Study The Influence Of Expression Of IL-15 By RNA Interference On An Asthma Mouse Model

IL15comes from various tissues. Most of tissues and cells could expressIL15,and monocaryon/macrophage is main resource. The main functional ofmonocaryon/macrophage is that could present the antigenic message to T cell,and promoted it secreted cytokines and mediator of inflammation precurosor.Well, IL15could induce T cell expressed cykines and theirs receptor,participate inflammation which make T cell activated and generation earlier,promote B cell secreted IG, enhanced NK cell cytotoxity and producedcytokines, modulated monocaryon/macrophage produce anteroinflammationcytokine. It is cleared that IL15could induce neutrophil expressed NFκB,and inhibited apoptosis of neutrophil and leukomonocyte. During inflammation,there had a certain degree neutrophilic leukocytosis, and released leukotriene,prostaglandin and platelet activating factor, participated moludatedinflammation of respiratory passage, but It is not identify the relationshipbetween IL15and asthma which is still on the stage of exploration.RNAi is a new gene blockage technology by introducted doublestrandedRNA which is high performance, definite, satefy and operated. It could highefficiency and specific to block the special gene expression, promotedegradation of target of mRNA, and lure the absence of expression of the definite gene, got the aim of post transcriptional gene silencing. We utilizedRNA interference to research gene function, signal transduction, relationship ofeach other and antivirus, antineoplaston. Although it was applicated in multudefields and clinical research, it is still on the stage of vitro cell line research inrespiratory system disease.Our aim is to discuse the effect of IL15on asthma and the influence afterinterfered it. In our experiment we design 21nt base sequence according toprinciple of design of RNAi. To utilize pSIRENRetroQZsGreenconstructionrecombinant plasmid of pSIRENRetroQZsGreenIL15siRNA(pSRZsiIL15)which can block the expression of IL15mRNA in the lung.,and discuss the effection of IL15siRNAin the lung tissue on an asthmamouse model.Method1. To establish the sequence of IL15siRNAthat is only one, amply andsequence identity which has synthetical expression plasmid pSRZsiIL15.2. To establish an asthma mouse model in accordance with reference.Oberserve the change of ethology on mouse, to determine the level ofIL4,INFγand IgE in sera, the change of eosinophils and neutrophil cellcounts in BALF, and the change of histopathology.3. Isolated culture spleen T lymphocyte of asthma mouse, and delivereddifference ration of pSRZsiIL15and Lipofectamine 2000 to T lymphocyte.We collected the cells after 72 hours, observed the results of transfected of Tlymphocyte by fluorescent microscope, adopted RTPCRto test the expressionof IL4mRNAand IL15mRNA,used ELISA to determine the change ofcytokines in lymphocyte culture supernatant such as IL4and IFNγ,compared the effects of different ration of pSRZsiIL15and Lipofectamine 2000.4. Based on the asthma mouse model, the mice were divided into normalcontrol group, asthma control group, hormonal therepy group, vetor group andRNAi group. To establish an asthma mouse model in accordance withreference. Among those: 3days before sensitized and 1 day before stimulatedwas delivered the ration of pSIRENRetroQZsGreenIL15siRNAandLipofectamine 2000 1/2. Killed the mice after transfected 24,48 and 72 hourspartly. At meantime, randomly get a part of fresh lung tissue and made frozensection to observe the respiratory transfected by confocal microscopy andIL15siRNAeffected on correlation factors in lung tissue. We determined thelevel of IL4,IL15,IFNγ,IgE in each group, detected the expression ofIL4mRNAand IL15mRNAby RTPCR,determined the expression of IL15in lung tissue by immunohistochemical method, observed the changes ofhistopathology in lung tissue and change of eosinophils and neutrophil cellscounts in BALF.Results1.Identity construced the pSRZsiIL15was our need by amplification andsequencing.2. We observed the ethology change of mouse, the level of IL4on asthmamouse model was higer than that of normal control group, the level of INFγwas lower than that of normal control group, at the same time, the level of IgEalso increased obviously, the eosinophils cell counts in BALF higher comparedto normal control group. Change of lung histopathology was typical that loss ofbronchial mucosa epithelium and was hurt. Eosinophils, leukomonocyte andlymphomonocyte infiltrated, mucous formed. That established an mouse model successfully.3. Interfered on T cell on asthma mouse model in vitro①After 48 hours observed that there had some green fluorescence proteinon asthma mouse T cell that indicate the succed in transfecting.