| Objective: The influence of NE on the proliferation and transformation of phenotype ofvascular smooth muscle cells and its mechanism were studied to supply a experimentaldata and base for studying the effects of the sympathetic nerves on the proliferation andtransformation of phenotype of VSMC.Methods:The vascular smooth muscle cells from aorta of healthy SD rats were incubated incontaining 20% bovine serum culture medium for propagation of the experimental cellsand the fifth generation of cultured cells was used to this experiment.The first experiment: The experimental cells were divided into three experimentalgroup and one control group:A:normal control group; B: treat with NE (5×10-5mol/L)group; C: treat with NE (5×10-5mol/L) and OX-LDL (50ug/ml) group; D:treat withOX-LDL (50ug/ml) group. The experimental cells of each group were cultured incontaining 0.5% serum for 24 hours. Then the culture media were discarded and threeexperimental groups were replaced by NE, NE plus OX-LDL and OX-LDL respectively,and the control group was replaced by serum-free medium for continuous incubation for24h. As for the experimental cells used for immunocytochemical staining, thebromodeoxyuridine BrDU (8×10-4mol/l) was added into the culture medium as sign dosagebefore 24h of accomplishing culture and others was used to detect the expression ofSM22aand HRG-1mRNA by RT-PCR.The second experiment: The experimental cells were divided into three experimentalgroup and two control group: A: 72h cotrol group; B:normal control group; C: treat withNE (5×10-5 mol/L) group; D: treat with NE (5×10-5 mol/L) and OX-LDL (50ug/ml) group; E: treat with OX-LDL (50ug/ml) group. The culture media of each group werereplaced into containing 20% serum media after cultured in containing 0.5% serum for 24hours. Then the culture media were discarded and three experimental groups were replacedby NE, NE plus OX-LDL and OX-LDL respectively, and the control group was replacedby serum-free medium for continuous incubation for 48h and the 72h control group wasstopped to culture.The experimental cells of each group was collected to detect theexpression of SM22a and HRG-1 mRNA by RT-PCR.The Third experiment: The experimental cells were divided into three experimentalgroup and one control group: A: normal control group; B: treat with a1-R- (10-4mol/l)and NE (5×10-4mol/l) group; C: treat withβ1-R- (10-4mol/l) and NE (5×10-4mol/l)group; D: treat with NE (5×10-4mol/l) group. The experimental cells of each group werecultured in containing 0.5% serum for 24 hours. Then the culture media were discardedand three experimental groups were replaced by a1-R- and NE,β1-R- and NE and NErespectively, and the control group was replaced by serum-free medium for continuousincubation for 24h. As for the experimental cells used for immunocytochemical staining,the bromodeoxyuridine BrDU (8×10-4mol/l) was added into the culture medium as signdosage before 24h of accomplishing culture and others was used to detect the expression ofSM22aand HRG-1mRNA by RT-PCR.Results:1.Effects of NE on the proliferation of vascular smooth muscleThe mRNA expression of HRG-1 was decreased after VSMC of contractilephenotype treated by NE and OX-LDL and a lot of proliferous cells labeled by Brdu can beseen. When VSMC of synthesize type treated with NE and OX-LD respectively, it can beseen that the mRNA expression of HRG-1 was up regulative by NE but not by OX-LDL.The results demonstrate that NE can regulate the proliferation of VSMC, however, effect ofOX-LDL on VSMC aren't reversed. The mRNA expression of HRG-1 in treat with a1-R-andβ1-R- groups is more than in treat with NE group, and the number of proliferous cellslabeled by Brdu is getting little. This results show that the effect of NE on proliferation ofVSMC come true by means of a1-R andβ1-R function. 2.Effects of NE on tansformation of phenotype of VSMCAll normal VSMC show the expression of SM22amRNA, but the SM22amRNAexpression had apparent decreased after VSMC of contractile type treated by NE andOX-LDL. However, when VSMC of synthesize type treated with NE and OX-LDLrespectively, the mRNA expression of SM22a was up regulative by NE but not byOX-LDL. The results demonstrate that NE can regulate transformation of phenotype ofVSMC, however, effect of OX-LDL on transformation of phenotype of VSMC isn'treversed.Conclusions;1. NE can promote the proliferation and transformation of phenotype of VSMC fromcontractile to synthesize type. It also can regulate the proliferation and phenotype ofsynthesize type VSMC. This demonstrates NE is reversible regulating VSMC as vascularnerve function.2. OX-LDL can promote proliferation and transformation of phenotype of contractile typeVSMC, but it was not seen as the effect of NE on the proliferation and transformation ofphenotype of VSMC, when VSMC treat with OX-LDL. This showed that effect ofOX-LDL on VSMC isn't reversed.3. It is completely interrupted the effect of NE on proliferation and transformation ofphenotype of VSMC after using a1-R and 1β-R, which shows that effects of NE onproliferation and transformation of phenotype of VSMC come true by means ofa1-R andβ1-R.4. It is necessary to make furthur research of the effect of NE on VSMC as vascular nervefunction.
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