Effection Of Inhibition Of Hypoxia-inducible Factor-1α(HIF-1α) On Glioma Cell Apoptosis

[Objective]:The research aims at simulating the survival hypoxia microenvironment of glioma to observe the shape of glioma cells under hypoxia by inverting the microscope. Examine1%O2concentration to glioma apoptosis, and use immunofluorescence and Western Blot technique to conduct quantitative detection of HIF-1α.Furthermore apply RNAi technology and gene silencing hypoxia-inducible factor HIF-la in vitro, and to observe the effect of the proliferation and apoptosis of gliomas under hypoxia. In order to further survey the glioma treatment regard HIF-las the target to provide experiment and theory gist.[Method]:l.By means of hypoxia incubator mimicking microenvironment and observing by inverting the microscope and t Hoechst staining and MTT detect the effect of hypoxia to proliferation and invasion of glioma cells; Culture4h-72h in the hypoxia(1%O2) incubator. To examine the expression T98G cell’s variation of4h-72h hypoxia-inducible factor-1under the hypoxia.2.Pass the normal T98G cell’s culture and count, and design a group of cell density gradient and dispose24h under hypoxia(concentration of1%O2) by Western blot quantitative analysis expression variation of HIF-1.3.Using lipofection to have HIF-1α SiRNA plasmid transfection into glioma cell line T98G cell, apply Western blot technology to the effectiveness of stabilizing cell interference after the infection of protein level detection.4. Select effective SiRNA plasmid transfection T98G and respectively set as interference group and blank control after screening. Under the oxygen concentration of1%O2and21%. cultivate24h,48h.72h.96h,120h.MTT.then detect the survival of each cell and proliferation situation. Flow cytometric examination of condition of cell apoptosis.[Results]:1. Cultivate glioma cell T98G under the oxygen concentration of1%and21%, and observe by inverting the microscope and Hoechst staining does not appear obvious cell apoptosis,MTT detection hypoxia group and normoxic group both appear proliferation state. Under the Oxygen concentration of1%, cultivate T98G cell line4h,6h,12h,24h(<24h),HIF-la protein expression increases rapidly, and24h,48h,72h(>24h),HIF-la protein expression declines.2. In the influence of different cell density affecting T98G cell HIF-la, among them there are85000cell/cm2density of T98G,HIF-1α express the highest. Excessive or low cell density both lead to the decrease of HIF-la expression.3. Liposomal method wraps HIF-la-SHRNA transfection T98G cell, inhibition rate of HIF-1α protein expression is higher after the screening of G418,even reach to92.15%.4.Hoechest staining and FCM examine the no interference group and control blank group under1%oxygen concentration, both have no obvious apoptosis. MTT detection discovery that hypoxia interference group has slower proliferation than hypoxic control group(p<0.05),and cells of normal oxygen interference group grow better than hypoxia interference group(p<0.05).[Conclusion]:1%hypoxia microenvironment cannot guide glioma cells to occur apoptosis; In the hypoxia microenvironment of1%02concentration, The hypoxia treatment of T98G cell is within24hours, and the protein expression of HIF-1α increases rapidly. T98G cells hypoxia treat24h,48h,72h, and its expression of HIF-1α reduces graduate.,its convergence degree reaches to95%under the hypoxia, the expression of HIF-1α is the highest and excessive or low cell density both will decrease the expression of HIF-1α. Interference with silence T98G cells cannot induce cell apoptosis of T98G, but can reduce the multiplication rate of T98G cells.