兰州大学机构库 >生命科学学院
PTEN重建联合VEGF基因敲除有效治疗人神经胶质瘤
Alternative TitleCombination of PTEN reconstruction and VEGF knockdown offered a potential gene therapeutic strategy for malignant gliomas.
王会霞
Thesis Advisor王新宇 ; 黄来强
2010-05-23
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword神经胶质瘤 PTEN VEGFsiRNA
Abstract胶质母细胞瘤是一种预后极差的高恶性颅内肿瘤。血管内皮生长因子(VEGF)过度表达,同时抑癌蛋白PTEN功能缺失是该肿瘤的两个重要特征。目前,肿瘤的联合治疗可通过抑制不同的细胞信号通路进而克服肿瘤的药物耐受性,成为一种很有前景的治疗策略。本文研究野生型PTEN蛋白重建联合VEGF siRNA对人神经胶质瘤细胞U251增殖的抑制作用及对其致瘤性的影响。结果如下:1)重组载体构建与稳定转染细胞系建立的结果。成功构建pRNATU6.1/Neo-VEGFsiRNA质粒,将PTEN 蛋白cDNA序列构建到四环素调控型的反转录病毒载体pRetro-on中,重组载体转染U251细胞。建立了PTEN稳转细胞系和PTEN与shRNA共表达的双稳转细胞系。2)VEGF siRNA干涉效率和PTEN诱导表达的结果。实时定量real-time PCR检测发现,细胞中VEGF的 mRNA水平下降了58.5%;ELISA分析,转染VEGF siRNA 后,培养基中VEGF的分泌量明显降低,表明VEGF siRNA 基因敲除效率显著。western blot检测PTEN蛋白成功得到诱导表达。3)PTEN重建与VEGF基因敲除对细胞增殖、细胞周期和细胞凋亡影响的结果。PTEN诱导表达后,AKT的磷酸化水平显著下降,而在VEGF siRNA表达组中,AKT的磷酸化水平变化并不明显。PTEN诱导组而非VEGF shRNA瞬时转染组显著抑制U251细胞的增殖;PTEN重建能够显著地增加G0/G1期细胞的比份,但VEGF shRNA的转染并未对U251细胞的周期造成明显的影响。这表明,PTEN 重建能够抑制U251细胞的增殖,将细胞周期阻滞在G0/G1期,促进细胞的凋亡。4)PTEN重建与VEGF基因敲除对细胞克隆形成与迁移能力的影响。瞬时转染的VEGF siRNA虽然不能抑制U251细胞的增殖和细胞周期进程,但在稳定转染细胞中却能够有效地抑制锚定不依赖性克隆形成能力和细胞迁移能力,而且,PTEN联合VEGF siRNA实验组抑制作用更明显。5)在体内肿瘤模型实验中,PTEN诱导组和VEGF siRNA组都能显著抑制U251细胞的增殖,并且在联合治疗组中没有观察到明显的瘤体组织。 综上所述,野生型PTEN重建联合VEGF siRNA证实是一种有效的人神经胶质瘤治疗方案。
Other AbstractGlioblastoma (GBM) is a highly malignant tumor with poor prognosis.High expression of vascular endothelial growth factor (VEGF) and the depletion of PTEN are two of the most important hallmarks of GBM.To date,combined therapies are becoming a promising strategy owning to its potential ability of overcoming tumor drug resistance produced in monotherapy via inhibiting different signaling pathways.So in present research, we want to determine whether combined gene therapy of wild-type PTEN reconstruction and VEGF siRNA is a more effective approach for inhibition of tumor growth and tumorigenicity of PTEN-null glioblastoma.1) The results of recombination vectors and the stable expression cell lines.siRNA sequences were synthesized and inserted between the BamHI and HindIII restriction sites of pRNATU6.1/Neo for shRNA expression.PTEN cDNA sequence was cloned into tetracycline-inducible retroviral expression vector pRetro-on.The recombinant construct was confirmed by both restriction enzyme analysis and sequencing.Stable PTEN inducible U251 cell line was selected with 1ug/mL puromycin and induced by 1ug/mL doxycycline.Double stable expression of PTEN and shRNA were selected by 600ug/mL G418.2) The efficiency of VEGF shRNA construct and the inducd expression of PTEN.The mRNA level of VEGF was tested by real-time PCR at 48h post-transfection.Protein expression of VEGF in the cell culture supernatant were determined by ELISA.We get a marked knockdown effect:58.5% and a significant reduction of VEGF secretion level.At 48h post-transfection and doxycycline treatment,the results of western blot showed that PTEN was successively induced.3) The results of cell proliferation,cell cycle and cell apoptosis.Cell proliferation was measured by MTT assay and phospho-Akt protein expression was detected by Western blot.Cell cycle was analyzed using flow cytometry and cell early apoptosis was detected by annexin V-PE staining.PTEN restoration could affect proliferation, arrest cell cycle at G0/G1 stage and promote apoptosis of U251 glioblastoma cells via inhibition of PI3K/AKT signaling pathway in vitro.4) The results of colony formation ability and cell migration.Colony formation ability was analysed by Anchorage-dependent and independent conony formation assay.Cell migration ability was measured using a wound-healing assay.In spite of the fact that transient transfection of VEGF shRNA failed to affect the growth and cell cycle progression of U251 cells,the VEGF siRNA seemed ...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221879
Collection生命科学学院
Recommended Citation
GB/T 7714
王会霞. PTEN重建联合VEGF基因敲除有效治疗人神经胶质瘤[D]. 兰州. 兰州大学,2010.
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