特定沉默胶质瘤U251细胞株的PDGF-B基因对细胞凋亡和增殖的影响研究  

The influences of PDGF-B gene of specific U251 silencing glioma cell lines on cell proliferation and apoptosis

作　　者：李令建 汤艳萍 

机构地区：[1]新疆维吾尔自治区职业病医院神经外科,新疆乌鲁木齐830000 [2]新疆维吾尔自治区职业病医院放射科磁共振,新疆乌鲁木齐830000

出　　处：《临床和实验医学杂志》2017年第8期756-760,共5页Journal of Clinical and Experimental Medicine

摘　　要：目的利用RNA干扰基因治疗技术,特定沉默胶质瘤U251细胞株的血小板衍生生长因子B(PDGF-B)基因,观察其对U251细胞株细胞凋亡和增殖的影响。方法用血清培养液(含10%胎牛血清DMEM培养基)体外培养人U251脑胶质瘤细胞系。随机分为3组,第1组为实验组:转染siRNA的细胞,第2组为阴性对照组:转染空白质粒的细胞;第3组为空白对照组:细胞常规培养不予以干预。利用脂质体将siRNA转染进入U251细胞,在细胞内形成双链siRNA,识别并降解PDGF-B mRNA。通过RT-PCR检测U251细胞中PDGF-B基因mRNA的表达水平,Western blot检测PDGF-B的蛋白表达,MTT法检测U251细胞的增殖情况,流式细胞学对比检测3组细胞的凋亡情况。结果利用脂质体将靶向PDGF-B基因siRNA转染进入U251细胞,48 h后利用RT-PCR检测PDGF-B基因mRNA的表达,结果显示与阴性对照组和空白对照组对比,转染组PDGF-B基因mRNA表达水平明显下降;转染72 h后,Western blot检测显示siRNA转染组PDGF-B蛋白表达明显抑制(抑制率>60%);MTT结果显示siRNA转染组U25细胞增殖较对照组明显降低;流式细胞学检测显示,与阴性对照组和空对照组相比,转染siRNA的细胞的实验组中,G1期细胞明显增多,S期细胞明显减少,提示降低PDGF-B在胶质瘤细胞的表达,能够使细胞周期阻滞在G1期,从而抑制胶质瘤细胞的增殖,并促进细胞的凋亡。结论构建靶向胶质瘤细胞PDGF-B的RNA干扰质粒并转染人胶质瘤U251细胞株后,通过体外试验观察可明显抑制U251细胞株PDGF的表达,其所产生的RNA干扰效应对人胶质瘤U251细胞株有明显的生长的抑制和促进凋亡作用。Objective To observe the influences of PDGF-B gene of specific U251 silencing glioma cell lines on cell proliferation and apoptosis by RNA interference of genetherapy technology. Methods Human U251 glioma cell lines were cultured in serum medium（containing10% fetal bovine serum in DMEM medium） in vitro. The cells were divided into three groups randomly. Group 1 were experimental group which were transfected with siRNA. Group 2 were negative control group which were transfected with empty plasmid; Group 3 were U251 cells without intervention. The siRNA were transfected into U251 cells by Liposomal,the cells formed a double-stranded siRNA to recognize and degrade PDGF-B mRNA. RT-PCR were used to detect the gene expression level of the PDGF-Band Western blot analysis to detect PDGF-B protein expression. U251 cell proliferation was detected by MTT. The apoptosis of three groups of cell were showed by the method of flow cytometry. Results siRNA of the PDGF-B gene were transfected into U251 cells by liposomal,and 48 hours later RT-PCR were used to detect the gene expression of PDGF-B. The results showed that PDGF-B gene expression in transfect group was significantly decreased than that of the negative control and blank control group. The analysis of Western blot 72 h after transfection showed that PDGF-B protein expression in siRNA transfect group was significantly inhibited（inhibition rate 60%）. MTT assay showed that in siRN Atransfect group U251 cell proliferation was significantly lower than that in the control group. Flow cytometric detection showed that G1 phase cells in the experimental group of cells transfect with the siRNA were increased significantly compared with the negative control group and blank control group,while S-phase cells were significantly reduced,which indicated that reduced expression of PDGF-B in glioma cell could effectively inhibit glioma cell mitosis and promote apoptosis. Conclusion When the RNA interference of U251 cells are constructed and the siRNA of the PDGF-B gene are transf

关 键 词：U251胶质瘤细胞 血小板衍生生长因子B RNA干扰 

分 类 号：R739.41[医药卫生—肿瘤]