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

作　　者：农晓琳[1] 李昊[1] 夏勇[1] 黎彦[1] 李佳荃[2] 杨亦萍[3] 

机构地区：[1]广西医科大学口腔医学院口腔颌面外科,南宁530021 [2]广西医科大学医学实验中心 [3]广西医科大学口腔医学院口腔病理科,南宁530021

出　　处：《中华口腔医学杂志》2011年第1期15-19,共5页Chinese Journal of Stomatology

基　　金：国家自然科学基金（30060082）;教育部“春晖计划”科研项目启动基金（教外司留[2003]593号）;广西自然科学基金（2010GXNSFD013047）;广西壮族自治区卫生厅重点科研项目（重200006、重200927）;广西研究生教育创新计划（2007重-16、2009105981003M174）

摘　　要：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.

关 键 词：RNA干扰 血管内皮生长因子 抗药性 肿瘤 舌肿瘤 

分 类 号：R739.86[医药卫生—肿瘤]