Screening and verification of plasma biomarkers for stable angina pectoris: a differential proteomic analysis / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare and analyze the differentially expressed plasma proteome between patients with stable angina pectoris (SAP) and healthy donors to identify the biomarkers for early diagnosis of SAP.</p><p><b>METHODS</b>Plasma samples from 60 patients with SAP and 60 healthy controls were collected. Twenty samples (100 mL each) randomly selected from each group were pooled and after removing high-abundance proteins from the pooled plasma, two-dimensional gel electrophoresis (2DE) was performed to isolate the total proteins. The protein spots with more than 2 fold changes were selected after 2D analysis using software, and the differentially expressed proteins were identified by MALDI TOF/TOF mass spectrometer. ELISA was performed to detect hemoglobin subunit delta (HBD) levels in 40 randomly selected samples from each group for verification of the results of 2DE.</p><p><b>RESULTS</b>A total of 7 differentially expressed proteins were found in plasma samples from patients with SAP, including 3 up regulated proteins (serum albumin, hemoglobin subunit alpha and hemoglobin subunit delta,) and 4 down?regulated ones (apolipoprotein L1, apolipoprotein C3, apolipoprotein E and complement C4B). ELISA results showed that HBD level was increased in SAP plasma, which was consistent with the results of 2DE.</p><p><b>CONCLUSION</b>Patients with SAP have different plasma protein profiles from those of healthy controls, and HBD may serve as a potential specific biomarker for early diagnosis of SAP.</p>

Identification of protein markers for gestational diabetes mellitus complicated by pregnancy-induced hypertensive syndrome / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the serum protein markers for the gestational diabetes mellitus (GDM) complicated by pregnancy-induced hypertensive (PIH) syndrome to provide a molecular biological basis for the screening, prevention and therapy of the related diseases.</p><p><b>METHODS</b>Serum samples were collected from the patients with GDM, PIH syndrome, and GDM complicated by PIH syndrome. IgG and albumins were removed from the samples before SDS -PAGE. The protein bands showing significant differences among the 3 samples were collected, digested and identified with mass spectrometry, and the function of the identified proteins was analyzed.</p><p><b>RESULTS</b>Three SDS-PAGE were performed in parallel to confirm the differentially expressed proteins. Mass spectrometry indicated that the proteins showing obvious differences among the 3 samples were haptoglobin, protein SMG8 and apoptosis-inducing factor-1.</p><p><b>CONCLUSIONS</b>The protein markers identified in GDM complicated by PIH syndrome may be integrated into the proteomic database of gestational metabolic diseases. Identification of the associated protein markers may provide significant experimental data for the prevention, diagnosis and therapy of the related diseases.</p>

Construction of a eukaryotic expression vector for alpha-1-antitrypsin and the localization of the expression product in NIH 3T3 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for alpha-1-antitrypsin (AAT) and detect its expression and localization in NIH 3T3 cells.</p><p><b>METHODS</b>The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding sequences for mouse AAT (GenBank accession No. NM_009243) were amplified by RT-PCR and cloned into hemagglutinin (HA)-tagged vector pcDNA3-HA. The construct was then transfected into NIH 3T3 cells, which were observed under fluorescence microscope.</p><p><b>RESULTS</b>The recombinant plasmid was verified by PCR, enzyme digestion and sequence analysis, and the fusion protein was highly expressed in NIH 3T3 cells. Under fluorescence microscope, the fusion protein was found to distribute mainly in the cytoplasm.</p><p><b>CONCLUSION</b>The expression vector for AAT-HA fusion protein has been successfully constructed and effectively expressed in mammalian cells to allow future functional study of AAT in mammalian cells.</p>

Extraction and two-dimensional liquid chromatographic fractionation of plasma membrane proteome of mouse liver cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To extract the plasma membrane proteins from mouse liver cells and investigate the approach for fractionating the protein mixtures by two-dimensional liquid chromatography.</p><p><b>METHODS</b>The plasma membrane of the liver cells from 10 mice was extracted by differential centrifugation and sucrose density-gradient centrifugation. The plasma membrane proteins were exchanged with the start buffer and separated by chromatofocusing in the first-dimensional fractionation. The final results were transformed into UV/pI maps using ProteoVue software.</p><p><b>RESULTS</b>We successfully extracted the plasma membrane proteins from mouse liver cells. Sixteen fractions between pH 8.5-4.0 were recovered in the first-dimensional chromatofocusing followed by 2D- chromatographic fractionation, and the results were displayed as UV/pI maps.</p><p><b>CONCLUSION</b>This approach for fractionating the mouse liver cell plasma membrane protein study provides the foundation for further studies on the functions of plasma membrane proteins and differential proteome of diseases.</p>

Expression and localization of endogenous C-reactive protein in THP-1 monocytes and LO2 hepatocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the expression and localization of endogenous C-reactive protein (CRP) in cells from different tissues under different conditions.</p><p><b>METHODS</b>Macrophages differentiated from THP-1 monocytes with phorbol ester (PMA) induction and human LO2 hepatocytes were stimulated with lipopolysaccharide (LPS). The culture supernatant of the LPS-stimulated THP-1 cells was collected and added into LO2 cell culture, and after incubation, the cells were lysed to extract the proteins for SDS-PAGE and Western blotting. The stimulated cells were also examined immunocytochemically for CRP expression.</p><p><b>RESULTS</b>Western blotting detected CRP in both of the unstimulated cell lysates, but in neither of the two cell supernatants. After LPS stimulation, CRP expression was significantly increased in the cell lysate of THP-1 cells with also a small amount present in the supernatant, but CRP expression and release in the LO2 cells showed no significant variation. Treatment of the LO2 cells with the culture supernatant of LPS-stimulated THP-1 cells resulted in positivity of CRP in the cell lysate and the culture supernatant. Immunocytochemistry identified CRP expression throughout the THP-1 cell body (most obvious in the nuclei), which increased after LPS stimulation. In LO2 hepatocytes, CRP expression was found only outside the nuclei and increased after stimulation with the culture supernatant of LPS-treated THP-1 cells, especially obvious around the membrane.</p><p><b>CONCLUSION</b>CRP can not be up-regulated directly by LPS treatment in LO2 cells, but can be induced by certain cytokines (IL-6) secreted from LPS-stimulated THP-1 cells. The localization of CRP represents the characteristics of secreted protein in LO2 cells, but in THP-1 cells, CRP is found mainly in the cell nuclei.</p>