ABSTRACT
ObjectiveTo study the influence of different culture conditions in vitro on phenotype, proliferation and cytoskeletal proteins expression of vascular smooth muscle cells(VSMCs). Methods The cultured VSMCs from rat aorta were divided into six groups: P2 control,P2 starvation,P4 control,P4 starvation,P6 control and P6 starvation. The proliferating cells were labeled by 5-bromodeoxyuridine (5-BrdU); The mRNA expression of smooth muscle 22 alpha (SM22α) was detected by reverse transcription-polymerase chain reaction method (RT-PCR); The cytoskeletal proteins including SMα-actin,β-Tubulin and Desmin were observed through immunohistochemical staining. Results With the increase of cell passage, cytoskeletal proteins expression of VSMCs decreased,cellular organs increased and secretory vesicles were abundant; in serum-free cultured cells mitochondria increased and electron density enhanced in cytoplasm of VSMCs.On the contrary the expression of SMα-actin decreased, and the expression of SMα-actin increased. The expression of β-Tublin and Desmin decreased more obviously, and at 6 passages failed to express. Conclusion The conditioned medium and serum-free had the different effects on the phenotype,proliferation and cytoskeleton of VSMCs in different passage, and there was internal relationship among them. The internal relationship played an important role in the maintaining of cell morphology, contractile function and vascular remodeling. The disappearance of expression of β-Tubulin and desmin might have important biological significance.
ABSTRACT
To study the mechanism of proliferous vascular disease as well as its prevention and treatment, an organic model was established with cultured aortas of rats, and the mechanism there-in invloved was probed. Immunostaining histology showed that smooth muscle cell (SMC) proliferation was observed in the aorta segments of rats, after their endothelia being injured and cultured in vitro with 20% fetal bovine serum. After being cultured for 5 days, various degrees of proliferation of SMC on cultured artery segments were observed by HE staining, and conspicuous plaques were developed after being cultured for 13 days. The proliferous SMC was also observed by Brdu labeling. RT-PCR examination showed that the mRNA expression of hypertension-related gene-1 (Hrg-1) and smooth muscle 22 alpha (SM22a) in the aortas decreased with the prolongation of culture time, and completely disappeared after being cultured for 13 days . But when cultured in vitro for ten days, the ET-1 content of supernatant and the proliferous SMC labeled by Brdu increased obviously and the expressions of Hrg-1 and SM22a decreased after the endothelium was destroyed. Compared with the injured endothelium groups, the proliferous SMC of injured endothelium plus BQ123 groups decreased visibly. The same significant differences between serum groups and serum-free groups were also observed. These results suggest that the culturing of rat aorta segments in vitro can induce the proliferation of SMC and the transform of phenotype from contractile type to synthetic type. The ET-1 and serum are the main factors in the proliferation of SMC and in the transform of phenotype. This organic model could serve as a good experimental platform for the researches into the mechanism of proliferous vascular disease as well as its prevention and treatment.