The Expression And Significance Of PTEN Protein In Autogenous Vein Grafts In Rats

ObjectiveOne of the effective therapeutic tool for the stenosis of peripheral artery is by - pass operation. Autogenous vein is the best transplanting materials, but the long term effect is not good because of restenosis. It has been evidenced by researches that the mechanism of restenosis is formed by neointima hyperplasia, and the main pathological basis is the proliferation and migration from tunica media to intima of vascular smooth muscle cells ( VSMC ). PTEN ( MMAC1/ TEP1) is a tumor suppressor gene founded in 1997, PTEN protein is composed of 403 amino acid residues, and its cytoplasm location is acquired by immuno-fluorescence microscope. PTEN is a lipid phosphatase whose major substrate is phosphatidylinositol -3,4,5 - triphosphate ( PIP3). PTEN decreases PIP3 plays a negative role during cell proliferation. At the same time. PTEN has the high homology with tensin and auxilin, inhibits FAK and ERK/MAPK pathway, may suppress cell proliferation, migration, spreading and adhesion formation. So in this experiment we research the expression of PTEN and the relationship between PTEN and neointima hyperplasia with the model of autogenous vein grafts in rats.MethodsAnimal model: An animal model of the autogenous vein graft through transplanting the jugular vein into abdominal aorta was established in 48 Wistar rats of 6 groups (body weight 250 50g, 3 months old, n =8). We carry out microsurgery operation, using 10 - 0 no - injure suturing, end - to - end anastomosisjugular vein into abdominal artery, checking on blood flow in stoma was unobstructed. The vein grafts were harvested at 1 day, 3 day, 7 day, 14 day, 21 day and 28 day after operation respectively. The jugular vein of opposite side were harvested as the control group.Histomorphology: The specimens were embeded in paraffin, made into paraffin section (4|xm) , stained by the methods of HE and Verhoeff. Morphology of tissue was observed in optics microscope. The intima thickness were measured by computer graph analysis system.Immunohistochemistry: The section were made with the same mothod a-bove. Then staining with antibody of PTEN and PCNA, using techique of immunohistochemistry (SABC method). The slide was observed in 400 optics microscope and calculated the positive cells of PTEN and PCNA respectively.Western blotting: The frozen specimens from different phase were treated by SDS - acrylamide gel electrophoresis (SDS — PAGE) , then immune reaction and result analysis, to get the level of PTEN protein.Statistical analysis: The result was analyzed wih computer using ANOVA by SPSS 11.0, statistical significance was defined at P<0.05.ResultsIn normal vein the vessel wall possess single layer of endothelial cell and 2~3 layers of VSMCs in tunica media. After the grafting operation, Intima andtunica media of vein graft were thickening, and after 3 days intima proliferatedmore faster. After 2 weeks proliferation reached to the highest point. From 2week, the extend of intimal hyperplastic became slow.The PTEN protein positive cells were cytoplasma stain. In normal vein it was high expression. It decreased significantly after vein transplantationion, almost no expression in 1 and 3 days, and increased again in 1 week gradually. Hie amount of positive cells increased to top in the 2 week and maintained a higher level to the 3 week. Then the expression restored to normal level in the 4 week. The PCNA positive cells was nucleus stain. In normal vein wasn't express , 1 day after vein transplantationion PCNA positive cells were detected, in-creased to top in the 7 day, then gradually decreased, and the amount of positive cells were less in the 4 week.The PTEN protein western blotting detection has the tendency similar to the immunohistochemistry. PTEN protein expressd in normal vein, 1 ~3 days after operation, PTEN didn't express. It expressed gradually after 3 days. The expression was obvious in the 2 ~ 3 week, then decreased to normal.Compared with the expression of PTEN and PCNA, PCNA was higher when intimal hyperplasia was serious and at the same time the expression of PTEN was lower. Intimal hyperplasia was inhabited when the expression of PTEN reached to higher level and the express of PCNA was restrained.DiscussionPTEN is a lipid phosphatase whose major substrate is phosphatidylinositol -3,4,5 - triphosphate (PIP3) , downstream of which is PKB/Akt pathway. The serine/threonine kinase Akt, when phosphorylated, protects cells from ap-optosis, Knowing that PTEN plays a negative role during cell proliferation. At the same time, PTEN has the high homology with tensin and auxilin, PTEN may suppress cell migration, spreading, and adhesion formation. As reported that TGF - p and IGF -1 can modulate the expression of PTEN .however, at present the specific mechanism is unclear. Proliferating cell nuclear antigen (PCNA) reside in nuclus of reproductive cycle cells, it is one of the markers of cell proliferation. PCNA express in Gl ~ S phase cells, almost no express in GO phase, coincident with DNA synthesis of VSMCs.In this experiment, we demonstrate that PTEN is important for the normal growth of vein tissue. In normal vein PTEN express and PCNA doesn't express, VSMCs present steady state. PTEN protein level were rapidly downregulated 1 day after vein transplantation and possess low degree for 1 week, indicate that the inhibit effect of growth and proliferation by PTEN were attenuated and PTEN play an important role in the early stage of VSMCs proliferation. In 1 week PCNA increased to top, VSMCs proliferation predominate in this period, however, PTEN already begin to increase. PTEN present a higher level in 2 week and...