| Objective:Endothelial progenitor cells (EPC) are vascular endothelial cell precursor cells and it plays an important role in post-ischemic angiogenesis. This study was to establish an in-vitro culture system of endothelial progenitor cells from cord blood and the intervention of melatonin on endothelial progenitor cells; to explore the optimal protocols and conditions of SELDI protein chip for screening protein expressions of cell protein samples of EPC after intervention of melatonin; and to isolate differential proteins by Tricine-SDS-PAGE, to analyze and identify differential expression proteins by FT-MS. So as to explore the role of melatonin invention on EPCs and its mechanism, to set up the research system from the cell culture and drug intervention to proteomics analysis methodology, and to provide insights for the regulation of angiogenesis and tumor suppressor.Methods:Using density gradient centrifugation to obtain MNs, and then culture them in the system developed by our lab. In this study, immunocytochemistry and flow cytometric analysis of CD144,CD146,vWF,CD34 and CD133 were performed. Culture the cells of 7d in the medium in gradient concentration of melatonin(1μM,5μM, 10μM) for 24h.Obtain the cells, proteins were extracted from cell samples by ultrasonic disruption, U9 cell disruption buffer and cell disruption buffer established in our lab, respectively. The concentrations of proteins were detected by BCA method, then protein samples were applied directly to the ProteinChips arrays using WCX magnetic bead and the bioprocessor, respectively, and protein samples were captured by Chips of WCX2, SAX2, IMAC-Cu and H50, respectively, their results were compared, and detailed protein expression differences were detected using the chip of WCX2.To isolate the proteins by Tricine-SDS-PAGE, and to analyze and identify the differential expression proteins at 53kDa by FT-MS according to the results of SELDI.Results:The first five days were latent period, cells became adherent, but the number of cells did not increase substantially. At the 6th day, the number of cells came up to 287+45(the average number under 10X10 visual field),and at the 9th day went to 282+46 (P>0.05). After being cultured for 12 days, the cells came into logarithmic phase,and the number of cells was 805.33±66.61 (P<0.05),and continued to increase to 1115±182 (P<0.05) at day 19. Apoptosis took place at day 23, the number of cells decreased to 265+615 (P<0.05). Immunocytochemistric analysis indicated that the cells were weakly positive for vWF, CD 144 and CD 146, and the percentage of CD34 in cobblestone-shaped cells was 88.98%+5.15%(P< 0.05), which of CD133 was 1.20%±1.44%(P<0.05), and that of VEGFR2 was 41.83%±3.23% (P<0.05) with flow cytometric analysis.24h after drug intervention, under optical microscope, low concentrations (1μM) of melatonin can promote cell growth; high concentration (10μM) of melatonin has the effect of inhibition and apoptosis. The concentrations of proteins isolated from cells using the methods mentioned above are:0.25±0.034μg/μL, 0.6±0.06μg/μL,1.02±0.077μg/μL, respectively; The method of Biochip Processor is simple, which requires less sample size and time; There is a good relationship between the protein peaks and protein concentrations in SELDI protein spectra; There are differences in the type of proteins captured by chips of WCX2, SAX2, H50, IMAC-Cu; In the limit of 1000-300 OOODa,87 protein expression differential peaks were detected by WCX2 chip, amount of which,17 show a tendency variation. The target proteins can be obtained by Tricine-SDS-PAGE, and analyzed and identified by FT-MS, the differential expression protein of 53kDa is a-tubulin.Conclusions:The method established by our lab for EPCs culture is effective. Under Optical microscope observation, EPCs are promoted by melatonin in low concentration, and are inhibited in proliferation by melatonin in high concentration. Comparing the methods of extracting proteins:self-prepared cell disruption buffer provides the highest protein concentration,which is better for further test; Bio-Chip Processor does better in application of sample; Not only could SELDI detect protein expression differences it can also reflect a relationship between the protein concentration and the peak value; Selecting an appropriate chip or combining use of different chips to detect the protein expression differences leads to more satisfactory results. We get 17 tendency differential expression peaks screened by SELDI. By Tricine-SDS-PAGE, we successfully isolated proteins, which was to be analyzed and identified by FT-MS. One of the tendency differential expression proteins at 53kDa was identified as a-tutulin. |
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