中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2013年
10期
778-783
,共6页
蒋文燕%康佳丽%王小霞%杨文娟%聂妙玲%周聪
蔣文燕%康佳麗%王小霞%楊文娟%聶妙玲%週聰
장문연%강가려%왕소하%양문연%섭묘령%주총
卵巢肿瘤%异种移植模型抗肿瘤试验%ρA GTP结合蛋白质%RNA,小分子干扰%基因疗法
卵巢腫瘤%異種移植模型抗腫瘤試驗%ρA GTP結閤蛋白質%RNA,小分子榦擾%基因療法
란소종류%이충이식모형항종류시험%ρA GTP결합단백질%RNA,소분자간우%기인요법
Ovarian neoplasms%Xenograft model antitumor assays%rhoA GTP-binding protein%RNA,small interfering%Gene therapy
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RhoA mRNA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) mm3,小于阴性对照组和空白对照组[分别为(1190 ±332)和(1101±396) mm3],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同缩多见,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,RhoA mRNA的表达水平为0.30±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,差异均有统计学意义(P＜0.01)；RhoA蛋白的表达水平为0.14±0.06,低于阴性对照组的0.78±0.14和空白对照组的0.75 ±0.13,分别比较,差异均有统计学意义(P＜0.01)；实验组肿瘤细胞的AI为(20.9±3.4)％,高于阴性对照组的(5.2±2.0)％和空白对照组的(6.0±2.1)％,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组织中RhoA mRNA和蛋白的表达水平及AI分别与空白对照组比较,差异均无统计学意义(P＞0.05).结论 靶向RhoA基因的shRNA在体内有效下调卵巢癌裸鼠皮下移植瘤组织中RhoA基因的表达,抑制卵巢癌裸鼠皮下移植瘤的生长,其机制可能与促进细胞凋亡有关.
目的 探討靶嚮RhoA基因的短髮夾狀RNA(shRNA)對卵巢上皮性癌(卵巢癌)裸鼠移植瘤的影響及可能的抗腫瘤作用機製.方法 建立卵巢癌裸鼠皮下移植瘤模型,應用隨機數字錶法將荷瘤裸鼠隨機分為3組,即實驗組(予攜帶靶嚮RhoA基因的shRNA慢病毒液治療)、陰性對照組(予攜帶針對隨機無關序列的shRNA慢病毒液治療)、空白對照組(予燐痠鹽緩遲液),比較3組裸鼠的移植瘤生長情況,包括移植瘤生長速度、腫瘤體積、腫瘤質量；光鏡下觀察3組裸鼠移植瘤及主要器官組織的病理形態學特徵.採用實時熒光定量PCR技術、免疫組化SP法和蛋白印跡法檢測移植瘤組織中RhoA mRNA和蛋白的錶達；脫氧覈苷痠末耑轉移酶介導的覈苷痠缺口末耑標記(TUNEL)法檢測3組裸鼠移植瘤細胞的凋亡情況[以凋亡指數(AI)錶示].結果 (1)自治療開始的第9天起,實驗組裸鼠移植瘤的生長速度滯後于陰性對照組和空白對照組,差異均有統計學意義(P＜0.01).治療結束後10d,實驗組裸鼠的移植瘤體積為(338±114) mm3,小于陰性對照組和空白對照組[分彆為(1190 ±332)和(1101±396) mm3],分彆比較,差異均有統計學意義(P＜0.01)；實驗組裸鼠平均腫瘤質量為(0.23 ±0.11)g,低于陰性對照組和空白對照組[分彆為(0.79 ±0.19)、(0.74±0.17)g],分彆比較,差異均有統計學意義(P＜0.01).而陰性對照組裸鼠移植瘤的生長速度、移植瘤體積、腫瘤質量分彆與空白對照組比較,差異均無統計學意義(P＞0.05).光鏡下觀察,實驗組腫瘤細胞呈大片壞死區域且覈同縮多見,而陰性對照組和空白對照組腫瘤細胞無明顯變化.(2)實驗組裸鼠移植瘤組織中,RhoA mRNA的錶達水平為0.30±0.05,低于陰性對照組的0.95±0.06和空白對照組的1.00±0.11,分彆比較,差異均有統計學意義(P＜0.01)；RhoA蛋白的錶達水平為0.14±0.06,低于陰性對照組的0.78±0.14和空白對照組的0.75 ±0.13,分彆比較,差異均有統計學意義(P＜0.01)；實驗組腫瘤細胞的AI為(20.9±3.4)％,高于陰性對照組的(5.2±2.0)％和空白對照組的(6.0±2.1)％,分彆比較,差異均有統計學意義(P＜0.01).而陰性對照組裸鼠移植瘤組織中RhoA mRNA和蛋白的錶達水平及AI分彆與空白對照組比較,差異均無統計學意義(P＞0.05).結論 靶嚮RhoA基因的shRNA在體內有效下調卵巢癌裸鼠皮下移植瘤組織中RhoA基因的錶達,抑製卵巢癌裸鼠皮下移植瘤的生長,其機製可能與促進細胞凋亡有關.
