Gene Expression Profiling Analysis Of Mouse Brain Microvascular Endothelial Cells Incubated With Glycuronoxylomannan Of Cryptococcus Neoformans

BackgroundsEach year up to 1 million cases of cryptococcus meningitis (CM) newly occur Each year about 620000 people died of the disease. Cryptococcosis is becoming more and more attention by people. C. neoformans as an important medical fungi is characterized by a polysaccharide capsule. It's traditionally thought that there are three main components of C.neoformans'extracellular polysaccharide, including glucuronoxylonmannan (GXM), galactoxylomannan (GalXM) and mannoproteins (MP). In recent years, owing to the application of serology technology, sugar composition analysis and nuclear magnetic resonance (NMR) technology, knowledge of the C. neoformans'capsular polysaccharide greatly deepened. GXM leads to different antigenicity between strains. It has been divided into 5 kinds of serotype——type A?B?C?D and AD. GXM is the main ingredient of capslar accounted for about 88% of the capsule's total mass. It is generally believed the toxic effects of capsular polysaccharide mainly in resistancing to phagocytosis and affecting the host's immune. The in vitro experiment has relatively sufficient evidence, but still do not know which part of it is more toxic. The distincive characteristic of cryptococcosis is that it's strongly addicted to central. More than 90% of cryptococcosis attrack the central nervous system, but the molecular mechanism of it is still unclear.Molecular mechanism of how C. neoformans crossing the blood-brain barrier has always been a research hotspot. The blood-brain barrier is mainly composed of cerebrovascular endothelial cells, astrocytes and pericytes. The three together form the reticular structure of tight junction. It is effectively isolating blood and cerebrospinal fluid and controling the material's exchange. Among three of them, the cerebrovascular endothelial cells play the most important role in maintaining blood-brain barrier function. Different from the endothelial cells of other tissues, cerebrovascular endothelial cells are in weak endocytosis, expressing specific ion and peptide transporters. And the tight junctions between cerebrovascular cells significantly reduce the permeability of small molecules. Some scholars have found that cerebrovascular endothelial cell function had changed after co-culturing with C. neoformans. This may be associated with cryptococcus hit central system capability. This study preliminary explored effects of GXM which is the main composition of capsule on brain microvascular endothelial cells.ObjectsTo explore whether cryptococcal capsular polysaccharide GXM can influence the gene expression of cerebrovascular endothelial cells and providing clues for the further study of the molecular mechanism about Cryptococcus' addiction to the central system.Methods1. The Mouse 12 x 135K Gene Expression Array manufactured by Roche NimbleGen was used to identify the differentially expressed genes of bEnd.3 cells after co-culturing with GXM in different levels. TopGO method is used to analyze the differentially expressed genes and infer gene function of these differences. We analyze differences in gene function by the GO classification and find information from the literature data.2. The changes of some important genes were found and then validated by RT-PCR.Results1. After comparison, we got 698 differentially expressed genes,including 402 up-regulated and 296 down-regulated in the GXM(90?g/mL) group and 985 differentially genes, including421 up-regulated and 564 down-regulated in the GXM (180?g/mL) group. Cryptococcal capsular polysaccharide GXM can regulate vascular endothelial cell gene expression. Genes relate to tight junction, cell transduction, cytoskeleton, metabolism of cell membrane componentsetc have been impacted.2. Considering literature and GO analysis results, we selected gene PIK3C2G and gene ADAMDEC1 and validated their expression levels by RT-PCR. Both genes are up-regulated and positively related to the GXM concentraio. The results were in line with the microarray data.ConclusionsCryptococcal capsular polysaccharide GXM can influence the gene expression of cerebrovascular endothelial cells. Increase in genetic PIK3C2G, ADAMDEC1 may relate to the ability of cryptococcus invaded blood brain barrier.