中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2009年
3期
214-218
,共5页
卵巢肿瘤%端粒%末端转移酶%启动区(遗传学)%肽碎片%胶原Ⅳ型%血管生成抑制剂%腺病毒科
卵巢腫瘤%耑粒%末耑轉移酶%啟動區(遺傳學)%肽碎片%膠原Ⅳ型%血管生成抑製劑%腺病毒科
란소종류%단립%말단전이매%계동구(유전학)%태쇄편%효원Ⅳ형%혈관생성억제제%선병독과
Ovarian neoplasms%Telomerase%Promoter regions ( genetics )%Peptidefragments%Collagen type Ⅳ%Angiogenesis inhibitors%Adenoviridae
目的 构建含hTERT基因核心启动子和canstatin基因的重组腺病毒载体AdhTERT-Can,探讨其对卵巢上皮性癌(卵巢癌)的抑制作用.方法 应用分子克隆技术构建重组腺病毒载体AdhTERT-Can,并采用酶切、电泳、测序方法进行鉴定.体外实验:卵巢癌细胞株HO8910PM细胞经脂质体介导转染AdhTERT-Can后,荧光显微镜观察HO8910PM细胞的转染情况,RT-PCR技术检测HO8910PM细胞中eanstatin mRNA的表达.体内实验:建立卵巢癌裸鼠异体移植瘤动物模型,随机分为3组,AdhTERT-Can组(尾静脉注射含canstatin基因的重组腺病毒载体AdhTERT-Can上清液)、Ad-Can组(尾静脉注射Ad-Can上清液)和空白对照组(尾静脉注射磷酸盐缓冲液),比较各组裸鼠治疗后的肿瘤体积.结果成功构建重组腺病毒载体AdhTERT-Can,并经相关鉴定证实.体外实验显示,AdhTERT-Can质粒转染HO8910PM细胞后,在荧光显微镜下观察可见约60%卵巢癌细胞发出绿色荧光;RT-PCR技术检测显示,HO8910PM细胞中有canstatin mRNA的表达.体内实验显示,自治疗后8 d开始,各组肿瘤生长出现明显筹异,AdhTERT-Can组肿瘤体积明显小于Ad-Can、空白对照组(P<0.01),且随着治疗时间的延长差异越来越明显;而Ad-Can组与空白对照组比较,差异则无统计学意义(P>0.05).治疗30 d后,各组均可见肿瘤破溃和囊性变,但以AdhTERT-Can组最为明显,Ad-Can组次之;病理检查可见,各组肿瘤细胞均可见液化和坏死,尤其以AdhTERT-Can组最为明显.结论本研究成功构建了重组腺病毒载体AdhTERT-Can,且其转染卵巢癌细胞的能力较强;AdhTERT-Can质粒通过尾静脉注射能明显抑制裸鼠体内卵巢癌的生长,显示r肿瘤基因治疗的靶向性.
目的 構建含hTERT基因覈心啟動子和canstatin基因的重組腺病毒載體AdhTERT-Can,探討其對卵巢上皮性癌(卵巢癌)的抑製作用.方法 應用分子剋隆技術構建重組腺病毒載體AdhTERT-Can,併採用酶切、電泳、測序方法進行鑒定.體外實驗:卵巢癌細胞株HO8910PM細胞經脂質體介導轉染AdhTERT-Can後,熒光顯微鏡觀察HO8910PM細胞的轉染情況,RT-PCR技術檢測HO8910PM細胞中eanstatin mRNA的錶達.體內實驗:建立卵巢癌裸鼠異體移植瘤動物模型,隨機分為3組,AdhTERT-Can組(尾靜脈註射含canstatin基因的重組腺病毒載體AdhTERT-Can上清液)、Ad-Can組(尾靜脈註射Ad-Can上清液)和空白對照組(尾靜脈註射燐痠鹽緩遲液),比較各組裸鼠治療後的腫瘤體積.結果成功構建重組腺病毒載體AdhTERT-Can,併經相關鑒定證實.體外實驗顯示,AdhTERT-Can質粒轉染HO8910PM細胞後,在熒光顯微鏡下觀察可見約60%卵巢癌細胞髮齣綠色熒光;RT-PCR技術檢測顯示,HO8910PM細胞中有canstatin mRNA的錶達.體內實驗顯示,自治療後8 d開始,各組腫瘤生長齣現明顯籌異,AdhTERT-Can組腫瘤體積明顯小于Ad-Can、空白對照組(P<0.01),且隨著治療時間的延長差異越來越明顯;而Ad-Can組與空白對照組比較,差異則無統計學意義(P>0.05).治療30 d後,各組均可見腫瘤破潰和囊性變,但以AdhTERT-Can組最為明顯,Ad-Can組次之;病理檢查可見,各組腫瘤細胞均可見液化和壞死,尤其以AdhTERT-Can組最為明顯.結論本研究成功構建瞭重組腺病毒載體AdhTERT-Can,且其轉染卵巢癌細胞的能力較彊;AdhTERT-Can質粒通過尾靜脈註射能明顯抑製裸鼠體內卵巢癌的生長,顯示r腫瘤基因治療的靶嚮性.
