Different Expression Of Cav1Impact Of U87Glioma Cell And Effects Of Extracellular ATP On Growth And Invasion Of U87Glioma Cell In Vitro

Background and Purpose:Gliomas (glioma) is the most common human intracranial tumors, the most importantfeatures of its biological characteristics of tumor cells to invade the normal braintissue,Around the so-called " satellite tumor" focus formed around the primary tumor, andoften to the invasion of the important functional areas of the brain, there is no reasonablypracticable method or technique to identify the boundaries of the tumor, the extent ofsurgical resection of the tumor has been very limited, and the risk of tumor recurrenceincreased with increasing pathological grade.More terrible is about50%of human glioma,glioblastoma (glioblastoma), and their characteristics is invasive, shorter time torecurrence and poor prognosis. Modern computed tomography, preoperative MRI(magnetic resonance) and other imaging technology is still difficult to accurately determinethe tumor boundary,Intraoperative use of an ordinary microscope, but the magnification isstill very limited, is still difficult to identify the tumor boundaries, and even if the residualtumor cells accounted for only1mm~3volume cell number will reach109, but even if only1%of the tumor tissue remaining, back to the the original number of tumor cells only needeight weeks. Therefore, the study of the mechanisms of glioma invasive growth hasimportant implications for clinical treatment of glioma patients.The purpose of this project was to investigate the macro-level mechanisms ofmalignant glioma invasion as a starting point, the Cav1(Caveolin-1), extracellular ATP(Adenosine Triphosphate, ATP) in the invasive growth and migration of glioma.Analysis ofdifferent Cav1expression in U87glioma growth and invasion differences, to provide newideas and strategies for the further study of glioma treatment, this study also used the timemicroscope imaging techniques, which can directly reflect the role of external factors U87glioma cells migration phenomenon, and protein chip technology of the extracellular ATP role in cell protein expression, and to lay the foundation for further study.Materials and research methods:(1)Immunohistochemical detection of U87human glioma cells in Cav1positiveexpression；(2)Build the Cav1slow virus particles upward or downward the expression of the U87glioma cell in Cav1；(3)The application of flow cytometry detection of Cav1overexpression andinterference on the impact of the proliferation of U87glioma cells, U87human glioma cellgrowth curve and MTT assay；(4)Cells "Wound Healing" assay and Transwell invasion assay method of analysis thedown-or up-regulation of Cav1in human U87glioma in vitro;Extracellular ATP on U87glioma cell proliferation assay kit with CCK-8weredetected by0.1%,1%,10%serum concentration gradient ATP role in the proliferation ofU87glioma cells;Extracellular ATP on the U87glioma cell cycle, experiments using flow cytometry todetect the growth cycle of the U87human glioma cells incubated for24hours underdifferent culture condition;Extracellular ATP on the U87cell invasion in vitro, experiments with Transwellinvasion assay without ATP as a control group under the same conditions by the addition of100μmol/L of ATP as a treatment group after24h of cultivation and processingchamber, the transmembrane cell counts.Extracellular ATP on the U87cell migration speed, the experiment to time themicroscope every two minutes, collected photos, the thermostat system can maintain thecell activity, observation extracellular ATP under the action of the U87glioma cellmigration impact.Extracellular ATP role in U87human glioma cells, protein phosphorylation levels, theuse of protein chip technology, an analysis of100μM ATP with10%FBS in DMEM-F12medium for the role of U87glioma cells cultured in vitro45min after the cell motility-related protein phosphorylation levels.Results:1、Cav1overexpression and interference with the proliferation invasion of U87glioma cell in vitro(1)Cav1positive expression in U87glioma cells.(2)Successfully constructed and U87glioma cells, Cav1overexpression andCav1siRNA.(3)The application of flow cytometry detection of Cav1overexpression andinterference on the impact of the proliferation of U87glioma cells, U87human glioma cellgrowth curve and MTT assay.