靶向RhoA基因的shRNA对卵巢上皮性癌裸鼠移植瘤的影响及作用机制  

Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivo

作　　者：蒋文燕[1] 康佳丽[1] 王小霞[1] 杨文娟[1] 聂妙玲[1] 周聪[1] 

机构地区：[1]广州医学院附属广州市第一人民医院妇产科,广州510180

出　　处：《中华妇产科杂志》2013年第10期778-783,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：广东省科学计划（20108031600185）

摘　　要：目的探讨靶向RhoA基因的短发夹状RNA（shRNA）对卵巢上皮性癌（卵巢癌）裸鼠移植瘤的影响及可能的抗肿瘤作用机制。方法建立卵巢癌裸鼠皮下移植瘤模型，应用随机数字表法将荷瘤裸鼠随机分为3组，即实验组（予携带靶向RhoA基因的shRNA慢病毒液治疗）、阴性对照组（予携带针对随机无关序列的shRNA慢病毒液治疗）、空白对照组（予磷酸盐缓冲液），比较3组裸鼠的移植瘤生长情况，包括移植瘤生长速度、肿瘤体积、肿瘤质量；光镜下观察3组裸鼠移植瘤及主要器官组织的病理形态学特征。采用实时荧光定量PCR技术、免疫组化SP法和蛋白印迹法检测移植瘤组织中RhoAmRNA和蛋白的表达；脱氧核苷酸末端转移酶介导的核苷酸缺口末端标记（TUNEL）法检测3组裸鼠移植瘤细胞的凋亡情况[以凋亡指数（AI）表示]。结果（1）自治疗开始的第9天起，实验组裸鼠移植瘤的生长速度滞后于阴性对照组和空白对照组，差异均有统计学意义（P〈0．01）。治疗结束后10d，实验组裸鼠的移植瘤体积为（338±114）mm^3，小于阴性对照组和空白对照组[分别为（1190±332）和（1101±396）mm^3]，分别比较，差异均有统计学意义（P〈0．01）；实验组裸鼠平均肿瘤质量为（0．23±0．11）g，低于阴性对照组和空白对照组[分别为（0．79±0．19）、（0．74±0．17）g]，分别比较，差异均有统计学意义（P〈0．01）。而阴性对照组裸鼠移植瘤的生长速度、移植瘤体积、肿瘤质量分别与空白对照组比较，差异均无统计学意义（P〉0．05）。光镜下观察，实验组肿瘤细胞呈大片坏死区域且核固缩多见，而阴性对照组和空白对照组肿瘤细胞无明显变化。（2）实验组裸鼠移植瘤组织中，RhoAmRNA的表达水平为0．304±0．05，低于阴性对照组的0．95±0．06和空白对照组的1．00±0．11，分别比较，差异均�Objective To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA（shRNA） on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism. Methods Human ovarian cancer cell line H08910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer. Tumor-bearing nude mice were assigned randomizely to three groups： Lenti RhoA-sh group, Lenti- negative control （ NC ） group and phosphate buffered saline （PBS） group, lentivirus mediated RhoA shRNA, negative control lentivirus and PBS were respectively injected in the three groups. Effects of treatment were observed by tumor growth curve, tumor volume , tumor weight, and tumor inhibition rate. Xenograft tissues and liver, spleen, lung, and renal tissues were examined by hematoxylin and eosin （HE） staining or were detected by streptavidin-perosidase（SP） immunochemical method. The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR, immunochemistry and Western blot assay. Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling（TUNEL） method and apoptotic index （AI） were counted. Results Compared with Lenti-NC group and PBS group, the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days （P 〈 0. 01 ）.Tumor volume （338 ± 114） cm^3 decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group（ 1190 ± 332 ） mm3 and PBS group （ 1101 ± 396 ） cm^3 （ P 〈 0.01 ）. Tumor weight （0. 23 ±0. 11）g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group （0. 79± 0. 19）g and PBS group （0. 74 ± 0. 17）g （P 〈 0. 01 ）. Real-time qPCR result shown that the expression of RhoA mRNA （0. 30 ± 0. 05 ） decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group （0. 95 ±0. 06）

关 键 词：卵巢肿瘤 异种移植模型抗肿瘤试验 pA GTP结合蛋白质 RNA 小分子干扰 基因疗法 

分 类 号：R737.31[医药卫生—肿瘤]