反义寡核苷酸对人淋巴瘤细胞系Namalwa细胞血管内皮生长因子表达影响的体外研究_临床医学论文.docVIP

反义寡核苷酸对人淋巴瘤细胞系Namalwa细胞血管内皮生长因子表达影响的体外研究_临床医学论文 反义寡核苷酸对人淋巴瘤细胞系Namalwa细胞血管内皮生长因子表达影响的体外研究_临床医学论文 作者：黎纬明，张敏，邹菁，童允洁，邹萍【摘要】 为了研究硫代磷酸化修饰的血管内皮生长因子（vascular endothelial growth factor, VEGF）反义寡核苷酸(antisense oligodeoxynucleotide, ASODN)对人淋巴瘤细胞系Namalwa细胞VEGF表达的影响， 将终浓度分别为5、10、20 μmol/L的VEGF ASODN和错义序列与人淋巴瘤细胞系Namalwa细胞分别孵育24、48小时，采用RT检测VEGF mRNA的表达，采用链酶菌抗生素蛋白－过氧化酶免疫组织化学法（streptavidin/peroxidase, SP法）检测VEGF的表达。结果表明： VEGF ASODN 3个浓度组（5、10和20 μmol/L）处理的Namalwa细胞VEGF mRNA的表达分别为1.38、0.96、0.57, 错义序列组和对照组分别为1.79、1.84。当加入20 μmol/L VEGF ASODN作用48小时后，细胞内VEGF蛋白水平显著减少，而错义序列组Namalwa细胞VEGF蛋白水平未见明显改变。结论： VEGF ASODN在体外能够抑制Namalwa细胞VEGF的表达。 【关键词】 淋巴瘤 The recent studies have shown that in nongher grading of NHL and highgrade transformation of lowgrade lymphoma［1］. Data from Potti et al［2］ indicated that signal transduction inhibition therapy using VEGF Bellamy et al［3］ reported that VEGF over expressed in human lymphoma cell line Namalwa cells. We used VEGF ASODN and scrambled sequence transfecting Namalwa cell respectively, and confirmed the significant inhibition of VEGF ASODN on the VEGF mRNA expression, thus provided experimental and theoretical basis for the antisense gene therapy of lymphoma. Materials and Methods Drugs and reagents VEGF ASODN sequence was designed based on the human VEGF mRNA +261 to +281 nucleotides (5 TGGCTTGAAGATGTACTCGAT3), and the scrambled (SCODN) sequence was 5TACGTAGTATGGTGTACGATC3［4］. All these were prepared in the suitable concentration with RPMI Co. The SP kits were purchased from Beijing Zhongshan Biotechnology Ltd. The TRIZOL kits were purchased from Gibco, cDNA firststrand synthesis reaction kits were the products of MBI Co (purchased from Jingmei Biotech company). The dNTP and DNA enzyme were the products of Huamei Company. Effect of Antisense Oligodeoxynucleotides on the Expression of Vascular Endothelial Growth Factor in Namalwa cell Cell culture and treatment of ASODN Human lymphoma cell line Namalwa cells were obtained from Shanghai Bioc