RNA干扰沉默低氧诱导因子-1a对低氧条件下人脑胶质瘤凋亡影响的前期研究

发布时间：2018-01-25 09:10

本文关键词： 低氧低氧诱导因子1 T98G细胞株 RNA干扰增殖凋亡　出处：《昆明医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：[目的]：本研究在于模拟脑胶质瘤所生存的低氧微环境,通过倒置显微镜观察胶质瘤细胞在低氧下的形态,检测1%氧浓度对脑胶质瘤细胞凋亡的影响,并应用免疫荧光及Western Blot技术对HIF-1a进行定量检测,检查低氧下不同时间梯度及细胞密度对HIF-1α表达的影响,并在体外实验中运用RNA干扰技术,沉默低氧诱导因子-1,观察其对低氧下胶质瘤的增殖、凋亡的影响。为进一步研究以HIF-1为靶点治疗胶质瘤提供实验和理论依据。 [方法]：1、通过低氧培养箱模拟低氧微环境,通过倒置显微镜下观察和Hoechest染色、MTT检测低氧对胶质瘤细胞增殖凋亡的影响；在低氧(1%02)培养箱中培养4h-72h。通过western blot检查T98G细胞在低氧条件下4h-72h低氧诱导因子-1a表达变化.2、通过平时的T98G细胞培养、计数,设计一组细胞密度梯度并于低氧(1%02浓度下)处理24h,通过Western blot定量分析HIF-1a表达变化。3、采用脂质体转染法进行HIF-1a SiRNA质粒转染胶质瘤细胞系T98G细胞,运用Western blot技术在蛋白水平检测感染后稳定细胞系干扰的有效性。4、选取有效的SiRNA质粒转染T98G细胞,分别设为干扰组和空白对照,于1%02和21%氧浓度下培养24h、48h、72h、96h、120h,MTT检测各组细胞存活及增殖情况；流式细胞仪检测细胞凋亡情况。 [结果]1、正常脑胶质瘤细胞T98G于1%氧浓度下培养与21%氧浓度下培养,倒置显微镜下观察和Hoechest染色未见明显细胞凋亡,MTT检测低氧组与常氧组均呈增殖状态。1%氧浓度培养T98G细胞株4h、6h、12h、24h(24h),HIF-1α蛋白表达快速升高,24h、48h、72h(24h),HIF-1a蛋白表达是逐渐下降的。2、在不同细胞密度对T98G细胞HIF-1a表达的影响中,其中8.5万个细胞/Cm2密度的T98G细胞HIF-1a表达最高,细胞密度过高或过低都会导致HIF-1α表达降低。3、脂质体法包裹HIF-1α-SHRNA转染T98G细胞,经过G418筛选后,HIF-1α蛋白表达抑制率较高,可达至92.15%。4、Hoechest染色、流式细胞仪检测1%氧浓度下下干扰组和空白对照组细胞均无明显凋亡,MTT检测发现,低氧干扰组较低氧对照组增殖缓慢(P0.05),常氧干扰组细胞较低氧干扰组生长好(P0.05)。 [结论]1%低氧微环境不能诱导脑胶质瘤细胞发生凋亡；在1%02浓度低氧微环境中,T98G细胞低氧处理在24小时以内,其HIF-1α蛋白的表达是快速升高,T98G细胞低氧处理24h、48h、72h,其HIF-1α的表达是逐渐降低的。在低氧下其汇合度至95%左右HIF-1α的表达最高,细胞密度过高或过低均会降低HIF-1α的表达。干扰沉默T98G细胞HIF-1α并不能诱导T98G细胞凋亡,却能降低T98G细胞的增殖速度。
[Abstract]:[Objective: the aim of this study was to simulate the hypoxic microenvironment of glioma, observe the morphology of glioma cells under hypoxia by inverted microscope, and detect the effect of 1% oxygen concentration on apoptosis of glioma cells. Immunofluorescence and Western Blot techniques were used to detect the effect of different time gradient and cell density on the expression of HIF-1 伪. In vitro, RNA interference technique was used to silence hypoxia inducible factor-1 (HIF-1) to observe the proliferation of hypoxic glioma. The effect of apoptosis provides experimental and theoretical basis for further study on the treatment of glioma with HIF-1 as the target. [Methods: the hypoxic microenvironment was simulated by hypoxia incubator. The effect of hypoxia on the proliferation and apoptosis of glioma cells was detected by inverted microscope and Hoechest staining. T98G cells were cultured in hypoxia incubator for 4h-72 h. The expression of hypoxia inducible factor-1a (HIF-1 a) in T98G cells was detected by western blot at 4h-72 h after hypoxia. T98G cells were cultured and counted. A group of cell density gradient was designed and treated with hypoxia for 24 hours. The changes of HIF-1a expression were quantitatively analyzed by Western blot. The HIF-1a SiRNA plasmid was transfected into glioma cell line T98G by liposome transfection. Western blot technique was used to detect the interference effectiveness of stable cell lines after infection at the protein level. The effective SiRNA plasmid was selected to transfect T98G cells. The cells were cultured for 24 h, 48 h, 72 h, 96 h and 120 h, respectively as interference group and blank control group. The survival and proliferation of the cells in each group were detected by MTT. Apoptosis was detected by flow cytometry. [Results: 1. Normal glioma cells T98G were cultured at 1% oxygen concentration and 21% oxygen concentration respectively. No apoptosis was observed under inverted microscope and Hoechest staining. The expression of HIF-1 伪 protein in T98G cells cultured with 1% oxygen concentration in hypoxia group and normoxic group was rapidly increased at 4 h, 12h, 12h, 12h, 24h and 24h, respectively. The expression of HIF-1a protein decreased gradually in T98G cells with different cell densities. The expression of HIF-1a was the highest in T98G cells with 85,000 cell / / cm ~ 2 density. Too high or too low cell density could lead to the decrease of HIF-1 伪 expression in T98G cells. HIF-1 伪 -SHRNA was transfected into T98G cells by liposome method, and the inhibition rate of HIF-1 伪 protein expression reached 92.15.4 after G418 screening. Hoechest staining, flow cytometry detection of 1% oxygen concentration under the interference group and blank control cells were not significantly apoptotic. The proliferation of cells in hypoxic interference group was slower than that in hypoxic control group, while that in normoxic interference group was better than that in hypoxic interference group. [Conclusion: 1% hypoxia microenvironment can not induce apoptosis of glioma cells. The expression of HIF-1 伪 protein in T98G cells treated with hypoxia for 24 hours was increased rapidly after hypoxia for 24 h or 72 h. The expression of HIF-1 伪 decreased gradually, and its confluence degree reached about 95% under hypoxia. The expression of HIF-1 伪 was the highest. Too high or too low cell density could decrease the expression of HIF-1 伪, and interfering with silencing HIF-1 伪 of T98G cells could not induce the apoptosis of T98G cells, but could decrease the proliferation rate of T98G cells.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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