The Expression Of CD44 In Murine Model Of Cryptococcal Meningitis

Background:Cryptococcus neoformans is pathogenic fungus which do seriously harm to human health. Cryptococcosis incidence is increasing year by year,90% of which violate the central nervous system, causing fatal meningoencephalitis. How Cryptococcus neoformans violate the central nervous system and the therapy of meningoencephalitis are highly valued.The premise of the cryptococcal central infection is that Cryptococcus neoformans must pass through the blood-brain barrier. At present, domestic and foreign scholars have done a lot of research around the interactions of Cryptococcus neoformans with cerebral vascular endothelial cell, discovered the Cryptococcus neoformans pass through the cerebral vascular endothelial cells by means of endocytosis and exocytosis.The latest study shows that the hyaluronic acid (HA) in Cryptococcus neoformans capsular is interacted with CD44 of the host which promote cryptococcal invasion of the host cell and reorganization of the cytoskeleton during Cryptococcus neoformans infected the brain vascular endothelial cells, which is also benefit that Cryptococcus neoformans pass across the cerebral vascular endothelial cells. CD44 play an important role in the course of cryptococcal infection.The study suggested CD44 is extremely broad distribution as an cell surface transmembrane glycoprotein and belongs to unclassified adhesion molecules. CD44 is closely related to the progress and transformation of many diseases, even becomes a target for treatment of many diseases as a cell surface adhesion receptors.At present, that domestic and foreign scholars study the mechanism of cryptococcal infection of the central nervous system passing through the blood-brain barrier is mainly concentrated on the cellular level, the researches are a few in animal models. The research group has successfully established a murine model of Cryptococcal meningitis. On this basis, the researches have been carried out on the pathogenesis of cryptococcal meningitis, and suggested the water content of the brain tissue have changes in animal models of Cryptococcal infection of the central nervous system,and that AQP4 protein also plays an important role in the regulation of cryptococcal meningitis in brain-liquid equilibrium of water, which build a good foundation in order to further study and explore the pathogenesis of Cryptococcal meningitis, We intend to study the changes of CD44 expression in animal models of cryptococcal meningitis,clarify CD44 is an important protein which be relation to is cryptococcal meningitis, we will study the interaction of Cryptococcus neoformans with the host at the molecular level by modern molecular biology techniques which is already very mature. We will detect expression of CD44 in murine model of cryptococcal meningitis by means of RT-PCR technique, Western-blotting technique and immunohistochemical technique and clarify CD44 plays an important role in the incidence of cryptococcal meningitis and we will research the mechanism of it,which may help to find a new direction in prevention and treatment of cryptococcal meningitis.Objective:1.The changes of CD44 expression of cryptococcal meningitis in murine brain were measured by RT-PCR technique, Western blotting and immunohistochemistry.2.Clarify CD44 is relate to cryptococcal meningitis and explore the possible pathogenesis of cryptococcal meningitis.Method:Part 1:1.The tissue samples of Cryptococcal meningitis in murine brain were removed from the-70℃refrigerator spar.2.The tissue sample in each group was transferred to 12×55mmm test tube, homogenized, was treatmented isopropyl alcohol,75% ethanol,20ulDEPC water, RNA of the organization. Were extracted3. The RNA samples diluted were reversely transcribed into RT-cDNA by adding MMLV 1ul, oligo (dT0.5ul, dNTPs 0.5ul,5×reverse transcriptase buffer 4ul, DEPC treated water lOul reaction.3.The cDNA 2ul prepared was treatmented by SYBRGreen Mix 32.5ul, the upstream primer F 0.5ul, the downstream primer R 0.5ul, ddH2O water 14.5ul, for PCR amplification.Part 2:1.The tissue samples Cryptococcal meningitis in murine brain were removed from the-70℃refrigerator spare.2.The samples were homogenized under ice-bath with13200rpm centrifuged and the supernatant, were reserved under-80℃condition. 3.The samples in a 10-fold dilution, calculated total size of the sample in 20μl volume, the volume of each sample was 40μg.4.The samples were packed after SDS-PAGE electrophoresis and transfer membrane and closed, then added the antibody and anti-anti-antibody, after coloration with ECL. put it into fixer, detected CD44 expression in the brain.Part 3:1.the tissue specimens cryptococcal meningitis in murine brain and in normal murine brain tissue were removed from the-70℃refrigerator spare.2.1mmunohistochemical staining:The specimens of each group were produced paraffin removal, then routine dewaxing and dehydration, added the antibody and anti-antibody as well as the coloration liquid.3.The results of slice were observed under microscopicand and taked the photographs.Results:Part 1:Expression of CD44 mRNA in murine model of cryptococcal meningitis compared with the control group,48h,4d,7d group were significantly different at 12h. (P<0.05), Expression of CD44 mRNA in murine brain compared with the control group had not significant change at the other time points.Part 2:The protein expression of CD44 brain in murine model of cryptococcal meningitis compared with the control group had not significant change at each time point, (p> 0.05), there is no statistical significancePart 3:The results show that CD44 were uniformly distributed in the brain cell membrane in the normal group, control group, the distribution of CD44 in murine brain had not significant change at 6h,12h. Cryptococcus neoformans induced CD44 in the brain to migrate to one side of the brain at 24h, the distribution of CD44 took place significant changes in the test group at 48h,72h, CD44 of the brain were largely distributed nearby meningeal side, while the distribution of CD44 was decreased significantly in the side of parenchyma. The same result is that the distribution of CD44 increased at one side, but the other decreased, which was also observed around the lesion.Conclusion:1.Expression of CD44 mRNA in murine model of cryptococcal meningitis was transiently increased. But protein expression of CD44 compared with the control group had not significant changes.2.CD44 migrated to the one side of the brain and aggregated nearby meninges after Cryptococcus neoformans invated the murine brain. the unbalanced distribution of CD44 were also observed around Lesions. The result Shows that the incidence of cryptococcal meningitis are closely related to CD44 molecules.