The Influence Of NE On VSMC Proliferation And Expression Of Cytoskeletal Proteins,SM22α

Objective: To investigate the influence of NE on phenotype switch,proliferation and expression of cytoskeletal proteins of VSMC, and the relationship between them.Methods:The vascular smooth muscle cells from aorta of healthy SD rats were incubated in containing 20% bovine serum culture medium for propagation of the experimental cells, then the second and the sixth generation of cultured cells were used to this experiments.These cells were divided into two parts: one was cultured in bovine serum culture medium and the other was cultured in serum-free medium. Every part has two subgroup: NE group and normal control group. Then experimental cells were treated differently for different detection methods.①The experimental cells of each group, used for immunocytochemical staining, were incubated in 24-well culture plate with a piece of 0.5*0.5 cm glass placed horizontally at the bottom of each well. Every group has 6 wells and every well contains 1 ml cell suspension (cell density: 5×104 /ml).②The VSMCs of each group, which were collected to detect the expression of SM22αmRAN by RT-PCR, were cultured in a 50ml culture flask. Every culture flask contains 4 ml cell suspension (cell density: 5×105 /ml). Every group has 2 culture flask. Vascular smooth muscle cells from each group were incubated in 12% fetal bovine serum culture medium at 37℃and 5% CO2 for 8 hours. After the passaged VSMCs sticking to the wall of the culture flask, Cells from each group were incubated in 0.5% serum for 24 hours. Then cells from each group were incubated in 12% fetal bovine serum culture medium for 72 hours. NE group cells were treated by DMEM containing NE (5×10-5mol/L) for 24 hours. Bromodeoxyuridine Brdu (8×10-4mol/L) was added to the culture medium as sign dosage 24 hrs before the cells were collected. The cells were fixed in 4% paraformaldehyde for 30 mins ready for immunocytochemical staining. Cells for RT-PCR were collected and storaged in -20℃and 4℃. Cells from serum-free medium group were incubated in 12% fetal bovine serum culture medium for 72 hours. Then they were incubated in serum-free medium for 48 hours. The cells of experimental group were incubated in DMEM containing NE (5×10-5mol/L) for 24 hours. The other procedure was as the group of serum medium group what has mentioned above. Results:一,Effects of NE on phenotype switch of VSMCSM22αis a marker gene of contractile phenotype VSMC. It is expressed in normal VSMC. With the increasing cell passage number, its expression was decreased gradually in VSMC, which was cultured in bovine serum culture medium in vitro. Study shows that the SM22αmRAN expression was decreased in VSMC treated by NE and was increased in VSMC of passage 2 or 6 incubated in serum-free medium.二,Effects of NE on VSMC proliferationWith the increasing cell passage number, positive cells labeled by Brdu were increased gradually. Positive cells in NE group were more than in control group in the same passage. When experimental cells were incubated in serum-free medium, proliferous cells were decreased.三,Effects of NE on the expression of VSMC cytoskeletonThe cytoskeletal proteins of SMα-actin,β-Tubulin and Desmin are present in normal VSMC. With the increasing cell passage number, the expression of these cytoskeletal proteins were decreased in vitro culture conditions. Sharp drop of the expression fromβ-Tubulin and Desmin can be observed and their expression can not be detected in cells of passage 6. Down regulation of the three cytoskeletal proteins could be observed in VSMC treated by NE.Conclusions:1.NE can promote VSMC phenotype switch from a contractile to a synthesized type.2.NE can inhibit the expression of cytoskeletal proteins of SMα-actin,β-Tubulin and Desmin.3.Change of cytoskeletal proteins and Phenotype switch,proliferation have close relationship. Casual relationship between them needs further study.