목적 탐토파향RhoA기인적단발협상RNA(shRNA)대란소상피성암(란소암)라서이식류적영향급가능적항종류작용궤제.방법 건립란소암라서피하이식류모형,응용수궤수자표법장하류라서수궤분위3조,즉실험조(여휴대파향RhoA기인적shRNA만병독액치료)、음성대조조(여휴대침대수궤무관서렬적shRNA만병독액치료)、공백대조조(여린산염완충액),비교3조라서적이식류생장정황,포괄이식류생장속도、종류체적、종류질량；광경하관찰3조라서이식류급주요기관조직적병리형태학특정.채용실시형광정량PCR기술、면역조화SP법화단백인적법검측이식류조직중RhoA mRNA화단백적표체；탈양핵감산말단전이매개도적핵감산결구말단표기(TUNEL)법검측3조라서이식류세포적조망정황[이조망지수(AI)표시].결과 (1)자치료개시적제9천기,실험조라서이식류적생장속도체후우음성대조조화공백대조조,차이균유통계학의의(P＜0.01).치료결속후10d,실험조라서적이식류체적위(338±114) mm3,소우음성대조조화공백대조조[분별위(1190 ±332)화(1101±396) mm3],분별비교,차이균유통계학의의(P＜0.01)；실험조라서평균종류질량위(0.23 ±0.11)g,저우음성대조조화공백대조조[분별위(0.79 ±0.19)、(0.74±0.17)g],분별비교,차이균유통계학의의(P＜0.01).이음성대조조라서이식류적생장속도、이식류체적、종류질량분별여공백대조조비교,차이균무통계학의의(P＞0.05).광경하관찰,실험조종류세포정대편배사구역차핵동축다견,이음성대조조화공백대조조종류세포무명현변화.(2)실험조라서이식류조직중,RhoA mRNA적표체수평위0.30±0.05,저우음성대조조적0.95±0.06화공백대조조적1.00±0.11,분별비교,차이균유통계학의의(P＜0.01)；RhoA단백적표체수평위0.14±0.06,저우음성대조조적0.78±0.14화공백대조조적0.75 ±0.13,분별비교,차이균유통계학의의(P＜0.01)；실험조종류세포적AI위(20.9±3.4)％,고우음성대조조적(5.2±2.0)％화공백대조조적(6.0±2.1)％,분별비교,차이균유통계학의의(P＜0.01).이음성대조조라서이식류조직중RhoA mRNA화단백적표체수평급AI분별여공백대조조비교,차이균무통계학의의(P＞0.05).결론 파향RhoA기인적shRNA재체내유효하조란소암라서피하이식류조직중RhoA기인적표체,억제란소암라서피하이식류적생장,기궤제가능여촉진세포조망유관.
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 HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer.Tumor-bearing nude mice were assigned randomizely to three groups:LentiRhoA-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) mm3 decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group(1190 ± 332)mm3 and PBS group (1101 ± 396) mm3 (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) and PBS group(1.00 ±0.11 ; P ＜0.01).Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P ＜ 0.01).TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) ％ compared with those in Lenti-NC group(5.2 ±±2.0)％ and PBS group(6.0 ±2.1)％ (P ＜0.01).Conclusion Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA,inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.
