| Object Over the last decade, evidence has accumulated for organization of the cell surface into lipid-based mocrodomains called lipid raft, which is heterogeneous and composed of structurally and functionally distict islands of the plasma membrane. Based on the special flask-shaped invaginations of the plasma membrane and biochemical marker of caveolae-caveolin, caveolae is the best characterized type of the lipid rafts. These rafts function as platforms for the attachment of proteins when membranes interact as a acceptor and ligand during signal transduction. It has been found that a wide range of pathogens including bacteria viruses, bacterial toxin and prion have evolved mechanisms of invading host cells in order to survive and replicate. This has been the important study field in microbiology. In order to study the function of lipid rafts, the premier work is to purity or extract and identity them. We have establish a method using non-ionic detergent for extracting lipid rafts. Moreover, we find that the infection of HPIV-3 and NDV on MDCK invole the role of lipid raft or caveolae. The new findings help us establish a new model for further studying the common feature of caveolae or lipid raft-mediated endocytic processes and the virous infection through caveolae or lipid rafts also for finding new targets for antiviral therapy.Method1,OptiPrep density fractionation of MDCKCarry out all operations at 0-4℃. MDCK were grown to confluence on tissue culture bottle. The control group was treated with 15 mMmethyl-β-cyclodextrin .The following processes were the same with the experiment group. The number of the cells of each group was about 2X108. Pelleted and resuspensed the cells in breaking buffer. Homogenized the cells with Dounce homogenizer and left them on ice for 30min. Then centrifuged the homogenate to remove the cellular nucleus and membrane fragments and added 1ml supernatant to 2ml OptiPrep, then put them to the bottom of the tube and overlayed with 2ml each 30%,25%,5% OptiPrep. Centrifuged at 100 000g for 5h at 4℃. Collected the samples from the top to the bottom of the centrifuge tube, and 9 parts in all. Every part was analyzed by Western blotting.2,Preliminary research on lipid rafts mediated HPIV-3 and NDV into MDCK(1)Using embryonated eggs to multiply the two viruses, then tested the viruses'valency and TCID50. The valency of the HPIV-3 was 1:640, and NDV was 1: 1280.The TCID50 of the HPIV-3 was 10-4.2/0.1ml, and the TCID50 of the NDV was 10-5.3/0.1ml.(2)In vitro viral invasion assays-----using the CPE as an index The cells grown on 24-well plates were treated with 5mmol/L,10 mmol/Land 15 mmol/L methyl-β-cyclodextrin respectively, 30min,37℃. Then infected the cells with 100TCID50/0.1ml HPIV-3 or NDV for 1.5h, and incubated for 4d. Observed the infectious cells which were treated with different concentrations day by day. Calculated the percentage of the infectious cells treated by methyl-β-cyclodextrin in statistical method.(3) In vitro viral invasion assays-----using the virus plaque as an index Prepare 0.75% agar in non-phenol RPMI-1640 and incubated in 50℃water bath. The cells grown on 24-well plates were treated with 5mmol/L,10 mmol/L,15 mmol/L methyl-β-cyclodextrin respectively 30min,37℃. Infected the cells with 10-5 HPIV-3 and 10-7 NDV for 1.5h and incubated for 3d. The 4th day put another agar on the first layer. Incubated cells lucifugally several hours or overnight. Calculated the quantity of plaques under inverted microscope,and analyzed in statistical method.(4) Carry on morphologic observation of the infected or uninfected cells with TEM.Results1. According to ultracentrifugation and wenstern blot, lipid rafts located at 25%-30% interface, and 5-7th tubes of the samples. The molecular weight of caveolin-1 was 24KD. However, the 5-7th tubes of the control group didn't find the caveolin-1.2. The infection of the two viruses could be inhibition by methyl-β-cyclodextrin, and the effect depended on the dose.3.Inhibition effect of 10 and 15 mmol/L methyl-β-cyclodextrin was significant by statistical analysis(p<0.01). Though it seemed that NDV infected the cells relaying on lipid rafts more than that of HPIV-3 by number, it was not significant by statistical analysis.4. There were many caveolae of different shapes under TEM. After NDV infecting the cells 30min the virus attached a caveolae-like pit on the surface of the cell. At the same a virus was coated in a vesicle in the cytoplasm.Conclusion We purified the lipid raft in mammalian cells successfully by density gradient centrifugation and identified it by western blot,Which was useful for doing research on the interaction of virus and lipid rafts in the future. Meanwhile, we proved that caveolae mediated the NDV into MDCK primarily. And HPIV-3 infected MDCK relied on lipid rafts.
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