The Research Of The Role Of Exo70 In Vascular Smooth Muscle Cell A7r5 Migration

Vascular smooth muscle cells(VSMC)is an important component of blood vessels in the mesolamella,its anomalouschanges plays an important role in the occurrence and development of cardiovascular disease such as in hypertension,atherosclerosis(AS),and restenosis after angioplasty.The exocyst complex is composed of eight subunits that are evolutionarily conserved from yeast to human.The exocyst complex is mainly involved in the exocytosis process.As a key subunit of the exocyst complex,Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane.However,Exo70 not only played a role in the exocytosis process but also involved in mammalian cells migration,such as Hela cells.In order to make clear the role of Exo70 played in the migration process that VSMC proliferationin themesolamella,then migrated to lining.In this experiment,the vascular smooth muscle cells A7r5 as the experimental research object.The experiment research the expression and localization of Exo70 in A7r5 cells,and changes of the migration and invasion ability of the A7r5 cells that the Exo70 function missing before and after,throgh cell biology,molecular biology and biochemistry experiment methods.The results are as follows:1.The expression of Exo70 in vascular smooth muscle cells A7r5Rt-pcr and immunofluorescence test results show that Exo70 do have expressed both at the mRNA level and protein level in A7r5 cells,and it’s mainly distributed in the cell cytoplasm.2.The subcellular localization of Exo70 in A7r5 cellsFirst,use of immunofluorescence double marking method to detected the Exo70 localization in A7r5 cells and interaction with what kind of cytoskeleton protein.Results show that the Exo70 and Actin are co-localization,but with the tublinpositioning phenomenon is not obvious.In order to further validate the results,after treated A7r5 cells 1h using cytochalasin B,Actin depolymerization,meanwhile the distribution of Exo70 presents the dispersionshape in the cytoplasm,the experiment is given to illustrate the Exo70 interaction with microfilament to play function.Finally,in order to make clear the location of Exo70 in A7r5 cells migration using immunofluorescence double labeling the Exo70 in scratch system.Results show that the Exo70 and Actin are co-localization at the edge of the migrating A7r5 cells.Use of immunofluorescence double labeling tested the location of Exo70 in A7r5 scratch system.Results show that the Exo70 colocalization with Actin in migration A7r5 cells edge.3.Exo70 role in vascular smooth muscle cells A7r5 migrationIn order to further clear the role of Exo70 played in the process of A7r5 cells transfer.First,using a technique called RNA interference to silence the Exo70 expression,and select the A7r5 cell lines that the Exo70 function defects:A7r5-rat Exoc7-D2 and A7r5-ratExoc7-K1 cells,then using the scratch healing experiments to detect the miagration ability of this three cells,compared with wild type A7r5.Results show that the miagration ability of A7r5 cells have obvious difference before and after the cell interference,and the A7r5-rat Exoc7-D2 cell expend the longest time for healing,followed by A7r5 ratExoc7-K1 cells,suggesting that the inhibition the expressed of Exo70 in A7r5 cells,the cells migration ability is lower than before.Further proofed that Exo70 expression levels were positively correlated with cell migration ability.4.Exo70 role in vascular smooth muscle cells A7r5invasionIn order to further clear the function of Exo70 in the process of A7r5 cellstransfer.Then,using Transwell experiment testing the change of the A7r5 cells invasive ability before and after the interference Exo70,cultivate the three cells in the small room,after 24 h,the results show that the amount of the A7r5-ratExoc7-D2 and A7r5-rat Exoc7-K1 cells was significantly less than A7r5 cells in the lower chamber.Illustrates that after inhibits the expressionof Exo70 in the A7r5 cells,the invasion capacityof cells is reduction.So the Exo70 play an important role in the process ofA7r5 cell migration and invasion.This study identified that Exo70 expression and localization characteristics in the VSMC and the function played in the process of VSMC migration,helps to further understand the pathogenesis of cardiovascular disease,offers a new potential therapeutic targets for the treatment of cardiovascular diseases.