Six1和TGF-β及VEGF-C在人喉鳞状细胞癌裸鼠移植瘤淋巴转移中的作用

Effect of Six1,TGF-β,VEGF-C that promoting tumor lymphangiogenesis in human laryngeal carcinoma xenografts in nude mice

目的：探讨Six1、转化生长因子β（TGF-β）在人喉鳞状细胞癌裸鼠移植瘤淋巴转移中对血管内皮生长因子C（VEGF-C）的作用。方法：利用基因干扰技术,分别沉默人喉鳞状细胞癌细胞内Six1和TGF-β基因的表达,制备Six1和TGF-β靶向siRNA转染至喉鳞状细胞癌细胞,筛选出阳性克隆细胞进行扩增,制备荷瘤裸鼠模型。致瘤小鼠随机分为5组：A组（未转染组）,B组（转染空载体组）,C组（Six1转染组）,D组（TGF-β转染组）,E组（Six1＋TGF-β转染组）。肿瘤生长到6～12mm时,将裸鼠脱颈处死,观察并记录各组肿瘤的体积及转移情况。Western blot及免疫组织化学检测各组Six1、TGF-β及VEGF-C蛋白的表达;RT-PCR检测各组Six1、TGF-β及VEGF-C mRNA的表达。结果：A、B组比较,平均肿瘤体积及转移只数无明显差异;同A组比较,C、D、E组中平均肿瘤体积无明显减轻,但转移只数减少;与C、D组比较,E组平均肿瘤体积无明显减轻,转移只数无明显减少。Western blot、免疫组织化学及RT-PCR结果显示,A、B组比较,VEGF-C蛋白及其mRNA表达无明显变化;与A组比较,C、D、E组中VEGF-C蛋白及其mRNA表达均出现减弱;与C、D组比较,VEGF-C蛋白及其mRNA的表达在E组未出现进一步降低。结论：Six1和TGF-β均能够调控VEGF-C mRNA及其蛋白的表达,进而调控人喉鳞状细胞癌的淋巴转移,提示Six1、TGF-β的变化可能成为临床抑制人喉鳞状细胞癌淋巴转移的潜在靶点。

Objective：To research the effect of Six1 ,TGF β,VEGF-C that promoting tumor lymphangiogenesis in human laryngeal carcinoma xenografts in nude mice. Method： Technology of RNA interference was used for si- lencing Six1 and TGF-β genes expression in laryngeal squamous cell, preparation Sixl-targeting and TGF-β-targe- ting siRNA for transfecting into laryngeal squamous cell carcinoma, sieve out positive clone cell and amplify. Pre- paring bearing cancer mice, the mice were divided into five groups, group A （untransfected）,group B（empty vec- tor）, group C（Sixl-siRNA）, group D（TGFβ-siRNA）, group E（Six1 ＋ TGF-β-siRNA）. 6 -- 12mm when the tumor has grown, the mice were sacrificed by cervical. The size of each tumor and metastasis were observed and recor- ded. The protein expression of Sixl,TGF-β and VEGF-C was determined by immunohistochemistry and Western blot. The mRNA of Six1 ,TGF-β and VEGF-C was determined by RT PCR. Result： The average tumor volume and the number of metastasis cases in group B have no statistically significant compared with group A. The average tumor volume in group C, group D and group E has no significantly reduced, but there is a clear reduction of the number of metastasis cases compared with group A. The average tumor volume and number of metastasis cases in group E has no significantly reduced compared with group C and group D. Both protein and mRNA expression of Six1, TGF-β and VEGF-C in group B had no significant difference compared with group A. In group C, group D and group E,both protein and mRNA expression of VEGF-C was decreased, difference has statistically significa-nt compared with group A. Both protein and mRNA expression of VEGF-C in group E had no significant differ- ence compared with group C and group D. Conclusion： Both Sixl and TGF-β can mediate tumor lymphangiogenesis and lymph node metastasis by mediate the expression of VEGF-C. Suggest that Sixl,TGF-β might be a potential therapeutic target for preventing lymph node metastasis of tumor.