Effectes Of Transfection With Antisense Integrin Eukaryotic Vectors And Static Magnetic Field Intervention On The Biological Behavior Of Human Artery Smooth Muscle Cells

The study aimed to investigate the roles of two important ECM protein fibronectin, laminin and their receptor integrin alpha5betal in regulating the VSMCs biological behaviors. And using gene blockade by antisense integrin vector transfection or physically intervening byInstitute of Cardiovascular disease FMMU 2002static magnetic field, we studied its effects on interaction between VSMCs and ECM and intracellular signal transduction, and provide a new therapeutic target for RS. [ METHODS]It was successfully improved in primary culture method of human umbilical artery smooth muscle cells(hUASMCs). The coated fibronectin(FN) or laminin(LN) were used as insoluble FN(iFN) or insoluble LN(iLN), while those added to the medium directly were used as soluble FN and LN(sFN and sLN). Under these two status, DNA and collagen synthesis of hLASMCs were determined by H-thymidine and H梡roline incorporation. Total viable cells was detected by MTT assay. Adhesion and Transwell migration assay were also conducted to test the ability of adhesion to FN and LN and migration on them. The expression and distribution of integrin alphaS.hetal and alpha-SM-actin were determined by single or double條abelled indirect immunofluore.scent staining. We further constructed the antisense eukaryotir vectors of integrin alpha5 and betal subunits and transfected hUASMCs, or intervened hLASMCs with static magnetic field(SMF) with different intensity. Transmission electron microscopy showed the ultrastructure of hUASMCs after transfection or exposure to SMF. The expression of integrin alphaS and betal and phosphorylation of focal adhesion kinese(FAK) of hUASMCs were determined by Western Blotting. Dot Boltting were performed to detect the mRNA level of integrin alphaS and betal. The cell cycle were analyzed by flow cytometry alter pmpidium iodide(PI) staining. Intracellular free calcium ion were loaded with fluorescent probe Fluo?/AM and detected by laser scanning confocal microscopy.[ RESULTS]1. Modification of primary culture method of human umbilical artery smooth muscle cells.The umbilical artery were cut into 3?mm artery rings and vertically attached in the culture flask. Do not turn over the flask to soak the artery rings in medium until 5-6 h later, which was longer than usual for better attachment and get ride of the flbroblasts contamination. The primary culture time were shortened by adding higher doses of bFGF and EGF than what reported. There was no rytotoxicity detected when NaHCOi was used. Identification of SMCs had better been done within the first three passages. With the above methods, we got the pure hUASMCs that were confirmed by immunocyto-chemistry and immunofluorescence staining of alpha桽M梐ctin. Transmission electron microscopy demonstrated the typical characteristics o( "dense patch" or "dense body" .2. Effects of FN and LN in different physical status on die biological behavior of hUASMCs and mechanism.Institute of Cardiovascular disease FMMU 2002121). Different physical status of FN and LN showed adverse roles in mediating the biological behavior of hUASMCs. In presence of iFN at 20,40,60,80 and 100 iig/mL, the absorbance value, adhesion percentage, migratory cell numbers were increased remarkably in a concentration-dependent manner. iLN also increased the cell adhesion and proliferation, but inhibit migration and collagen syntgesis at the same concentrations. In contrast, sFN and sLN displayed a concentration?dependent inhibition of cell adhesion and proliferation except that sLN could increase the migration and collagen synthesis of hUASMCs slightly. 2). hUASMCs were pretreated with the blocking antibody against integrin alpha5 P1D6 and betal P4C10. GRGDSP. tyrnsine kinase inhibitor genistein and calcium channel blocker nicardipine to explore the mechanism of FN?and LN-mediated effects. The strongest inhibition of adhesion and proliferation were genistein, then came to or P1D6 and nicardipine. While towards migration. GRGDSP, P4C10 was the first two powerful inhibitors...