Objective To study the biological activities ofthe nerve regeneration conditioned fluid (NRCF), especially to further separateand identify the protein bands of the relative molecular mass of (232-440)×103. Methods The silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve in 6 New Zealand white rabbits (weight, 1.8-2.5 kg). The proteins in NRCF were separated by the native-polycrylamide gel electrophoresis (Native-PAGE), the protein bands of the relative molecular mass of (232-440)×103 were analyzed by the Shotgun technique, liquid chromatography, and mass spectrometry. Results The Native-PAGE result showed that there was 1 protein band of the relative molecular mass over 669×103, (232-440)×103 and (140-232)×103,respectively, and 6 bands of the relative molecular mass of (67-140)×103.Besides, 54 proteins were identified with at least 2 distinct peptides in 1 protein band of the relative molecular mass of (232-440)×103, including 4 unnamed protein products, mainly at the isoelectric points of 5.5-8.0 and of the relative molecular mass of (10-40)×103. Based on their functions in the protein database, allthe identified proteins in this study were classified into the following 5 groups: conjugated protein (43%), transport protein (30%), enzyme (6%), signal transducer (4%), and molecular function-unknown protein (17%). At the subcellular localization of the identified proteins, there was mainly a secreted protein (63%), and the remaining proteins were localized in the membrane and cytoplasm. Conclusion Native-PAGE and the Shotgun technique can effectively separate and identify proteins from NRCF, and can identify the components of the protein band of the relative molecular mass of (232-440)×103 and provide basicinformation on the unnamed protein products in NRCF.
ObjectiveTo analyze the differences in proteins between aneurysm/dissection patients and healthy subjects, and subsequently figure out differential proteins related to medial degeneration of aortic aneurysm/dissection.MethodsAortic wall samples were collected from 6 male aortic aneurysm patients (an aortic aneurysm group, mean age 56.50±8.19 years), 6 male aortic dissection patients (an aortic dissection group, mean age 54.17±6.68 years) and 6 male healthy subjects (a normal group, mean age 40.50±9.31 years) between December 2019 and May 2020 in West China Hospital of Sichuan University. Quantitative proteomics was performed using tandem mass tag (TMT) techniques, followed by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.ResultsA total of 63 differential proteins were obtained both in the aortic aneurysm group and the aortic dissection group compared with the normal group, with 30 up-regulating and 33 down-regulating. The differential proteins were involved in multiple biological processes and clusted on peroxisome proliferators-activated receptor (PPAR) signaling pathway, extracellular matrix-receptor interaction signaling pathway and complement and coagulation cascades signaling pathway.ConclusionThe identified proteins may help to demonstrate new molecular mechanisms related to medial degeneration of aortic aneurysm/dissection.
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.
ObjectiveTo characterize proteomic profile in aqueous humor of patients with pathologic myopia (PM) using quantitative proteomic analysis, which may provide new clues to understand the mechanisms and possible treatments of PM.MethodsA cross-sectional study. From January 2019 to August 2019, aqueous humor samples (32 cataract patients) were collected for quantitative proteomic analysis using liquid chromatography tandem mass spectrometry at Tianjin Medical University Eye Hospital. There were 11 males and 21 females. They were 58-76 years old with an average age of 68.41±6.09 years old. Sixteen patients with PM were regarded as PM group, 16 patients without myopia were regarded as the control group. The aqueous humor samples (100-150 μl ) were collected from all patients before cataract surgery. Using protein quantification and non-labeled liquid chromatography tandem mass spectrometry analysis, differentially expressed proteins were obtained. Five different proteins were randomly selected for ELISA verification. The differentially expressed proteins were further analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes, which were validated using ELISA in the other twenty samples of each group.ResultsA total of 583 proteins were identified and 101 proteins were found to be differentially expressed, including 63 up-regulated proteins and 38 down-regulated proteins. ELISA verification results showed that the expression trend of the 5 differentially expressed proteins between the PM group and the control group was consistent with the results of Label-free quantitative proteomics analysis. The main classifications of these differentially expressed proteins were protein-binding activity modulator, defense/immunity protein, protein modifying enzyme, metabolite interconversion enzyme, extracellular matrix protein, transfer/carrier protein and so on. The bioinformatics analysis suggested that PM was closely associated with inflammation and immune interactions, and remodeling of extracellular matrix.ConclusionsCompared with the control group, the protein expression profile of PM patients' aqueous humor specimens has obvious changes. These differences indicate that PM is closely related to inflammation and immune interaction and extracellular matrix remodeling.
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.
ObjectiveTo analyze the protein expression changes in the retina of non-arteritic anterior ischemic optic neuropathy (NAION) in rats.MethodsThe rat NAION (rNAION) model was established by Rose Bengal and laser. Twenty Sprague-Dawley rats were randomly divided into 4 groups, the normal control group, the laser control group, the RB injection control group, and the rNAION model group, with 5 rats in each group. The right eye was used as the experimental eye. The retina was dissected at the third day after modeling. Enzyme digestion method was used for sample preparation and data collection was performed in a non-dependent collection mode. The data were quantitatively analyzed by SWATH quantitative mass spectrometry, searching for differential proteins and performing function and pathway analysis.ResultsCompared with the other three control groups, a total of 184 differential proteins were detected in the rNAION group (expression fold greater than 1.5 times and P＜0.05), including 99 up-regulated proteins and 85 down-regulated proteins. The expressions of glial fibrillary acidic protein, guanine nucleotide binding protein 4, laminin 1, 14-3-3γ protein YWHAG were increased. Whereas the expressions of Leucine-rich glioma-inactivated protein 1, secretory carrier-associated membrane protein 5, and Clathrin coat assembly protein AP180 were decreased. The differential proteins are mainly involved in biological processes such as nerve growth, energy metabolism, vesicle-mediated transport, the regulation of synaptic plasticity, apoptosis and inflammation. Pathway enrichment analysis showed that PI3K-Akt signaling pathway and complement and thrombin reaction pathway was related to the disease.ConclusionThe protein expressions of energy metabolism, nerve growth, synaptic vesicle transport and PI3K-Akt signaling pathway can regulate the neuronal regeneration and apoptosis in NAION.
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.
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.
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.