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目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。
Objective To investigate the effect of antisense oligonucleotide (ASODN) on the expression of vascular endothelial growth factor (VEGF) and the growth of endothelial cells in thyroid cancer cells. Methods ASODNs targeting VEGF were transfected into human myeloid thyroid carcinoma (TT) cells and ECV304 cells were prepared. The expression of SOD was compared with that of blank control group. The growth of the cells was observed by RT PCR. Immunocytochemistry was used to detect the expression of VEGF mRNA and protein in TT cells. The growth inhibition rate (TT) of TT and ECV304 cells were detected by tetrazolium blue method. Flow cytometry, acridine orange / ethidium bromide The apoptosis of ECV304 cells was detected by staining. Results The expression of VEGF mRNA and protein in TT cells of ASODN group was significantly lower than that of SODN group and control group (P <0.01), but there was no significant difference in IR (P> 0.05). There was also no significant difference in the IR of ECV304 cells > 0.05). However, the growth of ECV304 cells treated with TT cells in ASODN group was significantly inhibited (IR 0.21 ± 0.03,0.31 ± 0.01,0.42 ± 0.22, P <0.05) <0.01), accompanied by significant apoptosis, the above-mentioned effects in a concentration-dependent manner. Conclusion ASODN can specifically block the expression of VEGF in thyroid carcinoma cells, inhibit the growth of endothelial cells and interfere with tumor angiogenesis.