含hTERT基因核心启动子和canstatin基因重组腺病毒载体的构建及其对卵巢上皮性癌的抑制作用

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.

Reconstruction of adenovirus vector contained canstatin gene and hTERT gene core promoter and its anti-tumor effect in human ovarian cancer

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.