Effect Of Nelin Gene On Phenotypic Transtition Of Human Great Saphenous Vein Smooth Muscle Cells

ObjectiveTo investigate the effect of Nelin on phenotypic transition of smooth muscle cell(VSMC) and explore the role of Nelin in primary lower extremity varicose veins.Methods(1) The primary culture were done by digestive and explant attached method, and the cells were subcultured with0.25%trypsin, the cultured cells were identified by morphology observation、immunofluorescence.The survival percentage、viability and proliferation ability of cells were determined by trypan blue staining method、drawing growth curve.(2) Serum starvation method was used to reverse of synthetic VSMC to contractile phenotype and the reversion of contractile VSMC to synthetic phenotype was induced by recovering serum stimulation.We acquired different phenotype of VSMC.(3) Nelin mRNA expression was detected in different phenotype of VSMC by reverse transcription polymerase chain reaction (PCR).(4) VSMC was transfected by Nelin-siRNA to knock-down the expression of gene Nelin, and then observe the effects on cytoskeleton and contractibility by respectively coomassie brilliant blue dyeing and acetylcholine(ACh)-induction.Results (1) VSMC of primary culture grew out of the edge of tissue pieces after incubation for5-7days, which were fusiform、polygon or irregular. Their body was little but cytoplasm was abundan and had high refraction. The cells were connected by process and the karyon was oval with one or more nucleolus; The passage cells maintained "hill-valley" pattern of growth,with more cell process、higher transparency, and lower refraction; Positive staining of a-actin in cell cytoplasm was revealed by immunofluorescence; Cell survival percentage was97%.The growth curve of VSMC resembled "S" in shape.(2) The differential expression of Nelin mRNA in different phenotype of VSMC:Synthetic VSMC can be reversed to contractile phenotype induced by serum starvation.We observed the expressing activities of Nelin mRNA in the process. RT-PCR showed that the expression level of Nelin mRNA in VSMC Which were stimulated by serum and serum starvation24h、48h、72h and restore stimulation by serum for24h、48h、72h was0.1940+0.0971,0.7114+0.1265,0.7270+0.1026,0.7841±0.1240,0.3208+0.1389,0.2997+0.1168,0.1807+0.0845respectively. There was significant differences between serum starvation group and serum stimulation group(P<0.05), There was no significant differences between serum starvation groups and between serum stimulation groups(P<0.05).(3) The effect of Nelin expression on cytoskeleton structure:The cytoskeleton structure of VSMC was remodulated from discrete and fuzzy network to well arranged and dense bundles after serum starvation.After knocking-down the expression of gene Nelin by transfecting of Nelin-siRNA, this processe was blocked.(4) The relationship between Nelin expression and contraction:Acetylcholine (ACh) did induce contraction of contractile VSMC.The result shows that contraction of VSMC which was transfected of negative control siRNA induced by ACh was found. Cell length get shorten and intercellular space got narrow down.When the expressions of Nelin were downregulated by transfecting of Nelin-siRNA, contraction of VSMC induced by ACh was disappeared.Conclusion (1) The cultured VSMC is characterised by contractile phenotype and highly purified with good structure and function. Proliferation activity is higher after growth for3-6d.(2) The results demonstrated that Nelin was involved in VSMC cytoskeleton reorganization and played an important role for VSMC remaining contractile phenotype and phenotypic transition.