The Expression Of Chemokine Receptor CXCR4 In Human Glioma And The Building And Meaning For The Application Of Invasive Model-under Agarose Cell Migration In Vitro To The Glioma Cells

Obejective:Glioblastoma is one of the most common malignant tumors of CNS and the leading cause of cancer-related deaths worldwide.This high mortality is probably attributable to early metastasis. Recent studies indicate that tumor cells express chemokine receptors and may use chemokine-mediated mechanisms during the metastasis process.Among the chemokines and chemokine receptors identified to date,the membranous CXC chemokine receptor-4(CXCR4) and its ligand,stromal-cell-derived factor1α(SDF-la or CXCL12),has been found to play a key role in tumorigenicity,proliferation.metastasis and angiogenesis.Although the mechanisms underlying SDF-1a/CXCR4-mediated tumor invasion have been studied in many cancers.the role of SDF-la/CXCR4 in the process of GBM cells proliferation and migration remains largely unknown.Therefore,the expression of CXCR4 in glioma patients was detected by immunofluorescence in this study. Based on the results,the role and mechanism of SDF-1/CXCR4 axis in metastasis of GBM were further studied by under-agarose cell migration assay in vitro.Metheods:1. Cells culture.for U251 cells:The U251 cells were supplemented in the culture medium(RPMI1640 or DMEMfl2 for U251 cell) containing 10% foetal calf serum, 100unit/ml penicillin and streptomy. Under the condition as below:37℃,saturated humidity,and 5% CO2.Renew the medium every 3-4 days for U251 cells.2. Using indirect fluorescent staining assay.dying the U251 with anti-CXCR4 antibody, TRITC. Detected cell receptor CXCR4 in U251 glioma cells.3. Establishing an under-agarose migration assay to investigate the invasion ability of U251 under different concentrations of chemokine CXCL12.Divided into 6 experimental groups,chemokines concentrations were 50ng/ml,100ng/ml,200ng/ml,300ng/ml,400ng/ml,500ng/ml. After overnight in the cell incubator, cells migration were observed under fluorescence microscope.4. Flow cytometry (flow cytometry,FCM) detected the CXCR4 expression rate ofⅠ,Ⅱ,Ⅲ,Ⅳgrade glioma.Results:1. Staining of CXCR4 protein was identified in the cell membrane and/or cytoplasm of cancer cells. It showed orange red under fluorescence microscope.2. The distance cell migrated were 40.96±5.22μm,49.84±2.20μm,106.11±1.59 u m,165.36±1.19μm,245.87±0.87μm,252.45±1.11μm.Compared to the negative groups the cell migrated significantly in the 100-500ng/ml groups (P< 0.05);among chemotaxis groups,the differences were significant compared to each one,but there was no significant difference between 100ng/ml and 500ng/ml group.3. The results of FCM analyzing showed that the CXCR4 expression rate for the different grade gliomas were 21.36±2.70%,26.39±4.27,52.59±2.37%,56.23±1.24%,and the positive rate of CXCR4 were significant difference (P<0.05).Conclusions:1. CXCR4 chemokine receptor expressed in U251 glioma cells.2. The experiment successfully established the invasion model in vitro of under-agarose migration that was adopted to the glioma cells.It was significant to the measurement of the invasive ability in vitro of the glioma cells and the transfer meehanism and influence factors of the cell;CXCL12 had significant chemotaxis effect on glioma, tumor cell migrated directly along a concentration gradient of the chemokine.3. Chemokine receptor CXCR4 expression was positively correlated with glioma grade, With the level of malignancy increased CXCR4 expression increased too. Show that the expression of CXCR4 is proportional to the level of glioma,the higher the grade was the highlier the CXCR4 expressed.