The Correlation Between Filamin And Phenotype Switch Of VSMC

Backgroud:Filamin play an important role in differentiation and immigration and signal transduction of VSMC and is involved into the development,mature and aging of artery and the arteriosclerosis. Knockdown the expreesion of FLNmRNA in VSMC by RNAi in order to inhibit the expression of FLN protein in VSMC which may interrupt the switch of VSMC from Contractil phenotype to Synthetic phenotype under the pathologic status. It may give us some new pathway or target to prevent or treat the artherosclerosis.Materials and Method:Design three fragments of SiRNA which may induce the knockdown of FLNmRNA, then construct the plasmid to transfection the rabbit VSMC. Observe the GFP in VSMC to assess the efficacy of transferction by fluorescence microscope. Assess the expression of FLNmRNA and FLN with Fluorescent Quantitation Realtime PCR and Westernblot. Then culture the normal and transfected VSMC with ox-LDL. Proliferation of VSMC was observed by MTT and 3H-TdR incorporation. Cell cycle and Apoptosis were detected by flow cytometry. Immunofluofrescence method was used to observe the apoptosis, cystoskeleton and morphous of VSMC. The expression of some proteins was evaluated by immunohisto-chemical method and use electron microscope to observed the ultrastructural change of VSMC.Result:All the three SiRNA can knockdown the expression of FLNmRNA. And the most significant effect was seen in No.7-1 and 7-2. The expression of FLN in target groups were also decreased from 30 to 50%.Reproductive activity of transfected VSMC is lower than normal VSMC.But the ratio of apoptosis and necrosis is similar. There are no significant diference between two groups in their morphology, cytoskeleton and ulrastructure.After cultured with ox-LDL,reproductive activity of normal group was enhanced...