0X1 0’w ass ignificantlyh ighert hant hosep atientsat the stage III and stage fV (Plt;10-’)，while there were no statistical difference of expression between diffeent clinical stages for the other 9 proteins points. And there were no significant expression differences of the above10 proteins between the patients with and without jaundice following cholangiocarcinoma. Instead, another threeproteinsw ithr elativem olecularm asseso f7 .25 5X 1 03，12.36 4X 1 0’a nd1 5.8 73X 1 03w ered etectedt oh aved ifferentproteine xpressions.A nda llo fth em showedh ighe xpressionsin j aundiceg roup( Plt;10-5).C onclusion Thereare remarkable differences of the expressions of serum proteins in peripheral blood in patients with cholangiocarcinoma.T hep roteinp ointw itha r elativem olecularm asso f1 1.86 3X 1 0’m ayb ea p otentialb iomarkerfo re arlyd iagnosisof cholangiocarcinoma
Objective The article introduces the present status of the application of comparative proteomics in study of tumor marker. Methods This essay review the present status and advances of the application of comparative proteomics in study of tumor marker through refer considerable literatures about proteome, proteomics and tumor marker. Results Follow the study of human genome deepening; the paradox between the finiteness of genes’ number and stability of genes’ structure and the variety of the life phenomena is more conspicuous. Then, the study of proteomics was pushed to the advancing front of life science research. The application of comparative proteomics to tumor research becomes a hot spot nowadays. Conclusion Screening tumor marker via comparative proteomics is an extremely promising research.
ObjectiveTo establish two-dimensional electrophoresis profiles with high resolution and reproducibility from subcellular immortalized human endocervical cell (H8) and cervical cancer cell (Caski), and to identify the differential expressions of subcellular proteins (cytoplasmic, membranous and nuclear proteins). MethodsH8 cells and Caski cells were incubated, and subcellular proteins of H8 cells and Caski cells were extracted and separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). Then the selected differential protein spots were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database. ResultsWe obtained well-resolved, reproducible 2-DE patterns; 6 differentially expressed cytoplasmic proteins and 3 differentially expressed membranous proteins and 9 differentially expressed nuclear proteins were defined in 2-DE gels. ConclusionSubcellular proteins of cervical precancerous lesion and cervical cancer are separated and analyzed by means of 2-DE and MALDI-TOF-MS/MS. There are significant differences between H8 cells and Caski cells. These data may be valuable for research of cervical precancerous lesion and cervical cancer, or as diagnostic markers and therapeutic targets for cervical cancer.
ObjectiveTo explore the possible active mechanism of the basic fibroblast growth factor (bFGF) long circulation l iposome (LCL) (bFGF+LCL) on spinal cord traction injury in rats at the level of proteomics. MethodsTwenty Sprague Dawly rats were randomly divided into groups A and B, 10 rats in each group. The models of spinal cord traction injury was established at T12-L3 spines. The rats were not treated in group A, and the rats were treated with bFGF+LCL (20μg/ kg) in group B. At 3 weeks after operation, the rats were sacrificed for harvesting T13-L2 spinal tissue specimens. The protein was extracted and quantified in the spinal tissue firstly. The proteins from spinal tissue were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. The different expression profiling was established in each group, and the differentially expressed protein was determined by comparing the level of each spot with gel imaging software and manually. The proteins were identified by nano ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (NanoUPLC-ESI-MS/MS), and the proteins were classified. ResultsThe differentially expressed protein spots were found in 2 groups. Compared with group A, 4 spots were up-regulated and 6 were down-regulated in group B. NanoUPLC-ESI-MS/MS results showed that 18 significant proteins were identified in 26 differentially expressed proteins, including 4 apoptosis-related proteins, 3 nerve signal transduction related proteins, 7 proteins involved in metabolism, 1 unknown function protein, and 3 unnamed proteins. ConclusionThe differentially expressed proteins are found in spinal cord traction injury of rats treated with bFGF+LCL. bFGF+LCL can affect the proteins expression in rats with spinal cord traction injury. The possible active mechanism is that it has protective and repair effects on injured spinal cord by nerve signal transduction, and regulation of nerve cells apoptosis and metabolism.
The pathogenesis of diabetic retinopathy (DR) is complicated and has not yet been fully elucidated. To explore the pathogenesis of DR and the mechanism of drug action, proteomics through quantitative analysis techniques is very useful. It can analyzes differentially expressed proteins in the retina, vitreous fluid, aqueous humor, tears, and blood of DR patients and diabetic rats, and analyzes differentially expressed proteins after drug intervention. This paper is a review of the progress in proteomic research of DR in recent years.
ObjectiveTo detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE).MethodshRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins.ResultsCCK-8 kits detection results showed that the A value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P＜0.01), while there were no differences between 10 and 20 μmol/L 4-HNE-stimulated groups, and the A value of 50 μmol/L 4-HNE-stimulated groups was lower than negative control. A total of 2710 quantifiable proteins were identified by peoteomics, and 118 proteins were differentially expressed (fold change＞1.5, P＜0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport.ConclusionAfter stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation.
With the development of life sciences and informatics, bioinformatics is developing as an interdisciplinary subject. Its main application is the relationship between genes and proteins and their expression. With the help of genomics, proteomics, transcriptomics, and metabolomics, researchers introduce bioinformatics research methods into fundus disease research. A series of gratifying research results have been achieved including the screening of genetic susceptibility genes, the screening of diagnostic markers, and the exploration of pathogenesis. Genomics has the characteristics of high efficiency and accuracy. It has been used to detect new mutation sites in retinoblastoma and retinal pigment degeneration research, which helps to further improve the pathogenesis of retinal genetic diseases. Transcriptomics, proteomics, and metabolomics have high throughput characteristics. They are used to analyze changes in the expression profiles of RNA, proteins, and metabolites in intraocular fluid or isolated cells in disease states, which help to screen biomarkers and further elucidate the pathogenesis. With the advancement of technology, bioinformatics will provide new ideas for the study of ocular fundus diseases.