iNOS反义寡核苷酸对肿瘤血管生成抑制的研究

Inhibition of angiogenesis in cancer by iNOS antisense oligodeoxynucleotide

目的:探讨诱导型一氧化氮合成酶(induciblenitricoxidesynthase,iNOS)的反义寡核苷酸对肿瘤血管生成的抑制作用。方法:将人工合成的互补于iNOS基因翻译起始部位的反义-寡核苷酸(AS-ODN)及其错义-寡核苷酸(Mis-ODN)经脂质体介导转染入A549细胞后,了解其产生一氧化氮(nitricoxide,NO)的能力;将AS-ODN及其错义序列、空白对照分别在第1、4、7、12和18天注入接种有A549肺腺癌细胞的裸鼠的腹腔中,于第21天检测小鼠体内肿瘤的大小、肿瘤内血管密度(microvesseldensity,MVD)和肿瘤组织血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)表达的情况。结果:体外实验显示,iNOS的AS-ODN组和对照组相比,对A549细胞产生NO的抑制能力较强,并呈时间和剂量依赖性。在动物实验中,iNOS的AS-ODN对肿瘤的抑制作用较Mis-ODN和空白序列为强,差异均有统计学意义,F值分别为3·24和3·89,P值分别为0·044和0·032;AS-ODN组、Mis-ODN组及空白对照组中MVD的平均值分别为6·9±3·2、11·8±6·8和12·9±5·4,AS-ODN组MVD较Mis-ODN组和空白组明显降低,差异均有统计学意义,F值分别为5·53和7·63,P值分别为0·006和0·001;同时其VEGF的表达阳性率较Mis-ODN组明显下降,u=-2·457,P=0·017。结论:iN-OS的AS-ODN可降低肿瘤细胞产生NO的能力,在动物体内可降低肿瘤VEGF的表达和新生血管的生成,抑制肿瘤的生长。

OBJESTIVE： To investigate the effect of antiangiogenic activity induced by iNOS antisense oligodeoxynucleotide（AS-ODN）. METHODS： A549 cell was transfected with a synthetic of oligodeoxynucleotide complementary to the beginning region of translation of iNOS. Then the production of NO was measured. AS-ODN and its control sequences, mismatch oligodeoxynucleotide（Mis-ODN） and saline solution were delivered into nude mice intraperitoneally at day 1, 4, 7, 12,18after A549 tumor cell implantation. On day 10 the mice were sacrificed and wight of tumors were measured. Microvessel density（MVD） and expression of vascular endothelial growth factor（VEGF） was detected via immumohistochemistry method. RESULTS： NO of AS-ODN group was lower than that of Mis-ODN and in saline solution group in a timedependent and dose-dependent manner. Weight of tumor in AS-ODN was lower than that in Mis-ODN and saline solution group,F=3.24,3.89, P=0. 044,0. 032. MVD of AS-ODN group, Mis-ODN group, and saline solution group were 6.9±3.2,11.8±6.8, and 12.9±5.4, respectively, F= 5.53,7.63, P = 0. 006, 0. 001; Positive expression rate of VEGF protein in ASODN treated mice was significantly lower than those treated by Mis-ODN, u=-2.457,P=0.017. CONCLUSIONS：iNOS AS-ODN can reduce the production of NO in tumor cells, down-regulate expression of VEGF and suppress angiogenesis in vivo.