The Study Of The Protein Of HUVEC Coculturing With Cryptococcus Neoformans By Proteome Screening

Cryptococcus neoformans is one of the most important mandical fungus, which may infect all the organs of the body with the increasing morbidity of immunosuppressive states such as acquired immunodeficiency syndrome (AIDS) and after bone marrow transplantation (BMT). Especially the meningitis or meningoencephalitis caused by Cryptococcus neoformans striking the Central Nervous System is a lifethreatening infection with huge mortality. Althougt the mechanism of the infection remains to know, some studies show that Cryptococcus neoformans may be inhaled as spore, crossing the air-blood barrier even the blood-brain barrier into the CNS. As vascular endothelial cell is the most important part of the barrier, the study of Cryptococcus neoformans invading vascular endothelial cell may elucidate the mechanism of the infection. The invading is a very complicated network regulation process. Lots of genes and proteins are involved in. Studying on a single gene or protein can not completely reveal the mechanism. In consideration of this, gene chips were widely used in similar research. Through this technology, we can easily get the profile of gene alteration between normal endothelial cell and invaded endothelial cell. But there are many conflicting consequences between the abundance of mRNA and proteins level. Because genes performed their functions through proteins, studying on the protein profile is very necessary and important in the research. The high-throughput proteomic technology will conduce to comprehensively understand of the process of Cryptococcus neoformans passing across the barrier.Objective and Methods:We selected human umbilical vascular epithelial cell (HUVEC) as the cell model. Cryptococcus neoformans wild strain B3501,capsule depletion strain Cap60 and melanin depletion strain Mel- are used as the test fungi.HUVEC was incubated with these fungi in 30min,1h,2h,3h,4h,5h separately.The activity of the cells was measured by CCK-8 and the apoptosis ratio of HUVEC was detected by flow cytometry(FCM).We select the appropriate test fungus in these three fungi and incubating time according to the results.In this study, we used 2-D protein electrophoresis technology to obtain protein alteration profile between normal HUVEC and the cell incubated with Cryptococcus neoformans. We selected some proteins which may be involved in the invading process, then observe its expression level in different stage of invading. We try to find some proteins that may play an important role in the process of invading, and we observed the contribution of these proteins in the different stage of invading.The different expression proteins between normal and the invaded cells were obtained after 2-D electrophoresis and been identified by MALDI-TOF MS technology. Two proteins (Calpactin I light chain and Peroxiredoxin 1) were selected as research targets from these different expression proteins. Fluorescence quantitative PCR was used to observe the expression level of these two proteins in the different stage of invading.Results:The results of CCK-8 were as following: the inhibition ratioes were 40%,40%,38%,30%,24%and 20% after coculturing HUVEC with the strain B3501 at 30min ,1 hour,2 hours, 3 hours, 4 hours and 5 hours respectively. The results of strain Cap60 were 29%,79%,43%,40%,30% and 21%respectively. The results of strain Mel- were 35%,48%,59%,77%,27% and 18% respectively.The results of FCM were as following: the apoptosis ratioes were 5.0%, 4.8%, 2. 9%and 2. 6% after coculturing HUVEC with strain B3501 at 30min,1 hour , 2 hours and 3 hours. The results of strain Cap60 were 28.7%, 42.9%, 13.1%and 8.1% respectively. The results of strain Mel- were 13.0 % , 18.7 % , 19.3 % and 24.2 % respectively. According to the results,we picked Cryptococcus neoformans wild strain B3501 as the test fungus and 4 hours as the appropriate incubating time.The sliver stained gels of 2-D electrophoresis were scanned at 256 grayscale and 8 bits degree. After analyzed by ImageMaster 2D Elite 3.10, 1025±38 spots in normal HUVEC cells and 921±33 spots in test cells were detected. Normalized gel of HUVEC cells was selected as reference gel. After Spots matching with other gels, the unstably developed difference expression spots between the three times electrophoresis was precluded from analysis. There are 82.89% and 79.58% of the spots could be matched for reference and test gels. Difference analysis showed that 19 credible protein spots were differentially expressed between reference and test cells. In these spots, five protein spots were only expressed in reference cells, three were only expressed in test cells. In other 11 spots, one spot was higher expressed and ten spots were lower expressed in test cells.Using MALDI-TOF MS technology, fifteen spots were identified by PMF, including enzymes, cell signal proteins, b n inding proteins, metabolism related proteins.FQ-PCR results: (1) The mRNA quantities of S100A10 was increased gradually in HUVEC, HUVEC1h, HUVEC2h, HUVEC4hcells, and the difference was statistically significant (P<0.01) except that between HUVEC and HUVEC1h cells (P>0.05).(2) The mRNA quantity of PrxⅠwas decreased with the increasing incubating time. There was significant difference in mRNA quantity among each groups (P<0.01).Discussing:S100A10 , Calpactin I light chain, p11 is one of the S100 family, the largest group of EF-hand signalling proteins in humans. The S100 proteins are small acidic proteins (10–12 kDa) comprise at least 25 members which found exclusively in vertebrates. Each S100 protein has generally been shown to have a preference for expression in one particular tissue or cell type and have 25–65% identity at the amino acid level. Binding with calcium, the S100 family are proposed to have intracellular and extracellular roles in the regulation of many diverse processes such as protein phosphorylation, cell growth and motility, cell-cycle regulation, transcription, differentiation and cell survival . S100A10 can form a heterotetramer complex with its target protain annexin A2,another member of the S100 family. Within the cell, The heterotetramer with annexin A2 is localized at the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton.The complex have great effect on the cytoskeleton which support endocytosis, exocytosis, direction growth,adhesion, migration of the cells. Xiaoxuan Fan found annexin bridging serve as both ligand and receptor in promoting phagocytosis.In the research, we found the expression of S100A10 was increased gradually and the difference was statistically significant ,which suggest that the endocytosis or exocytosis of HUVEC may play a important role in the process of Cryptococcus neoformans passing across the endothelium barrier.PrxⅠ, (1-Cys peroxiredoxin, 1-cysPrx) is a member of novel antioxidant protein family able to reduce phospholipid hydroperoxides in vitro by using glutathione as a reductant. Oxidant-mediated injury to cells likely involves peroxidation of cell membrane-associated phospholipids. Thus, the ability to reduce phospholipid hydroperoxides may be of primary importance to protect cells against oxidative stress.PrxⅠis an inducible protein that it can be up-regulated by oxidative stress, and the increment of PrxⅠis correlated with the oxidative stress. Yan Wang found adenovirus-mediated transfer of the 1-cys peroxiredoxin gene to mouse lung protects against hyperoxic injury.Otherwise, PrxⅠmay promote generation and proliferation of cell. The upregulation of 1-cysPrx expression may be beneficial to the corneal wound-healing process. Our research found the expression of PrxⅠwas decreased with the increasing incubating time suggested that the tolerance to oxidative stress of HUVEC may be decreased in the same time. All of these were demonstrated that apoptosis of HUVEC induced by PrxⅠmay plays an important role in the invading process.