实时荧光定量RT-PCR检测犬乳腺肿瘤血管内皮生长因子(VEGF)基因方法的建立及初步应用

Development and application of canine mammary gland tumor VEGF gene using real-time RT-PCR

为了准确地分析血管内皮生长因子(Vascular endothelial growth factor,VEGF)在犬乳腺肿瘤形成过程中的作用,试验设计了针对犬VEGF基因的特异性引物,提取乳腺肿瘤组织RNA并扩增出目的片段,将PCR产物克隆到pGEM-T Easy载体,经质粒PCR扩增、酶切和测序鉴定重组质粒,构建标准曲线等,成功建立了乳腺肿瘤VEGF基因实时荧光定量PCR检测方法。运用实时荧光定量PCR检测了40例不同的犬乳腺肿瘤病例、2例正常乳腺组织。结果发现:在犬恶性乳腺肿瘤组织中,VEGF基因表达量明显高于良性乳腺肿瘤和正常乳腺组织(P<0.001);在伴有淋巴结转移的犬乳腺癌组织中,VEGF基因表达量明显高于没有发现转移的乳腺癌组织(P<0.01),但VEGF基因的表达量与肿瘤的大小和发病动物年龄无关(P>0.05)。VEGF基因在犬乳腺癌组织中的表达量远高于其在正常乳腺和良性乳腺肿瘤组织中的表达,且与淋巴结是否发生转移相关。因此,VEGF可作为犬乳腺癌转移、复发的预测指标之一。

To analyze accurately the role of vascular endothelial growth factor（VEGF） with the canine mammary gland tumor,a method of real-time PCR to examine canine mammary tumor VEGF gene expression was established.Total RNA was extracted from tumor sample and the fragments of target gene were amplified by RT-PCR with special primer.PCR product was cloned via pGEM-T Easy vector and plasmid extracted.The success of recombinant plasmid was identified by PCR amplified and restriction enzyme analysis.40 canine mammary gland tumors,2 normal mammary gland were investigated by real-time RT-quantitative PCR.VEGF gene expression level in canine malignant mammary tumor was much higher than that in benign mammary tumor（P〈0.001）;Similarly,the positive expression of VEGF in mammary cancer with lymph node metastasis was much higher than that in mammary cancer without lymph node metastasis（P〈0.01）,but the expression of VEGF was not correlated with tumor size and patient′ age（P〉0.05）.In conclusion,the expression of VEGF in canine malignant mammary tumor was significantly elevated than in benign and normal mammary tissues and was related with lymph node metastasis.It may be a marker to predict the metastasis and recurrence of canine mammary cancer.