shRNA沉默肾癌786-0和OS-RC-2细胞株中HIF-2α的表达对VEGF的影响

发布时间：2018-02-07 10:07

本文关键词： HIF-2α VEGF 免疫组化 786-0 OS-RC-2细胞 shRNA 肾癌　出处：《兰州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究低氧诱导因子2a (hypoxia-inducible factors-2α, HIF-2α)和血管内皮生长因子(vascular endothelial growth factor, VEGF)在肾细胞癌(renal cell carcinoma, RCC)中的表达及临床意义。构建在哺乳动物细胞中表达的低氧诱导因子-2的短发夹RNA的表达质粒,在体外模拟肿瘤常氧和低氧状态下研究对VEGF表达的影响。探讨在786-0和OS-RC-2细胞株中HIF-2a对VEGF的表达影响,HIF-2α可能为肾透明细胞癌(clear cell renal cell carcinoma, CCRCC)潜在的新效靶。方法：选用免疫组织化学方法,检测HIF-2a蛋白和VEGF蛋白的表达,其中76例RCC组织,12例癌旁组织,研究与RCC的相关性。构建HIF-2a的重组载体质粒psiHIV-U6, shRNA-1、2、3、4为重组质粒。转染至786-0细胞,常氧条件培养48h后提取总RNA逆转录cDNA后Real time-PCR筛选沉默效率最强的shRNA载体质粒。该质粒psiHIV-U6送公司测序鉴定。实验设空白、阴性对照组和实验组即重组质粒组,每组均设常氧组和低氧组,共6组,空白对照组不做任何处理,阴性对照组转染空质粒,实验组转染重组质粒psiHIV-U6,细胞缺氧微环境通过含150umol/L氯化钴(Cocl2)的完全培养基模拟。实时定量PCR分析HIF-2a shRNA重组质粒转染786-0和OS-RC-2细胞后常氧及低氧状态下细胞的HIF-2a mRNA表达,流式细胞仪分析凋亡变化,蛋白免疫印迹方法测定VEGF蛋白的变化。实验数据进行了spssl8.2统计学软件处理,计算资料以均值±标准差表示,临床特点指标采用x2检验和秩和检验,Spearman用来等级相关分析,组间比较应用方差分析,以P0.05有显著性差异。结果:HIF-2α和VEGF在正常肾组织中不表达,而在RCC组织中表达较强。HIF-2α与肿瘤组织的病理类型,病理分级及临床分期有显著相关性,VEGF与肿瘤组织病理分级及临床分期有显著相关性。DNA测序分析证实了重组质粒psiHIV-U6构建成功,在786-0细胞株中shRNA-4沉默HIF-2a mRNA的效率最强,shRNA-4可用于后续实验。HIF-2a重组质粒shRNA-4成功转染786-0和OS-RC-2细胞,转染成功的细胞发出绿色荧光,与对照组的细胞形态不同。RT-PCR分析显示：786-0细胞和OS-RC-2细胞中,在阴性对照组中,HIF-2a mRNA的表达低氧环境较高。实验组在低氧微环境下HIF-2α mRNA的表达量较高。低氧环境下较常氧环境下HIF-2α mRNA的表达量无明显变化。转染HIF-2α shRNA-4重组质粒在低氧环境下能诱导786-0和OS-RC-2细胞凋亡增加。采用Quantity one软件进行灰度值分析,阴性对照组低氧组VEGF蛋白表达高于常氧组,实验组VEGF蛋白表达量同时下降,且常氧较低氧环境下VEGF蛋白下降显著。结论:HIF-2α和VEGF与肾透明细胞癌的生长密切相关,HIF-2α的研究可能成为治疗CCRCC患者潜在的效靶,为下一步研究提供了基础。
[Abstract]:Objective: to study the expression and clinical significance of hypoxia-inducible factors-2 伪 (HIF-2 伪) and vascular endothelial growth factor vascular endothelial growth factor (VEGF) in renal cell carcinoma (RCC). The expression plasmid of hairpin RNA, The effect of HIF-2a on the expression of VEGF in 786-0 and OS-RC-2 cell lines was studied in vitro under simulated hypoxic and normoxic conditions. To explore the effect of HIF-2 伪 on VEGF expression in 786-0 and OS-RC-2 cell lines, HIF-2 伪 may be a potential new target for clear cell renal cell carcinoma (CCRCCs). Methods: immunohistochemical method was used to detect the expression of HIF-2a protein and VEGF protein, including 76 cases of RCC tissues and 12 cases of adjacent tissues, to study the correlation with RCC. The recombinant plasmid psiHIV-U6 of HIF-2a and shRNA-1m2m3O4 were constructed and transfected into 786-0 cells. After the total RNA reverse transcription cDNA was extracted under normoxic condition for 48 h, Real time-PCR was used to screen the shRNA vector plasmid with the best silencing efficiency. The plasmid psiHIV-U6 was sent to the company for sequencing. The experiment was blank, the negative control group and the experimental group were the recombinant plasmid group. Each group was divided into normoxic group and hypoxic group, six groups, blank control group did not do any treatment, negative control group transfected empty plasmid, In the experimental group, the recombinant plasmid psiHIV-1 U6 was transfected, and the cell anoxic microenvironment was simulated by a complete medium containing 150umoll / L cobalt chloride Cocl2.Real-time quantitative PCR was used to analyze the expression of HIF-2a mRNA in the cells transfected with HIF-2a shRNA recombinant plasmid 786-0 and OS-RC-2 cells in normoxic and hypoxic state. The changes of apoptosis were analyzed by flow cytometry and the changes of VEGF protein were measured by Western blot. The experimental data were processed by spssl8.2 software and the calculated data were expressed as mean 卤standard deviation. The clinical characteristics were analyzed by Spearman and x2 test and rank sum test respectively. ANOVA was used in the comparison between groups. There was significant difference between the two groups (P0.05). Results there was no expression of HIF-2 伪 and VEGF in normal renal tissues, but strong expression of .HIF-2 伪 in RCC tissues and pathological types of tumor tissues. There was a significant correlation between the pathological grade and clinical stage. DNA sequencing confirmed the successful construction of recombinant plasmid psiHIV-U6. In 786-0 cell line, shRNA-4 silencing HIF-2a mRNA was the most efficient, which could be used in further experiment. HIF-2a recombinant plasmid shRNA-4 was successfully transfected into 786-0 and OS-RC-2 cells. The cell morphology was different from that in the control group. RT-PCR analysis showed that, in the OS-RC-2 cells and the cell lines, In the negative control group, the expression of HIF-2a mRNA was higher in hypoxic environment, the expression of HIF-2 伪 mRNA in experimental group was higher in hypoxic microenvironment, but the expression of HIF-2 伪 mRNA in hypoxic environment had no significant change. The recombinant plasmid transfected with HIF-2 伪 shRNA-4 was transfected with HIF-2a. The apoptosis of 786-0 and OS-RC-2 cells was increased in hypoxic environment. The gray value of 786-0 and OS-RC-2 cells was analyzed by Quantity one software. The expression of VEGF protein in hypoxic group was higher than that in normoxic group. The expression of VEGF protein in experimental group was decreased at the same time, and the VEGF protein in normoxic group was significantly lower than that in hypoxic group. Conclusion the study on the relationship between the growth of clear cell carcinoma of kidney and the growth of HIF-2 伪 and VEGF may be a potential target for the treatment of CCRCC patients, which may provide a basis for further research.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

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