Construction Of SiRNA System Targeted To Mannose Receptor Of Macrophage And Its Effects On Binding Of Oligochitosan In Macrophages

[Background & Objective] Chitin is one of the most abundant biological natrue resources, comprising the horny substance in the exoskeletons of crabs, shrimp, and insects as well as fungi. Chitin as well as its partially deacetylated product-chitosan, have many kinds of physiological functions, including antitumor activity, antimicrobial activity, antifungal activity, immuno-enhangcing effects. However, their high insolubility and high viscosity in neutral aqueous solutions restrict their application. Recent studies on chitosan have attract interest for oligochitosan which is not only water-soluble, non-toxic, biocompatible, but also possess versatile functional properties .Our former experiment observed the binding of fluorescence labeled mannan, one of the ligand of mannose receptor, in RAW264.7 macrophage and found that oligochitosan can competitively inhibit the binding of mannan on macrophage with other ligand of mannose receptor and cut down the binding by 44% .[Experimental procedures]1. The construction of plasmid PGCsi-U6/Neo/DsRed.Design the siRNA with the online software provided by Ambion corporation. Then synthesize DNA oligos which contains siRNA sequences of target gene. Linearize the plasmid with BamH â…  and Hind â…¢ ,and ligate the DNA oligos into the linearizedplasmid, sequencing to confirm the ligation. Transform the plasmid into E.coli DH5α for amplification, then purefy the plasmid and transfect the RAW264.7 cells. We detect the express of RFP by fluorescence microscope and the express of mannose by RT-PCR on the level of mRNA and by flow cytometry on the level of protein.2. The labling of oligochitosan with FITCDissolve oligochitosan in the buffer which contains 0.2 M CH₃COOH and 0.1 MCH₃COONa. Add NaOH until pH 7.7, then add distilled water to obtain 1% oligochitosandilution. Add FITC diluted with methanol into 10 ml oligochitosan dilution and put it onto the rocking bed with thespeed of 150 round per minute to shake for 24 hours at the temperature of 4℃. Centifuge and transfer the clarified supernatant to a bag filter which trapping 1000 MWt, put the bag filter into the container full of PBS, protect from light, reacts for up to 3 days. Cryodesiccate and dilute it with c-PBS. The labeling efficiency is determined by the ratio of OD 495nm and OD 280nm.3. The detection of the intensity of fluorescence by flow cytometryAdd FITC labeled oligochitosan into the culture flask and incubate for certain time. Scrape the cells and move the cells to a 1.5 ml microcentrifuge tube. Centrifuge and move the supernatant, resuspend the cells with c-PBS. Detect the intensity of fluorescence by flow cytometry and analyse the data with the software of Cell Quest.[Results] (1) We successfully constructed siRNA system targeted to mannose receptor of macrophage, plasmid named Mrc1-1, Mrc1-2, Mrc1-3 and another plasmid named Non as a negative control. (2)Plasmid Mrcl-1, Mrcl-2, Mrcl-3 had successfully inhibit the expression of mannose receptor on macrophage cell line RAW264.7. (3)The binding of oligochitosan in macrophage has declined notablely because of the treatment of macrophage with Mrcl-2 plasmid.[Conclusions] Oligochitosan can bind the macrophage through mannose receptor.