The Effect Of New Gene VSTMl-v2on Klebsiella Pneumonia Infections

Objective:With the rapid progress of Genomics and Proteomics,the importantresponsbility of life science facing with is how to translate the information of genome sequence into the information of genome fuction, and understand moleculemechanism of vital movement,improve hunman health. Based on the whole genome they selected the coding genes of the potential cytokines according to the expression and structural features. Human VSTM1（V-set and transmembrane domain containing1） is such a gene,which is located on chromosome19q13.42, without any functional report.VSTM1has five forms of expression, VSTM1-v1andVSTM1-v2are the main forms.And VSTM1-v2is a glycoprotein of classical secretion,which is mainly expressed in immune tissues and cells. Our department has cooperated with Peking University in research and development of new genes,and screeninged of VSTM-1which is associated with inflammation by nested PCR. Klebsiella pneumoniae is one of the most prevalent causal pathogen in hopital.When airframe immunity is low, it can cause a variety of infections. But withthe wide application of cephalosporin antibiotics, clinical isolates of Klebsiella pneumoniae are in the increasingly serious multidrug resistant to antimicrobial agents,which becomes the control difficulty now. In this paper,through the establishment of the model of the Klebsiella pneumoniae pneumonia of C57BL/6mice, weresearched on the effect of VSTM1-v2on inflammation, and its in-depth study has important theoretical significance and potential clinical value.Methods: Pathogenic strains: Klebsiella pneumoniae, isolated in the patient’ssputum of K. Pneumoniae infections. After the bateria was cultured on the the plate ofLB, we choose single colony and put it into the LB cultivation. According to therelation between the growth of bacteria and OD values, we have dedicated that thebacteria is the most active which has been cultured for7to11hours. Animal Model: After the Klebsiella pneumoniae cultured for7to11hours, we dissolved a singlecolony in the sterile saline and adjust the concerntration at107cfu/ml and then inject20ul into the mouse trachea to induce pneumonia. Animal treatment: Total88C57BL/6J mice were randomly divided into an VSTM1-v2-treated group (40mice), asaline control group (40mice),and a normal group(8mice). The normal group werewithout any treatment.Each mice of the VSTM1-v2-treated group was injected100μlVSTM1-v2(1ng/μl) through the vena caudalis, whereas each mice in the saline controlgroup was injected the equal volume of normal saline through the same route. After anhour, in the both two groups tracheotomy was done and injected20μl K. pneumoniae(1.0×107CFU/ml). Detection method: Mice were randomly euthanized and dissected tosuccessively collect the lung tissue and blood serum at the different time point of3h,6h,12h and24h after the injection of K. pneumoni.And then we observed theappearance of lung tissue, lung HE staining and immunohistochemical stainingrespectively, did Real-time quantitative PCR to detect inflammatory cytokine andwestern-blot to check out chemokines GRO(belongs to the intercrine alpha (chemokineCxC) family) protein expression， then we assayed the activity of MPO and iNOS,andcollected blood serum to do cytometric bead array to detect the change of cytokines.Results:We compared VSTM1-v2treated group with saline control group,the results are asfollow:1.In the VSTM1-v2-treated group, pathology report showed that the lungs were redand lung marking was more clear than the saline control group, Klebsiella-inducedpneumonia inflammation was reduced; VSTM1-v2alleviate the acute inflammatoryresponse caused by Klebsiella pneumoniae by inhibiting neutrophil invasion from HEstaining view, immunohistochemical staining showed the decrease of IL-6and TNF-аcytokines.2.The cytokines IL-6,IL-1β, TNF-α, CXCR2and MIP-2mRNA poorlyexpressed,which was detected by Real-time quantitative PCR3.The expression of GRO was reduced by western-blot analysis shown.4.Flow cytometry bead array that the cytokines IL-6, IL-10, IL-12, IFN-γ, TNF-α, andMCP-1of peripheral blood were obviously decreased in the VSTM1-v2-treated group.5. The activity of MPO and iNOS were evidently reduced.Conclusion: In the acute inflammatory reaction caused by Klebsiella pneumoniae,VSTM1-v2can significantly reduce a variety of inflammation related cytokines.