短发夹RNA干扰血管内皮生长因子对舌癌耐药细胞移植瘤的影响

In vivo short hairpin RNA interference of vascular endothelial growth factor on drug-resistant tongue cancer cells

目的 研究短发夹RNA（short hairpin RNA,shRNA）干扰血管内皮生长因子（vascular endothelial growth factor,VEGF）的表达对人舌癌耐药细胞Tca-顺铂（Cisplatin,DDP）移植瘤的影响及可能机制.方法 以人舌癌Tca8113细胞系为亲本诱导建立耐药Tca-DDP细胞系,将其接种于裸鼠背侧皮下,建立移植瘤模型.模型动物分为4组,即空白对照组（非转染）、空载体组（转染空质粒载体）、无关片段组（转染无关对照质粒）、干扰质粒组（转染靶向VEGF的shRNA表达载体）,每组6只,每3天以脂质体为载体作瘤体内及瘤周多点注射.观测移植瘤生长曲线,转染l0次后取瘤体称重,免疫组化法检测移植瘤微血管密度,原位杂交法检测移植瘤VEGFmRNA,免疫组化技术检测移植瘤VEGF、多药耐药蛋白P糖蛋白（P-glycoprotein,P-gp）、B细胞淋巴瘤-白血病因子2（B cell lymphoma/leukemia-2,bcl-2）及胞外信号调节激酶2蛋白（extracellular signal-regulated kinase 2,ERK-2）.结果 干扰质粒组的瘤体生长明显受到抑制（P＜0.05）,移植瘤重量（0.4781±0.0860）g及微血管密度（7.35±1.31）个/视野,低于其他3组（P＜0.05）,且VEGF的mRNA及蛋白、P-gp、bcl-2及ERK-2的相对表达量分别为（0.0767±0.0234）、（0.1301±0.0433）、（0.1517±0.0184）、（0.1218±0.0251）及（0.1178±0.0291）,表达均显著减少（P＜0.05）.结论 RNA干扰抑制VEGF的表达,可抑制人舌癌Tca8113耐药细胞移植瘤的生长及血管生成,下调P-gp、bcl-2及ERK-2表达水平,提示靶向抑制VEGF可能是治疗耐药舌癌途径之一.

Objective To study the in vivo interference effects of vascular endothelial growth factor （VEGF） short hairpin RNA （shRNA） on xenografts of drug-resistant tongue cancer cells. Methods Drugresistant tongue caner cells Tca/Cisplatin（DDP） were injected subcutaneously into nude mice to establish xenograft models, which were randomly divided into non-transfected group, mock control group, control group transfected with scrambled sequence plasmid, interference group transfected with VEGF-shRNA expression plasmid. Liposome-mediated plasmid transfection was done in the latter three groups every three days. Xenografts were observed and tumor growth curve was measured. After the 10th transfection, tumors were anatomized and weigh. Microvessel density was detected by immunohistochemical staining. In situhybridization assay was used to test VEGF mRNA, and immunohistochemistry to test VEGF, P-glycoprotein （ P-gp）, B cell lymphoma/leukemia-2 （ bcl-2 ） and extracellular signal-regultaed kinase 2 （ERK-2） protein. Results Tumor growth in VEGF-shRNA interference group was significantly slow. Tumor weight was （0.4781 ±0.0860） g, microvessel density （7.35 ± 1.31）/view, VEGF mRNA（0.0767 ±0.0234）, VEGF protein （0.1301 ± 0.0433 ）, P-gp （0.1517 ± 0.0184 ）, bcl-2 （ 0.1218 ± 0.0251 ） and ERK-2 protein （0.1178 ±0.0291 ） in VEGF-shRNA interference group; all of them were less than those in the other three groups（ P 〈 0.05 ）. Conclusions Inhibition targeting VEGF may become a potential therapy for drugresistant tongue cancer.