②After transfected to T cell, the expression of IL15is significant lowerin RNAi group than that of in asthma control group. It suggested thatpSRZsiIL15was transfected into T cell successfully, and specific blocked thehe expression of IL15got the gene silencing.③The level of IL4was lower in RNAi group than that of asthma controlgroup in lymphocyte culture supernatant, so does in the expression of IL4mRNA. In lymphocyte culture supernatant, no change on level of IFNγof Th1cytokine which indicated that IL15has no regulated or micro modulated.④It could get the best specificity gene silence when the ration ofpSRZsiIL15andLipofectamine 2000 is 1:2 which had advangtage of theretion of 1/1 and 1/3.4. pSRZsiIL15Lipinterfered on asthma mouse model in vivo afterrespiratory transfection.①After transfected 24 hours, we could observed that there has some greenfluorescent material on bronchial mucosa epithelialis and around lung tissueunder the confocal microscopy. And that suggested that respiratory transfectedsuccesfuly.②In asthma group eosinophils and neutrophils cell counts, as well as thelevel of IL15was significant heighten. After delivered pSRZsiIL15,it coulddescend eosinophils and neutrophils cell counts in RNAi group, especially aftertransfected 4872hours which could get a stable level, and the level of IL15is positive correlation with eosinophils and neutrophils cell counts.③The level of IgE in RNAi group is lower than asthma control group. Atmeantime, that of 48 hours and 72 hours transgected was significant lower than24 hours transfected. In the experiment the change of 48 hours transfected wassimilar to that of 72 hours, that was pSRZsiIL15Lipeffected on the level ofIgE got a stable level after respiratory transfected in vivo.④There is no change on levels of INFγin sera in RNAi group. level ofIL4in RNAi group is lower than asthma control group. At meantime, that of48 hours and 72 hours transgected was significant lower than 24 hourstransfected. In the experiment the change of 48 hours transfected was similar tothat 72 hours, that was pSRZsiIL15Lipeffected on the level of IL4got astable level after respiratory transfected in vivo.⑤Level of IL15in RNAi group is lower than asthma control group. Atmeantime, that of 48 hours and 72 hours transfected was significant lower than24 hours transfected. In the experiment the change of 48 hours transfected wassimilar to that 72 hours, that was pSRZsiIL15Lipeffected on IL15got astable gene silencing level after respiratory transfected in vivo.⑥The positive staining cell counts was lower than asthma control group,The positive staining cell counts was similar to normal control group aftertransfected 48 hours and 72 hours. pSRZsiIL15Lipcould lessen eosinophilsleak out in BALF and inflammation cell infiltrating in lung tissue.⑦pSRZsiIL15Lipcould cut down the expression of IL4mRNAin lungtissue after airway drop in it. But there was no difference in every time intervalthat paralleled to the change of sera. At meantime, pSRZsiIL15Lipcould cutdown the expression of IL15mRNAin lung tissue especially after transfected 48 hours and 72 hours. That could got a stable the gene silencing level aftertranfected 4872hours.Conclusion1 Succeeded in preparation in asthma mouse model by OVA sensitizedand stimulated.2 Succeeded in identification on amplification and sequencing ofpSRZsiIL15.3 It could get the best specificity gene silence when the ration ofpSRZsiIL15andLipofectamine 2000 is 1:24 During the course of asthma IL15main effects is promoted theflammation.5 After respiratory transfection, pSRZsiIL15Lipcould get a stable thegene silencing level after tranfected 4872hours.6 pSRZsiIL15Lipcould lessen eosinophils inflammation and correctthe disbalance of Th1/Th2 on asthma mouse in lung tissue.7 Thus airway inhaled pSRZsiIL15Lipcould be one of effectivemethods to cure asthma.