목적 구건함hTERT기인핵심계동자화canstatin기인적중조선병독재체AdhTERT-Can,탐토기대란소상피성암(란소암)적억제작용.방법 응용분자극륭기술구건중조선병독재체AdhTERT-Can,병채용매절、전영、측서방법진행감정.체외실험:란소암세포주HO8910PM세포경지질체개도전염AdhTERT-Can후,형광현미경관찰HO8910PM세포적전염정황,RT-PCR기술검측HO8910PM세포중eanstatin mRNA적표체.체내실험:건립란소암라서이체이식류동물모형,수궤분위3조,AdhTERT-Can조(미정맥주사함canstatin기인적중조선병독재체AdhTERT-Can상청액)、Ad-Can조(미정맥주사Ad-Can상청액)화공백대조조(미정맥주사린산염완충액),비교각조라서치료후적종류체적.결과성공구건중조선병독재체AdhTERT-Can,병경상관감정증실.체외실험현시,AdhTERT-Can질립전염HO8910PM세포후,재형광현미경하관찰가견약60%란소암세포발출록색형광;RT-PCR기술검측현시,HO8910PM세포중유canstatin mRNA적표체.체내실험현시,자치료후8 d개시,각조종류생장출현명현주이,AdhTERT-Can조종류체적명현소우Ad-Can、공백대조조(P<0.01),차수착치료시간적연장차이월래월명현;이Ad-Can조여공백대조조비교,차이칙무통계학의의(P>0.05).치료30 d후,각조균가견종류파궤화낭성변,단이AdhTERT-Can조최위명현,Ad-Can조차지;병리검사가견,각조종류세포균가견액화화배사,우기이AdhTERT-Can조최위명현.결론본연구성공구건료중조선병독재체AdhTERT-Can,차기전염란소암세포적능력교강;AdhTERT-Can질립통과미정맥주사능명현억제라서체내란소암적생장,현시r종류기인치료적파향성.
Objective To study anti-tumor effects on human ovarian cancer xenografted tumors in mice by constructing adenoviral expression vector containing canstatin gene and hTERT gene core promoter (AdhTERT-Can). Methods AdhTERT-Can vector was constructed and identified by means of enzyme cutting, electrophoresis and sequencing. Then, transfected into HO8910PM cells by means of lipofectamine and confirmed by green fluorescence protein(GFP) expression under laser confocus microscope. The mRNA expression of canstatin gene was tested by RT-PCR. Human ovarian cancer xenografted tumor models in nude mice were established and randomly divided into AdhTERT-Can group received viral supematant solution of AdhTERT-Can by tail vein injection, Ad-Can groups received viral supernatant solution of Ad-Can by tail vein injection, and control groups received phosphate buffer solution (PBS). The volume of tumors were measured and compared in each group to evaluated the anti-tumor efficacy. Results All the constructed vectors of AdhTERT-Can were verified by enzymed digestion. There were green fluorescence from 60% of HO8910PM cells transfected by AdhTERT-Can under laser confocus microscope, and the mRNA expression of canstafin gene in HO8910PM cells were also verified by RT-PCR The growth of tumor in AdhTERT-Can group was significantly inhibited compared with those in Ad-Can groups and control groups from the 8th day (P <0.01 ). However, there were not significant difference between Ad-Can group and PBS group (P> 0.05). On the 30th day, the tumors showed liquefaction necrosis and cystic degeneration in each group, especially in AdhTERT-Can group. Conclusions The recombinant adenovirus vector of AdhTERT-Can has been constructed successfully and could steady express in ovarian cancer cell lines HO8910PM. The results shown that it could inhibit significantly the growth of human ovarian cancer xenografted tumors in mice and shown to be the target of gene therapy.
