| [Background and objective]Cryptococcus neoformans is the major fungus that can cause clinical Cryptococcosis,especially in immunocompromised patient.The infection starts with inhalation the desiccated yeast cells of Cryptococcus neoformans to the pulmonary alveolus,it cause asymptomatic or mildly infection in immunocompetent human being.Furthermore Cryptococcus neoformans is prominently neurotropic,it can dissemination to the Central nervous system lead to a life treatend cryptococcal meningitis,therefore,immune cells like macrophages plays an important role of whether the Cryptococcus neoformans spread into the central nervous system become a fatal meningitis or not.micro RNA(mi RNA)are non coding RNA compose of 19 to 24 nucleotide,that regulate many of its target gene at a posttranscriptional level.One of them is micro RNA-155,it's participate in regulation of both inflammation and tumour progress.micro RNA-155 is regulate by mediators of inflammation like TNF-a and lipopolysaccharide through innate immune.micro RNA-155 is express in many activated immune cells,including pulmonary alveoli macrophages which is one of the decisive factor of whether Cryptococcus neoformans spread into the central nervous system,and express in other immune cells like T lymphocytes,B-lymphocytes and dendritic cell.It is known that many target gene transcription product of micro RNA-155 including pro-apoptotic and anti-inflammatory proteins,like the Fas-associated death domain protein,Ik B kinase and SOCS-1.The suppressor of cytokine signaling including CIS and SOCS1-7,is a family of immunosuppressive molecule.They all produce by cells and can inhibit the transfer process of cytokines for feedback,it can mediated a lot of kinds of cytokines,and take part in regulate a lot of kinds of inflammatory disease in human.Among them,SOCS-1,also called SSI-1(STAT-induced STATinhibitor-1)can induce by many cytokines,it is an important negative mediator,and it can inhibit cytokines including TNF-?,IL-6 and IFN-?.In the past,studies have found that in the macrophages inflammatory response of lipopolysaccharide and virus induced,can detected up regulate of micro RNA-155.In Cryptococcus neoformans induced inflammatory response of which we concern about,whether can micro RNA-155 affect the suppressor of cytokinesignaling-1 or not,is it affect cytokines like TNF-? and IL-6 are still undefined.In this experiment,we use heat inactivated Cryptococcus neoformans induced mononuclear macrophage RAW264.7,to observe the regulation of mi R-155 aiming SOCS1 and its regulating characteristics,to analysis if there is a mi R-155 target SOCS1 immune regulation mechanism in inflammatory response in Cryptococcus neoformans induced mononuclear macrophage,searching if it take a role in the pathogenesis of Cryptococcus neoformans.Part one: The expression level of mi R-155 and TNF-? ? IL-6 in Cryptococcus neoformans induced macrophagesTo determine whether level of micro RNA-155 of mononuclear macrophage induced by Cryptococcus neoformans is influence the cytokines or not.we use Cryptococcus neoformans(WM148)to incubate with mononuclear macrophage(RAW264.7),gather and test the cells and the supernate in 0?3?6?9 and 12 hours respectively.test micro RNA-155 through real time PCR,and test cytokines TNF-? and IL-6 through ELISA,and observe the change regular pattern.Result:After incubate Cryptococcus neoformans with mononuclear macrophage for 0?3?6?9 and 12 hours respectively,we find that expression of micro RNA-155 is lifting sharp(P<0.05),meanwhile the cytokines TNF-? and IL-6expressing time dependent increase,though it is not so dramatic at the laterperiod.Part two: The gene and protein level of SOCS1 in Cryptococcus neoformans induced macrophagesSOCS1 is a major gene target of micro RNA-155,to clear that if SOCS1 is a regulator in the Cryptococcus neoformans induced mononuclear macrophage,we incubate Cryptococcus neoformans with mononuclear macrophage for 0?3?6?9 and 12 hours respectively and collect the cells to test the gene expression through real time PCR and to test the protein expression through Western blot.Result: After incubate Cryptococcus neoformans with mononuclear macrophage for 0?3?6?9 and 12 hours respectively,we find that gene expression of SOCS1 is markedly increased(P<0.05),but the protein expression is not obvious.Part three: expression of SOCS1 after inhibited the micro RNA-155 of macrophage through transfectionTo further discover whether micro RNA-155 influence its target gene SOCS1 or not when mononuclear macrophage is incubated with Cryptococcus neoformans,we inhibited micro RNA-155 through transfection the si RNA of mi R-155 in RAW264.7,and then incubated RAW264.7 with Cryptococcus neoformans again,and collect the cells in 0?3 and 9 hours to test the gene expression through real time PCR and to test the protein expression through Western blot.Result: after inhibited micro RNA-155,mononuclear macrophage is incubated with Cryptococcus neoformans and collect the cells in 3 and 9 hours,the gene and protein level of SOCS1 are both increase noticeably compare with the 0 hour.[Conclusions] Our research shows that when incubate mononuclear macrophage with Cryptococcus neoformans,it will stimulate the release of cytokines like TNF-? and IL-6,and to trigger the activation of series mechanism of negative feedback.The gene of SOCS1 is activated under the stimulation of cytokines,but we find out that the protein level of SOCS1 did not increased markedly,it means that there is something inhibit the activated SOCS1 gene being transcription in some signaling pathway exist.It is because of this mechanism existing that in the early period of inflammation,it can effectively avoid the negative regulation to be over produce and the macrophage may not be able to function normally enough to resist the pathogenic bacteria.Our experiment inhibited micro RNA-155 gene expression to observe the regulation of micro RNA-155 aiming its target gene SOCS1.we find out that when transfection si RNA to inhibited micro RNA-155 gene expression,then after 3 and 9 hours of incubate with Cryptococcus neoformans,both expression of the gene level and the protein level are rising,that means that it somehow lower the suppression of SOCS1 when inhibiting micro RNA-155.To sum up,our experiment shows that in the early phase of macrophage in flammation when Cryptococcus neoformans was inhalation in the pulmonary alveoli,the cytokines like TNF-a ? IL-6 induces SOCS-1 can cause a negative regulation of inflammation to regulate a suitable inflamation.SOCS-1 is one of the target gene of mi R-155,in the macrophage inflammation induced by Cryptococcus neoformans,SOCS-1 is down regulate by mi R-155,this prevent the negative regulation over regulate in the early phase of inflammation,that can't function a normal resistance to pathogenic bacteria.It is because these factors all links with another that makes the immune response moderate and normal. |
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