| Objective:By comparing the concentration of uPAR and the GPI-PLD enzyme activity of healthy people and glioma patients' cerebrospinal fluid(CSF) investigate the relationship between both of uPAR/GPI-PLD and glioma invasion.Transfecting GPI-PLD cDNA to U251 and to detect the effect of the overexpression of glycosyl-phosphatidylinositol-specific phospholipase D(GPI-PLD) gene on the GPI anchored protein uPAR.Observing the change of invasion of pcDNA3.1(+) /GPI-PLD transfected U251,measuring the influence of GPI-PLD gene on glioma's growth, apoptosis and invasion,to start a new way to treat glioma.Method:1.Measuring the concentration of uPAR and the GPI-PLD enzyme activity of 30 healthy people and 15 glioma patients' cerebrospinal fluid;2.The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/GPI-PLD produced by our laboratory was transfected into U251 cell by lipid-media transfection. Untransfected U251 and U251 transfected with pcDNA3.1(+) were used as control. After screening G418,the single clone was got.Expression of GPI-PLD mRNA in U251 was detected by RT-PCR.MTT was employed to detect proliferation of three groups;Morphologieal assessment of apoptosis was examined by flow cytometry; GPI-PLD activity levels of cells,are detemined through TritonX-114 Phase Partitioning by using GPI anehored Placental the alkaline phosphatase(PLAP), produced by our laboratory,as substrate,the GPI anchored PLAP detected by spectrophotometrie method;the GPI anchored uPAR was detected by ELISA method.Results:1.GPI-PLD enzyme activity of glioma patients and healthy people' cerebrospinal fluid(CSF) are 30.7%±4.6%and 44.1%±4.4%,separately;the concentration of uPAR are 2.4±0.6(ng/ml),3.6±0.5(ng/ml),the difference has significance(P＜0.05).2.RT-PCR results and GPI-PLD activities showed that the U251 cell line with stably over-expression of GPI-PLD gene was constructed.(1) Compared U251 cell and pcDNA3.1(+)/U251 cell,expression of GPI-PLD mRNA in pcDNA3.1(+)/GPI-PLD/U251 cell was significantly up-regulated.(2) GPI-PLD activities in pcDNA3.1(+)/GPI-PLD/U251 cell were obviously increased.Compared with U251 or pcDNA3.1(+)/U251 cell,GPI-PLD activities in three groups were 5.9 %±2.6%,5.7%±3.0%,13.1%±5.6%,separately.(3) After GPI-PLD gene transfected into U251,the GPI anchored protein uPAR were released by GPI-PLD,on the group culture supernatants uPAR level in pcDNA3.1(+)/GPI-PLD/U251 cell compared with U251 cell or pcDNA3.1(+)/U251 cell obviously increased,the difference has significance(P＜0.01).3.The effect of overexpression of GPI-PLD gene on U251 cells.A.Proliferative capacity in pcDNA3.1(+)/GPI-PLD/U251 compared with U251 or pcDNA3.1(+)/U251 cell obviously decreased.B.comparing with U251 or pcDNA3.1(+)/U251 cell,pcDNA3.1(+)/GPI-PLD/U251 did not present increasing in apoptosis,but has difference in cell growth cycle,the percentage of S stage cells in U251,pcDNA3.1(+)/U251,pcDNA3.1(+)/GPI-PLD/U251 are 17.1%,16.46%,9.33%,separately.C.After 24h vitro invasion test and count the number of cells invaded cross the matrigel in 10 visual fields found that the invasion rate of pcDNA3.1(+)/GPI-PLD/U251 cell were significant higher comparing with U251 or pcDNA3.1(+)/U251 cell.The results imply that GPI-PLD can decompose GPI anchor and lead to the release of uPAR from the surface of U251,as a result,inhabit both of locating and the function of degrading matrix and the signal transduction mediated by uPA-uPAR.Conclusion:1.The activity of GPI-PLD and the concentration of uPAR in cerebrospinal fluid of 15 patients with glioma were significantly lower than in 30 health adults.2.Successfully constructed the stable U251 cell line with over-expression of GPI-PLD gene.3.It was proved that GPI-PLD gene effectively decreased U251 vitro invasion and influenced the cell circle.
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