The Influence Of The Ligand Of DC-SIGN On Adhesion Capacity Of Dendritic Cells In Patients With Pulmonary Tuberculosis And BCG

ObjectiveDC-SIGN is located at the surface of dendritic cells, it is a immune molecule found in recent years and closely related to tuberculosis. The studies in vitro of healthy people have shown that Mycobacterium tuberculosis can adhere to the DC-SIGN molecule by ManLAM , following the opening of several mechanisms for suppression of anti-tuberculosis host immune response: to suppress the maturation of the immature dendritic cells ; to induct the dendritic cells around that has not been infected into a state of semi-mature; to increase the expression of immunosuppressive factors such as IL-10. In this study,we explore the influence of the ligand named ManLAM of DC-SIGN on adhesion capacity of dendritic cells in patients with pulmonary tuberculosis and BCG ,and further study the role of DC-SIGN molecule in the course of TB occurred .Methods:1. To construct the E. coli - mycobacteria shuttle expression vector containing EGFP. We double digest the plasmids of pEGFP and pHSP70-LA73 with the restriction endonuclease named KpnⅠand XbaⅠto prepare the target gene and the vector fragment, connect them with T4 ligase at room temperature, then translate them into competent E. coli DH5αprepared and screen recombinants on LB solid medium containing 30μg/ml kanamycin.2. To mark BCG with green fluorescent protein.The recombinant plasmids were transformed into competent BCG cells with electroporation instrument (eppendorf), then the positive recombinants were cultured to logarithmic phase. They were heat-induced so that the green fluorescent protein was expressed, thus BCG was marked.3. To study the influence of ManLAM on adhesion capacity of dendritic cells in patients with pulmonary tuberculosis and BCGThe peripheral blood mononuclear cells of the study objects were separated, the dendritic cells were induced to grow , the surface expression of DC-SIGN was detected by flow cytometry. After pre-incubated with the different concentrations of ManLAM, add the marked BCG by the ratio of 5:1 , they were incubated together and then detected the green fluorescence , thus the rate of adhesion was measured.Results1.The strips after digestion are correct , the mycobacterium shuttle plasmid is constructed successfully, named pHSP70-EGFP.2.To observe bacteria smear with inverted fluorescence microscope, the bacteria can be seen strong green fluorescent issued to prove that green fluorescent protein is expressed in BCG, BCG is marked successfully.3.The DC-SIGN expression rate of the surface of peripheral blood dendritic cell in patients with tuberculosis is (86.69±3.66)%, significantly higher than the healthy control group. The adhesion rate of peripheral blood dendritic cell in patients with tuberculosis and BCG marked is (89.84±1.57)%, which continues to drop by increasing the concentrations of ManLAM.Conclusion1.The pHSP70-EGFP shuttle plasmid can be used in the BCG to express green fluorescent protein, thereby BCG is marked.2. The dendritic cells in patients with pulmonary tuberculosis can bind BCG primarily through attaching to ManLAM, that also prove DC-SIGN is the major Mycobacterium tuberculosis receptor on the surface of dendritic cells in patients with pulmonary tuberculosis.