携带人肿瘤抑素Tumstatin基因的重组腺相关病毒载体的构建及其在乳腺癌细胞中的表达研究

Construction of Recombinant AAV Vector Mediated Tumstatin Gene and the Detection of Its Expression in Breast Cancer Cell Line MCF-7

目的构建携带人肿瘤抑素Tumstatin基因的重组腺相关病毒载体,研究其在乳腺癌细胞MCF-7中的表达。方法以人胚肾293细胞为材料,用RT-PCR方法获取人肿瘤抑素Tumstatin基因,将其克隆入pAAV-MCS载体中形成重组载体pAAV-Tum质粒。用脂质体介导法将重组载体导入乳腺癌细胞株(MCF-7)建立一株稳定的表达Tumstatin基因的乳腺癌细胞株MCF/AAV-Tum,并用RT-PCR检测该细胞系中Tumstatin基因的表达。结果成功构建了pAAV-Tum重组腺相关病毒载体,筛选出乳腺癌细胞株MCF/AAV-Tum,该细胞能表达Tumstatin基因。结论构建的pAAV-Tum重组载体能在MCF-7细胞中表达,为其后的肿瘤抗血管生成治疗研究奠定了基础。

Objective To construct the recombinant adeno-associated virus（rAAV） vector containing Tumstatin gene,and to detect its expression in the MCF-7 cells.Methods The cDNA fragment of Tumstatin was obtained by a reverse transcriptasepolymerase chain reaction（RT-PCR） with total RNA extracted from 293 embryonic kidney cells.The RT-PCR product was cloned into pAAV-MCS vector,and then the pAAV-Tum plasmid was obtained.Transfection of pAAV-Tum into MCF-7 cells was performed by using lipofectin.An MCF-7 cell line that could stably express Tumstatin gene was established,and then mRNA level of pAAV-Tum was detected by RT-PCR. Results AAV vector,which contained Tumstatin gene,was constructed successfully.An MCF-7 cell line that could stably express Tumstatin gene was established. Conclusions The recombinant vector of pAAV-Tum can express in MCF-7 cell,which will lay a foundation for its anti-angiogenesis therapy of tumor.