The Study Of Single Molecule Force For Gene Polymorphisms Of ICAM-1and Its Ligand Mac-1

Objective: In this study, we constructed4mutations of ICAM-1,whichare GK, GE, RK and RE. The cell adhesion assay had been used to observe theadhesion of the4mutant ICAM-1s and human PBMCs. To further investigatethe anti-inflammatory mechanism of atherosclerosis, we then carried out asingle-molecule study of ICAM-1/Mac-1interaction force microscopy byAFM.Methods:1Construction of recombinant ICAM-1-pEGFP-N1plasmids.HUVEC were isolated by0.1%type I collagenase digestion for15-20minat37℃, and cultured in low serum endothelial cell medium. The total RNAwas extracted from cultured HUVEC. As cDNA of ICAM-1to be a template,apair of PCR primers that one end contains BamHI and the other containsHindIII restriction sites were designed and synthesized.The coding region ofwild type ICAM-1(ICAM-1-GK) was then amplified by RT-PCR using theprimers. As ICAM-1-GK to be the template, site-directed mutagenesis ICAM-1-GE,ICAM-1-RK,ICAM-1-RE were successfully obtained using overlapextension PCR and comfirmed by DNA sequencing. The coding region of4mutations of ICAM-1were inserted into the pEGFP-N1vector to construct therecombinant ICAM-1-pEGFP-N1plasmids.2Establish CHO cell line which constantly express ICAM-1mutations.The recombinant ICAM-1-pEGFP-N1plasmids were transfected intoCHO cells by Effectene Transfection Reagent and G418was added to obtainCHO cell lines which constantly expression ICAM-1mutations. Theexpression level of ICAM-1was determined by flow cytometry and the resultswere show as MFI and percentage of green fluorescent positive cells (%gate).3. Cell adhesion assay. ICAM-1were inoculated in48-well plates, sub-experiment:①.Controlgroup②ICAM-1-GK-GFP-CHO;③. ICAM-1-GE-GFP-CHO;④ICAM-1-RK-GFP-CHO;⑤ICAM-1-RE-GFP-CHO. Human PBMCs were isolated bylymphocyte separation medium and stained with fluorescent wheat germagglutinin (WGA). After washing with PBS to remove excess WGA, thestained PBMCs were added into the ICAM-1constant expression CHO cellsand incubated30minutes at37℃.And then the number of adhensive PBMCswere counted under fluorescence microscope.4Measurement of single-molecule force of ICAM-1/Mac-1.The AFM tip was first cleaned according to standard procedures.Then itwas transferred to a solution of1.0%(v/v) MPTMS in toluene, incubated for2hours at room temperature to be silanized. A polyethylene glycol spacercontaining an amino-reactive group at one end and athiol-reactive group at theother end, was used to link Mac-1to the silanized AFM tips in order to keepthe immobilized Mac-1in a flexible state.Recombinant ICAM-1-pEGFP-N1plasmids were transfected into CHOand COS-7cells respectively. To achieve single-molecule force of ICAM-1/Mac-1, the Mac-1-coated AFM tip was directed to the ICAM-1-expressingcells with the help of the optical and fluorescence imaging of cells with thecombined AFM/fluorescence microscopy system.Under optimized experimentconditions, the AFM tip repeatedly contacted the CHO cell surfaces andrecorded1000force curves for each cell, the results were assessed withstatistics analysis.Results:1Wild type ICAM-1and site-directed mutations-ICAM-1-GE,ICAM-1-RK,ICAM-1-RE were successfully obtained.2DNA sequencing and double restriction enzyme digestion showed successfulconstruction of recombinant ICAM-1-pEGFP-N1plasmids. The transfectedCHO was observed that the green fluorescent protein was located on the cellmembrane by laser confocal microscopy(×1000).3Establish CHO cell line which constantly express4mutations of ICAM-1. The expression level of ICAM1was determined by flow cytometry and theresults of MFI and%gate showed that there was no significant differenceamong variant mutations of ICAM-1.4Compared with the control cells, the number of adhesive PBMCs in CHOcells expressing variant ICAM-1increased significantly(P<0.05). While therewas no significant difference among variant mutations of ICAM-1.(P>0.05)5The results of single-molecule force measured by AFM: there was nosignificant difference between CHO and COS-7cells(P>0.05),there was nosignificant difference in ICAM-1-SNPs(P>0.05), there was no interaction ofcelltypes with genotypes(P>0.05), and the binding probability of variantmutations of ICAM-1were lower significantly after blocking CD11b (P <0.05).Conclusion:1The plasmid fusing with GFP doesn't affect the location of ICAM-1.2G241R and K369E SNPs of ICAM-1doesn't affect the single moleculeforce with Mac-1.3We can choose both CHO and COS-7cell line to study the single moleculeforce of ICAM-1.