血管内皮生长因子-C干扰对人类结肠癌裸鼠移植瘤模型淋巴管生成的影响

Effect of VEGF-C interfering on the lymphangiogenesis of a mouse model bearing human colon cancer

目的构建携带有RNA干扰片段的腺病毒载体Ad5F35-VEGF-C siRNA-EGFP，探讨人类结肠癌BALB／c裸鼠皮下移植瘤模型瘤内注射腺病毒载体Ad5F35-VEGF-C siRNA-EGFP抑制血管内皮生长因子-C（VEGF-C）表达、结肠癌生长以及结肠癌淋巴管生成的效果。方法选择针对人类VEGF-C mRNA的特异性siRNA靶序列，设计合成其相应的双链DNA，利用BamHI、HindⅢ双酶切插入pDC316-EGFP-U6载体后构建穿梭质粒pDC316-VEGF-C siRNA-EGFP-U6，再与骨架质粒pBHGF35共转染293细胞，同源重组，酶切鉴定，包装扩增后得到重组腺病毒Ad5F35-VEGF-C siRNA-EGFP-U6。裸鼠皮下注射LoVo细胞构建人类结肠癌皮下移植瘤模型24只，随机分为4组（每组6只）：以腺病毒、空病毒、裸siRNA以及PBS分别进行瘤内原位注射干预，计算肿瘤体积变化并绘制生长曲线，使用抗LYVE-1单抗和抗CD34单抗分别染色瘤内的血管和淋巴管，检测瘤内淋巴管和血管生成情况，计算微血管密度（MVD），微淋巴管密度（LVD）。结果腺病毒组肿瘤体积明显小于其他各组，腺病毒组与PBS对照组LVD分别为8．47±2．1，17．35±4．7（P〈0．05），腺病毒组与PBS对照组MVD分别为22．65±6．04，23．19±7．63（P〉0．05）。结论实验设计并合成的腺病毒载体介导的VEGF-C siRNA，在人类结肠癌裸鼠移植瘤模型中，瘤内注射腺病毒载体能显著抑制移植瘤的生长和肿瘤淋巴管生成，而对肿瘤血管生长无显著影响。

Objective To construct recombinant adenovirus containing small interfering RNA （siRNA） targeting human VEGF-C mRNA, then to study the inhibitory effects of adenovirus-mediated VEGF- C siRNA on growth and lymphangiogenesis of human colon cancer in BABL/c nude mice model.Methods In vitro： the specific siRNA sequence targeting human VEGF-C mRNA was selected .The homologous doublestrand DNA was designed and synthesized. After such DNA was inserted into pDC316-EGFP-U6 by BamHI and HindⅢ, pDC316-VEGF-C siRNA-EGFP-U6 was obtained, then it was co-transfected into 293 cells with the bone plasmid pBHGF35. After generated by homologous recombination, Ad5F35-VEGF-C siRNA- EGFP-U6 was obtained, and it was packaged and amplified in 293 cells. The human colon cancer model was established in nude mice by hypo-injection LoVo cells. They were divided randomly into four groups （n=6）： adenovirus,virus without target gene, single siRNA and PBS control group. Injecting intervention intra-tumorly in each groups, calculating the tumors volume and drawing the growth curve, calculating micro-vascular density （MVD） and micro-lymphatic density （LVD） by staining respectively of the lymphatic and vascular with anti-LYVE-1 monoclonal antibody and anti-CD34 monoclonal antibody were completed. Results Adenovirus group tumor sizes were smaller than other groups. The LVD were 8.47±2.1 and 17.35±4.7 （P 〈0.05） in adenovirus group and the PBS control group.The MVD were 22.65±6.04 and 23.19±7.63 （P 〉0.05） in adenovirus group and the PBS control group respectively. Conclusion The adenovirus-mediated VEGF-C siRNA can significantly inhibit the growth and lymphangiogenesis of human colon cancer in nude mice model, but have no effect on microangium vessels growth.