摘要
目的:血管内皮生长因子能促进血管内皮细胞增生和血管形成,而RNA干扰技术可高效特异地导致转录后基因沉默现象。实验针对人血管内皮生长因子基因化学合成小分子干扰RNA,观察其对胶质瘤U251细胞的体外干扰效应。方法:实验于2006-09/2007-02在中国科学院上海生物化学研究所分子生物学国家重点实验室完成。①实验材料:人类U251胶质母细胞瘤细胞株由上海市肿瘤研究所朱景德教授惠赠。非特异性小分子干扰RNA,绿色荧光标记的FAMnegativecontrolsiRNA(上海吉玛公司)。②实验方法:选择基因序列号为NM0033761的血管内皮生长因子基因cDNA序列上的3个位点设计3条不同序列的小分子干扰RNA,并应用BLAST技术排除其他同源基因。采用脂质体法转染U251细胞,以200nmol/L进行转染,以转染非特异性小分子干扰RNA作为阴性对照组,以未加入小分子干扰RNA作为空白对照组。48h后RT-PCR筛选最佳小分子干扰RNA,将抑制率最高的小分子干扰RNA再分别按50,100,200,300nmol/L进行转染。③实验评估:荧光显微镜及流式细胞仪检测转染效率,RT-PCR法半定量检测浓度梯度下小分子干扰RNA对U251细胞血管内皮生长因子mRNA表达的抑制效果。结果:①荧光显微镜及流式细胞术检测转染效率:镜下可见胞质内的绿色荧光,同一视野下细胞核呈蓝染,转染效率达95%以上。②RT-PCR检测血管内皮生长因子mRNA的表达水平:不同序列的小分子干扰RNA以200nmol/L转染细胞后血管内皮生长因子mRNA的表达水平均有所下调,以小分子干扰RNA1转染组最为明显(P<0.01),抑制率大于70%,选为最佳特异性小分子干扰RNA。U251细胞转染50,100,200,300nmol/L的小分子干扰RNA148h后,随着转染浓度的升高,小分子干扰RNA1的抑制效果逐渐增强,各浓度转染组血管内皮生长因子mRNA的表达水平可下调16%～73%。结论:以血管内皮生长因子为靶基因、化学修饰合成的特异性小分子干扰RNA,在体外可有效抑制目的基因mRNA的表达。
AIM： Vascular endothelial growth factor （VEGF） plays a key role in promoting vasiformation and endothelial cell proliferation. RNA interference is an efficient and specific post-transcriptional gene silencing mechanism. This study was designed to investigate the inhibition effect of small interference RNA （siRNA） synthesized chemically on targeting human VEGF expressed by glioma cells U251 in vitro. METHODS： The study was carded out in the National Key Laboratory of Molecular Biology, Shanghai Biochemistry Institute of Chinese Academy of Sciences from September 2006 to February 2007.①Experiment material： U251 human glioblastma cells were given by Professor Zhu JD of Shanghai Tumor Research Institute. Nonspecific siRNA and FAM negative control siRNA marked by green fluorescence were from Shanghai GenePharma Company. ②Experiment method： Three pairs of siRNAs targeting 3 different sites of VEGF gene cDNA （NM0033761） were designed and synthesized chemically in vitro after the other homologous genes were depleted by the BLAST technology. These siRNAs were transfected into U251 cells by 200 nmol/L using lipoplast method, while nonspecific siRNA as negative control and those without siRNA as blank control. The most effective siRNA was selected after 48 hours by RT-PCR and then those with the highest inhibition ratio were transfected into U251 cells by 50, 100, 200, 300 nmol/L, respectively.③ Experiment evaluation： The transfection efficiency was detected by fluorescent microscopy and flow cytometry. The inhibition effects of the siRNAs by different concentration gradients on VEGF mRNA expression were semi-quantitatively detected by RT-PCR. RESULTS： ①Transfection efficiency detected by fluorescent microscopy and flow cytometry： Green fluorescence in the cells was seen cleady in FAM-siRNA transfected group and blue nuclei were found in the same visual field under the fluorescent microscope, the transfection efficiency was over 95%. ②The expression of VEGF mRNA decreased in deferent degrees after various sequences of siRNAs transfected into U251 cells by 200 nmol/L. The most effective siRNA1 could cause VEGF inhibition by over 70% （P 〈 0.01 ）. The expression of VEGF mRNA decreased by 16%-73% after the siRNA1 transfected into U251 cells by deferent concentrations for 48 hours. The inhibition effect was enhanced with the increasing concentration of transfection. CONCLUSION： The synthesized chemically siRNAs on targeting VEGF can effectively inhibit the expression of VEGF mRNA in vitro.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第36期7219-7222,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30371459)
上海市科学技术发展基金资助项目(034047)~~
