Zhang Hai-yan, Gao Da-xin, Li Ping, Ren Lu-ping, Cao Cui-ping, Liu Guo-liang
Department of Endocrinology, the First Affiliated Hospital, China Medical University, Shenyang 110001, China.
Zhonghua Nei Ke Za Zhi. 2005 Apr;44(4):280-4.
To investigate the inhibitive effect of antisense oligonucleotide (ASODN) on vascular endothelial growth factor (VEGF) expression and endothelial cell growth in thyroid carcinoma.
Targeted ASODN of VEGF was designed and synthesized, then transfected to TT (medullary thyroid carcinoma) cell line and the culture supernatant was collected in which ECV304 (endothelial cell line) was seeded. At the same time positive control [sense oligonucleotides (SODN) group] and normal control were set for comparison. Cell growth condition was observed under microscope. RT-PCR and immuocytochemistry were used for detection of VEGF mRNA and protein expression in TT cells. MTT assay was used for cell growth inhibition ratio (IR) of TT and ECV304 cells, flow cytometry (FCM) for apoptotic index (AI) of ECV304 cells and acridine orange/ethidium bromide (AO/EB) staining for apoptotic morphology of ECV304 cells.
As compared with positive and normal control groups, VEGF mRNA and protein expressions in TT cells of ASODN transfection groups were obviously decreased (P < 0.01). Cell growth was not influenced apparently in ECV304 cells with direct ASODN administration, but ECV304 cell growth in ASODN conditioned medium was significantly inhibited and IR (0.21 +/- 0.03, 0.31 +/- 0.01, 0.42 +/- 0.22) was significantly higher than that of SODN group (0.05 +/- 0.03, P < 0.01), with the presence of apparent apoptosis. The effect mentioned above was in a dose-dependent manner.
ASODN can suppress endothelial cell growth and inhibit tumor angiogenesis possibly by specifically blocking VEGF expression in thyroid carcinoma.
探讨反义寡核苷酸（ASODN）对甲状腺癌血管内皮生长因子（VEGF）表达及内皮细胞生长的抑制作用。
设计并合成针对VEGF的靶向ASODN，转染至TT（甲状腺髓样癌）细胞系，收集培养上清液并接种ECV304（内皮细胞系）。同时设置阳性对照[正义寡核苷酸（SODN）组]和正常对照进行比较。在显微镜下观察细胞生长情况。采用RT-PCR和免疫细胞化学检测TT细胞中VEGF mRNA和蛋白表达。采用MTT法检测TT和ECV304细胞的生长抑制率（IR），流式细胞术（FCM）检测ECV304细胞的凋亡指数（AI），吖啶橙/溴化乙锭（AO/EB）染色观察ECV304细胞的凋亡形态。
与阳性对照组和正常对照组相比，ASODN转染组TT细胞中VEGF mRNA和蛋白表达明显降低（P<0.01）。直接给予ASODN对ECV304细胞生长无明显影响，但ASODN条件培养基中的ECV304细胞生长受到显著抑制，IR（0.21±0.03，0.31±0.01，0.42±0.22）明显高于SODN组（0.05±0.03，P<0.01），且存在明显凋亡。上述作用呈剂量依赖性。
ASODN可能通过特异性阻断甲状腺癌中VEGF的表达来抑制内皮细胞生长并抑制肿瘤血管生成。
