Biological Characteristics Change In The Smooth Muscle Cell Of The Great Saphenous Vein By Down-regulation Of Osteopontin

PART I Primary culture and identification of smooth muscle cellsfrom saphenous veinsObjective This experiment was designed to culture and supply enough smooth muscle cells (VSMC) from saphenous vein for the flowing experiments.Methods VSMC were cultured by explant outgrowth from unused portions of human saphenous veins (3～8cm/once) harvested for coronary bypass surgery. Cells were cultured in DMEM. The morphology and proliferation of cells were observed with inverted phase contrast microscope. The type of cells was evaluated by immunocytochemistry with a-smooth muscle actin antibody.Results After 4～5 weeks, the primary cells were passed 3～5 times and the number of cells reached 8～10×10~6. Cells were observed by inverted phase contrast microscope exhibiting the grow characteristics of VSMC, and were demonstrated by immunocytochemistry method.Conclusion VSMC from saphenous veins were obtained by primary culture method. They can be cultured successfully and be amplified to a sufficient number for the following experiments. PART II Downregulating osteopontin inhibits the proliferation and migration of VSMC of saphenous veinsObjective Proliferation and migration of VSMC is the pathological base of intimal hyperplasia. This study was designed to investigate the effect of down-regulating osteopontin(OPN) in proliferation and migration of VSMC of Saphenous veins and discuss the possible mechanism.Methods OPN expression was assessed by real time PCR, Western blotting analysis and ELISA after OPN siRNA transfection; Cell proliferation and migration was assessed by MTT and Transwell methods. Further more, Western blotting analysis was performed to detect matrix metalloproteinases (MMP-2, MMP-9) expression.Results After transfection, expression of OPN mRNA and protein decreased significantly in experimental group compared to control group. MMP-2, MMP-9 protein decreased in experimental group and the ability of proliferation and migration was inhibited.Conclusion OPN plays an important role in proliferation and migration of VSMC by regulating the expression of MMP-2, MMP-9. PART III Downregulating osteopontin reduces angiotensin II -induced inflammatory activation in VSMCObjective The inflammatory activation characteristics of VSMC is the pathological base of atherosclerosis. This study was designed to investigate the effect of down-regulating OPN in angiotensin II (Ang II )-induced inflammatory activation in VSMC.Methods OPN expression was assessed by real time PCR, Western blotting analysis and ELISA after OPN siRNA transfection; Cells were stimulated with Ang II to activate the inflammatory pathway, then inflammatory activation was assessed by determining the release of interleukin-6 and activation of nuclear factor kappa B (NFkB) and activator protein-1 (AP-1).Results After transfection, expression of OPN mRNA and protein decreased significantly in experimental group compared to control group. Pro-inflammatory activation of NFkB and AP-1 was abolished and IL-6 release was reduced by OPN down-regulation.Conclusion OPN plays an important role in Ang II induced inflammatory activation in VSMC. The finding further supports OPN can serve as a potential target for atherosclerotic therapy. PART IV Downregulating osteopontin increases calcification of VSMC of saphenous veinsObjective The calcified characteristics of VSMC is the pathological base of calcification of vein graft and atherosclerosis plaque. This study was designed to investigate the effect of down-regulating OPN in calcification of VSMC of Saphenous veins.Methods OPN expression was assessed by real time PCR, Western blotting analysis and ELISA after OPN siRNA transfection; calcification was induced by 6-glycerophosphate, then calcium deposition and alkaline phosphate(ALP) activity was measured.Results After transfection, expression of OPN mRNA and protein decreased significantly in experimental group compared to control group. The ability of calcification was significantly boomed in experimental group; however, ALP activity was no difference between groups.Conclusion Down-modulation of OPN increases calcium deposition, and OPN inhibits calcification by playing its role in down-stream of ALP. Further study is warranted for new drugs which suppress epitopes concerning proliferation, mobility, inflammation and wouldn't affect epitopes concerning anti-calcified function.