(4)By Wound Healing assay, in U87cells containing10%FBS DMEM in the role ofdownstream scratch test, Cav1siRNA expression in U87glioma cell migration force issignificantly stronger than in U87cells Cav1overexpression; but containing0.1%FBSculture base in Cav1overexpression of migration and strong in Cav1interferenceexpression. This shows that the role of Cav1on the migration of glioma by the externalculture of the environmental impact.(5)Transwell experiments, containing10%FBS medium Cav1siRNA expression inU87glioma cell invasion force enhancement, and overexpression of Cav1in U87gliomacell invasiveness was decreased (Figure3); but in containing0.1%FBS medium Cav1siRNA expression, in U87glioma cell invasion ability actually was decreased, while Cav1overexpression in U87glioma cell invasion enhanced. This shows that the role of Cav1theinvasive ability of glioma by the external environment impact.2、Effects of Extracellular Adenosine Triphosphate on Growth and Invasion ofU87Glioma Cell in vitro(1)CCK-8assay kit were detected by0.1%,1%,10%serum concentration gradient ofATP under the action of the proliferation of U87glioma cells. The results showed thatlower concentrations of ATP (100μM,200μM,500μM) had no significant effect inthe proliferation of U87glioma cells in vitro extracellular, but a higher concentration ofATP (5000μM) cells was significantly inhibited.(2)Extracellular ATP on the U87glioma cell cycle by flow cytometry to detect thegrowth cycle of the U87human glioma cells incubated for24h under different culturecondition. The results showed that the treated group (100μMATP) and control group hadno significant effect on U87glioma cell growth cycle.(3)Extracellular ATP on the invasion of U87cells in vitro using Transwell invasion assay, indicating that a certain concentration of ATP can significantly enhance the U87glioma cell invasion ability.(4)Extracellular ATP on the U87cell migration speed, experimental time microscopeevery2minutes acquisition photos, the results found in the culture medium,100μmol/L ATP can significantly enhance the athletic ability of the U87glioma cells, so thatenhanced migration.(5)Extracellular ATP acting on the U87human glioma cells, protein phosphorylationlevels of the experimental protein microarray analysis and cell motility-related proteinphosphorylation levels.100μM extracellular ATP acting on the in vitro U87glioma cells45min after the VASP (Ab-238) and Rac1/cdc42(Ab-71) signaling pathway activation.Conclusion:This study is the role of Cav1in the U87glioma cell growth and invasion; as well asthe role of extracellular ATP on the U87human glioma cell proliferation, migration andinvasion; extracellular ATP role under visual observation of cell movement series device toobserve the effects of extracellular ATP U87glioma cell migration in vitro. The followingconclusions:(1)Cav1expression in high grade gliomas were significantly higher, the higher theexpression of Cav1the worse the prognosis of the patient; Cav1U87human glioma cellproliferation.(2)Cav1positive expression in U87human glioma cell growth, cell differences in therole of U87human glioma cell invasion and migration and is subject to the environmentalimpact of tumor cell growth. Cav1overexpression inhibited U87glioma cell invasion ofextracellular nutritionally adequate. In the case of the relative lack of nutrition, Cav1overexpression enhanced the U87glioma cell invasion.(3)The lower of the extracellular concentration of ATP (100μM,200μM,500μM) had no significant effect in the proliferation of U87glioma cells in vitro, but a higherconcentration of ATP (5000μM) cells was significantly inhibited.(4)Extracellular ATP (100μM) significantly enhanced the invasive ability of U87glioma cells.(5)Use the Time microscope and observe the device, you can visually see a certainconcentration of extracellular ATP (100μM) can enhance the ability of the U87glioma cell migration.(6)The protein chip technology that ATP role in U87glioma cells can significantlyenhance the VASP (Ab-238), and Rac1/cdc42(Ab-71) signaling pathway activation.Studies have pointed out that the glioma cells to extracellular ATP degradation ratemuch lower than the glial cells and glioma cells in which the microenvironment of the ATPconcentration; extracellular ATP concentration in normal brain tissue cells toxic effects,and glioma cells not only tolerated, and extracellular ATP can promote the growth andinvasiveness of glioma cells. This research explored the role and expression of Cav1in theU87glioma invasion, the role of extracellular ATP on the U87glioma cells, a clearmechanism of invasive growth of glioma, protein chip technology research further explorethe mechanism of glioma invasion provides a new way of thinking.